102, capillary pressure drives the fluid 120 into the proximal portion 106 of the first porous element 101 (FIG. 1B). A fluid front 122 continues to advance from the proximal portion 106 to the distal portion 108 of the first porous element 101. Notably, because the first porous element 101 and the second porous element 110 comprise different materials exhibiting different flow characteristics, the fluid 120 flows to the distal portion 108 of the first porous element 101 before wetting the second porous element 110 (FIGS. 1C and 1D). Once the fluid front 122 reaches the distal portion 108 of the first element, the capillary exerted on the fluid 120 is altered, and the fluid 120 can flow into the second porous element 110 (FIGS. 1E-1G). The fluid 120 will flow into the second porous element 110 until the first porous element 101 and the second porous element 110 are fully saturated.

Using Darcy's law to model the flow of fluid through device 100 would incorrectly predict that the fully saturated wetting front would move into the second porous element 110 before reaching the distal portion 108 of the first porous element 101. However, Richards equation accurately predicts the fluid will only enter the second porous element 110 once the partially saturating fluid front 122 reaches the distal portion 108 of the first porous element 101 by incorporating the difference of material, and thus capillary pressures, of the first porous element 101 and the second porous element 110.

III. Select Embodiments of Pressure-Based Microfluidic Elution Devices

FIG. 2A is a top view of an elution-based microfluidic device 200 configured in accordance with an embodiment of the present technology. As shown in FIG. 2A, the device 200 includes a first porous element 201 and a second porous element 210 positioned across the first porous element 201 such that an overlapping region 215 exists between the first porous element 201 and the second porous element 210. FIG. 2B is an enlarged view of the overlapping region 215 taken along the cross-section indicated in FIG. 2A.

Referring to FIG. 2A, the first porous element 201 includes a proximal portion 202 configured to receive one or more fluids. The proximal portion 202 can optionally be coupled to a separate porous element (not shown), such as a source pad (e.g., glass fiber, nitrocellulose, cellulose, etc.) that is positioned in fluid communication with the first porous element 201 and configured to receive a volume of fluid from a fluid source (e.g., a transfer pipette). The first porous element 201 further includes a distal portion 208 in fluid communication with a waste pad 203. The waste pad 203 is positioned to receive all or a portion of the delivered fluid after it travels through the first porous element 201. In some embodiments, the waste pad 203 can be configured to drive fluid flow from the proximal portion 206 of the first porous element 201 towards the waste pad 203. For example, the waste pad 203 can comprise a material having an appropriate capillary-flow pressure profile to promote fluid flow along the first porous element 201 (e.g., cellulose, nitrocellulose, etc.). In some embodiments, however, the device 200 does not include a waste pad 203.

The second porous element 210 includes a proximal portion 214 and a distal portion 216. The proximal portion 214 can include a separate input region 212 configured to receive and/or release one or more fluids onto the proximal portion 214. In other embodiments, however, the proximal portion 214 does not include a separate input region 212, and fluid can be delivered directly to the proximal portion 214 (e.g., via pipette transfer). As best seen in FIG. 2B, the proximal portion 214 overlaps with the first porous element 201 on a first side of the first porous element 201. The distal portion 216 overlaps with the first porous element 201 on a second side of the first porous element 201 opposite the first side. Accordingly, for a fluid to travel from the proximal portion 214 to the distal portion 216 of the second porous element 210, the fluid must flow through the overlapping region of the first fluid portion, as indicated by the arrow in FIG. 2B.

The overlapping portion of the first porous element 201 and the overlapping portion of the second porous element 210 together define the overlapping region 215. As described above, the proximal portion 214 of the second porous element 210 is positioned on top of and in direct contact with the first porous element 201. As such, the overlapping portion of the first porous element 201 is in fluid communication with the overlapping portion of the proximal portion 214. In other embodiments, the first porous element 201 can be on top of the proximal portion 214 of the second porous element 210. The distal portion 216 of the second porous element 210 is positioned underneath and in direct contact with the first porous element 201. As such, the overlapping portion of the first porous element 201 is also in fluid communication with the overlapping portion of the distal portion 216. In other embodiments, the first porous element 201 can be underneath the distal portion 216 of the second porous element 210.

In some embodiments, the overlapping portions of the first and second porous elements are not in direct contact but are nevertheless configured for fluid communication. For example, a thin separation material or membrane (not shown) can be positioned between the overlapping portions of the first and second membrane. In some embodiments, the separation material can have one or more openings that are sized and shaped to more precisely control the contact between the first and second porous elements 201, 210.

The first porous element 201 and the second porous element 210 can be selected based on their respective flow pressure properties, as exhibited in water retention curves (“WRC”). Suitable materials can include, for example, glass fiber, nitrocellulose, cellulose, etc. In addition to the material used, other variables that affect flow pressure include average pore size and contact angle. In the embodiment shown in FIGS. 2A-2B, the first porous element 201 is made of a first porous material having a first average pore size, and the second porous element 210 is made of a second porous material having a second average pore size greater than the first average pore size. In the embodiment shown in FIG. 2A, for example, the first porous element 201 is made of nitrocellulose, and the second porous element 210 is made of glass fiber. In other embodiments, however, the first and second porous elements 201, 210 can be made of other porous materials so long as the WRC of the second porous element 210 is shifted to the left of the WRC of the first porous element 201.

FIGS. 3A-3D are time-lapse views of the elution-based microfluidic device 200 during various stages of fluid delivery. Beginning with FIG. 3A, a first fluid 320 can be added to the input portion 202 of the first porous element 201 (e.g., by placing the input portion 202 into a well 307). The first fluid 320 then begins wicking distally through the first porous element 201 towards the overlapping region 215. Because of the relative flow properties of the first and second porous elements 201, 210 (e.g., the WRC's of the first and second porous elements), the negative fluid pressure magnitude within the overlapping portion 215 is such that the wicking front of the first fluid 320 passes through the overlapping region 215 without wetting (or without substantially wetting) the overlapping portion of the second porous element 210, as shown in FIG. 3B. As used herein, the phrase “without wetting” refers to substantially no transfer of fluid between the first and second porous elements 201, 210. As such, the first fluid 320 will continue to imbibe the first porous element 201 only, thereby leaving the second porous element 210 substantially dry.

Once the distal portion 208 and/or waste pad 203 of the first porous element 201 becomes sufficiently saturated, a second fluid 322 can be delivered to the input region 212 of the second porous element 210 (e.g., via transfer using a pipette 305). Addition of the second fluid 322 to the second porous element 210 lowers the magnitude of the negative pressure at the overlapping region 215, thereby allowing the second fluid 322 to imbibe distally along the second porous element 210. As shown in FIGS. 3C and 3D, as the second fluid 322 passes through the overlapping region 215, the second fluid 322 mixes with first fluid 320 and pulls a volume of the first fluid 320 distally along the distal portion 216 of the second porous element 210. Additional details of multi-component microfluidic systems configured to elute a captured sample are described in U.S. Patent App. Publication No. 2016/0310942, titled “PRESSURE-BASED CONTROL OF FLUID VOLUMES AND ASSOCIATED DEVICES, SYSTEMS, AND METHODS,” the disclosure of which is incorporated by reference herein in its entirety.

IV. Select Embodiments of Pressure-Based Stopped-Flow Microfluidic Devices

FIG. 4 illustrates a microfluidic device 400 configured in accordance with select embodiments of the present technology. As illustrated, the device 400 includes a first porous membrane/element 401 and a second porous membrane/element 410. A proximal end of the first porous element 401 is fluidly coupled to a fluid inlet pad 402, which is configured to receive a first fluid. A distal end of the first porous element 401 is fluidly coupled to a waste pad 403. The second porous element 410 includes a body portion 414 and a plurality of legs 416a-c extending from the body portion 414. The second porous element 410 can also have a fluid inlet port 412 configured to receive and/or house a second fluid for delivery to the body portion 414.

The first porous element 401 can comprise any material suitable for lateral flow microfluidic devices. For example, the first porous element 401 can comprise cellulose, nitrocellulose, glass fiber, and/or other materials known in the art. In addition, the first porous element 401 can have various pore sizes and dimensions to affect the flow of fluid therethrough. The first porous element 401 can be configured to receive about 0.1 mL to about 50 mL of first fluid. For example, the first porous element 401 can be configured to receive about 0.1 mL, about 0.5 mL, about 1.0 mL about, about 2.0 mL, about 3.0 mL, about 4.0 mL, about 5.0 mL, about 10 mL, about 20 mL, about 30 mL, about 40 mL, and/or about 50 mL of the first fluid. The first porous element 401 can be fluidly coupled to the fluid inlet pad 402. In some embodiments, the fluid inlet pad 402 is integral with the first porous element 401 and is configured to directly receive a first fluid (e.g., from a pipette or well). In some embodiments, the fluid inlet pad 402 is separate from but fluidly coupled to the first porous element 401. The fluid inlet pad 402 can include a sample port (not shown) for receiving the first fluid. Regardless of the configuration of the fluid inlet pad 402, when a first fluid is added to the fluid inlet pad 402, the first fluid travels distally along the first porous element 401 towards the waste pad 403. As will be described in greater detail below, the first fluid flows from the fluid inlet pad 402 to the waste pad 403 without wetting and/or entering one or more of the plurality of legs 416a-c.

The waste pad 403 can be any structure suitable for receiving fluid from the first porous element 401. For example, the waste pad 403 can be a receiving structure comprising cellulose and/or nitrocellulose that is positioned in fluid communication with the first porous element 401 and capable of receiving fluid flowing through the first porous element 401. In some embodiments, however, the device 400 does not include a separate waste pad 403 and fluid simply flows to a distal end portion of the first porous element 401.

The second porous element 410 can comprise any material suitable for lateral flow microfluidic devices. For example, the second porous element 410 can comprise cellulose, nitrocellulose, glass fiber, and/or other materials known in the art. In addition, the second porous element 410 can have various pore sizes and dimensions to affect the flow of fluid therethrough. In some embodiments, the second porous element 410 comprises a different material than the first porous element 401. In other embodiments, the second porous element 410 comprises the same material as the first porous element 401, but with different characteristics to affect the capillary pressure exerted on the first fluid. In some embodiments, the first porous element 401 and the second porous element 410 can be selected based on their respective flow pressure properties, as exhibited in WRCs. For example, in some embodiments the first porous element 401 is made of a first porous material having a first average pore size, and the second porous element 410 is made of a second porous material having a second average pore size greater than the first average pore size. In some embodiments, the first porous element 401 is made of nitrocellulose, and the second porous element 410 is made of glass fiber. In other embodiments, however, the first and second porous elements 401, 410 can be made of other porous materials so long as the WRC of the second porous element 410 is shifted to the left of the WRC of the first porous element 401.

The second porous element 410 includes a body portion 414. As illustrated, the body portion 414 extends generally parallel to the first porous element 401, although any number of configurations are possible without deviating from the scope of the present technology. A first leg 416a, a second leg 416b, and a third leg 416c extend from the body portion 414 (collectively referred to as the “legs 416a-c”). The legs 416a-c overlap with the first porous element 401 at varying positions along the length of the first porous element 401, thereby creating overlapping portions 415a-c. For example, the illustrated embodiment depicts the legs 416a-c extending over the top of the first porous element 401, although other configurations are possible. For example, in some embodiments, the legs 416a-c extend underneath the first porous element 401 to create the overlapping portions 415a-c. In some embodiments, one or more of the legs 416a-c extend over the top of the first porous element 401 while one or more of the legs 416a-c extend underneath the first porous element 401. In yet other embodiments, one or more of the legs 416a-c do not overlap with the first porous element 401, but rather are fluidly coupled to the first porous element 401 at a distal end of each of the respective legs 416a-c.

As illustrated, the first leg 416a overlaps with a proximal portion of the first porous element 401 (e.g., a first region of the first porous element 401) to form the first overlapping portion 415a, the second leg 416b overlaps with a medial portion of the first porous element 416 (e.g., a third region of the first porous element 401) to form the second overlapping portion 415b, and the third leg 416c overlaps with a distal portion of the first porous element 416 (e.g., a second region of the first porous element 401) to form the third overlapping portion 415c. As a result, the first porous element 401 includes a first intermediate region 404a between the first overlapping portion 415a and the second overlapping portion 415b. Likewise, the first porous element 401 includes a second intermediate region 404b between the second overlapping portion 415b and the third overlapping portion 415c.

In some embodiments, the legs 416a-c are in direct contact with the overlapping portions 415a-c of the first porous element 401. In some embodiments, the legs 416a-c and the overlapping portions 415a-c of the first porous element 401 are not in direct contact but are nevertheless configured for fluid communication. For example, a thin separation material or membrane (not shown) can be positioned between the overlapping portions of the first and second porous elements 401, 410. In some embodiments, the separation material can have one or more openings that are sized and shaped to more precisely control the contact between the first and second porous elements 401, 410.

The legs 416a-c are configured to deliver fluid from the body portion 414 to the overlapping portions 415a-c of the first porous element 401. For example, when a second fluid is released into the fluid inlet port 412, the second fluid flows through the body portion 414 and into the legs 416a-c. As will be described in greater detail below with respect to FIGS. 5A-E, the second fluid flows into the first porous element 401 at the overlapping portions 415a-c.

The legs 416a-c can be integral to the body portion 414 or can otherwise be fluidly coupled to the body portion 414 such that fluid can flow between the body portion 414 and the legs 416a-c. As one skilled in the art will appreciate, the second porous element 410 can include greater or fewer legs 416a-c than depicted in FIG. 4. For example, the second porous element 410 can include one, two, three, four, five, six, seven, eight, or more legs. As will be apparent following the description below, the number of legs can determine the number of regions exhibiting stopped flow along the first porous element 401.

FIGS. 5A-E are time-lapse views of the device 400 following delivery of a first fluid 520 to the fluid inlet pad 402 and a second fluid 522 to the fluid inlet port 412. As illustrated in FIG. 5A-5C, a first fluid 520 is added to the fluid inlet pad 402 fluidly coupled to the first porous element 401. The first fluid 520 flows through the first porous element 401 via capillary flow with a partially saturated wetting front. Due at least in part to the pressure differential between the first porous element 401 and the legs 416a-c, the first fluid 520 does not substantially wet the legs 416a-c as it flows through the plurality of overlapping portions 415a-c. Rather, the wetting front of the first fluid 522 travels distally into the waste pad 403.

Following partial or complete saturation of the waste pad 403 (or at another time deemed appropriate by a user), a second fluid 522 is added to the second porous element 410 via the fluid inlet port 412 (FIG. 5D). The second fluid 522 flows through the body portion 414 and into each leg 416a-c. Unlike the first fluid 520, the pressure exerted on the second fluid 522 permits the second fluid 522 to flow from each of the legs 416a-c into the respective overlapping portions 415a-c of the first porous element 401. In some embodiments, this occurs because, even when the first porous element 401 is wetted by the first fluid 520, the second porous element 410 still exerts a greater pressure on the second fluid 522 than the first porous element 401, and the second fluid 522 simply flows from the higher-pressure legs 416a-c into the lower pressure overlapping portions 515a-c. Accordingly, the second fluid 522 mixes with the first fluid in the first porous element 401 at each of the overlapping portions 415a-c. The mixture of the first fluid 520 and the second fluid 522 slowly expands outward from the overlapping portions 415a-c in both a proximal and distal direction along the first porous element 401 (FIG. 5E).

Following mixture of the second fluid 522 and the first fluid 520 at the overlapping portions 415a-c, the pressure exerted on the first fluid 520 along the intermediate regions 404a and 404b of the first porous element 401 is also altered because there is now equivalent saturation upstream and downstream from the intermediate regions 404a-b. For example, the first fluid 520a within the first intermediate region 404a is no longer subject to the negative capillary pressure drawing the first fluid towards the waste pad 403. Rather, the mixture of the first fluid 520 and the second fluid 522 at the first overlapping portion 515a provides a first flow pressure against the first fluid 520a in the first intermediate region 404a (e.g., pushing the first fluid 520a in a distal direction), while the mixture of the first fluid 520 and the second fluid 522 at the second overlapping portion 515b provides a second flow pressure against the first fluid 520 in the first intermediate region 404a (e.g., pushing the first fluid 520a in a proximal direction). The first and second pressures created at the overlapping portions 415a-b are substantially the same in magnitude but opposite in direction. Accordingly, because substantially equal and opposite flow pressures are exerted on the first fluid 520a in the first intermediate region 404a, the first fluid 520a ceases to flow (e.g., flow is substantially stopped in the first intermediate region 404a). Likewise, the first fluid 520b within the second intermediate region 404b is also no longer subject to the negative capillary pressure drawing the first fluid 520 towards the waste pad 403. Rather, the mixture of the first fluid 520 and the second fluid 522 at the second overlapping portion 415b provides a first flow pressure against the first fluid 520b in the second intermediate region 404b (e.g., pushing the first fluid 520b in a distal direction), while the mixture of the first fluid 520 and the second fluid 522 at the third overlapping portion 515c provides a second flow pressure against the first fluid 520b in the second intermediate region 404b (e.g., pushing the first fluid 520b in a proximal direction). The first and second pressures created at the overlapping portions 415b-c are substantially the same in magnitude but opposing in direction. Accordingly, because substantially equal and opposite flow pressures are exerted on the first fluid 520b in the second intermediate region 404b, the first fluid 520b ceases to flow (e.g., flow is substantially stopped in the second intermediate region 404b). Accordingly, following delivery of the second fluid 522, flow is substantially stopped in the first and second intermediate regions 404a-c. For the same reason, flow is also stopped in the second overlapping portion 415b. Accordingly, the second porous element 410 can act as a flow control element to control the flow of the first fluid 520a along the length of the first porous element 401, with the first leg 416a and the third leg 416c acting as flow control legs and the second leg 416b acting as a reagent delivery leg.

In some embodiments, the first porous element 401 can include a capture zone having one or more immobilized capture molecules. The immobilized capture molecules can be configured to selectively bind one or more components of a biological sample included within the first fluid sample. For example, in one embodiment, the capture molecules can be chitosan configured to selectively bind DNA. In another embodiment, the capture molecules are oligonucleotides complementary to a target nucleic acid sequence. In yet another embodiment, the capture molecules are antibodies or antibody fragments configured to selectively bind specific proteins. As one skilled in the art will appreciate, however, the devices described herein can include any number of capture molecules and, accordingly, the present technology is not limited to the capture molecules expressly discussed herein.

The capture molecules (e.g., the capture zone) can be immobilized on the first porous element 401 at the second overlapping portion 415b. Accordingly, as the first fluid 520 flows distally along the first porous element (FIGS. 5A-C), the first fluid 520 will flow through the capture zone, and target components of the first fluid 520 will be selectively captured and retained at the capture zone. Following capture of the target components at the second overlapping portion 415b, the target components can be amplified, visualized, or otherwise treated or altered.

FIG. 6 illustrates another embodiment of a stopped-flow lateral flow device 600 configured in accordance with the present technology. Device 600 is similar to device 400 in a number of aspects. For example, device 600 includes a first porous element 601 and a second porous element 610. A proximal end of the first porous element 601 is fluidly coupled to a fluid inlet pad 602, which is configured to receive a first fluid. A distal end of the first porous element 601 is fluidly coupled to a waste pad 603. The second porous element 610 includes a body portion 614 and a plurality of legs 616a-c extending from the body portion 614. As illustrated, the first leg 616a partially overlaps with a proximal portion of the first porous element 601 to form the first overlapping portion 615a, the second leg 616b overlaps with a medial portion of the first porous element 616 to form the second overlapping portion 615b, and the third leg 616c partially overlaps with a distal portion of the first porous element 616 to form the third overlapping portion 615c. The first and second legs 616a-b are illustrated as only partially overlapping the first porous element 601, although in some embodiments the first and second legs 616a-b can extend over the full width of the first porous element 601, like the second leg 615b. In yet other embodiments, the second legs a-b do not overlap with the first porous element 601 but are nevertheless in fluid communication with the first porous element 601. The second porous element 610 can also have a fluid inlet port 612 configured to receive or house a second fluid for delivery to the body portion 614. Moreover, the device 600 can include a capture zone at overlapping portion 615b to selectively capture target components of the first fluid.

As illustrated in FIG. 6, the second leg 616b includes dried reaction components 630. In some embodiments, the dried reaction components 630 can be lyophilized reagents (e.g., amplification reagents). The dried reaction components 630 are positioned upstream of the overlapping portion 615b and are configured to be delivered to the overlapping portion 615b to interact with the captured target components.

FIGS. 7A-D are time-lapse views of the device 600 following delivery of a first fluid 620 to the fluid inlet pad 602 and a second fluid 622 to the fluid inlet port 612. Similar to the process described above with respect to FIGS. 5A-5C, a first fluid 720 is added to the fluid inlet pad 602 and flows through the first porous element 601 via capillary flow with a partially saturated wetting front. Due at least in part to the pressure differential between the first porous element 601 and the legs 616a-c, the first fluid 720 does not substantially wet the first legs 616a-c as it flows through the plurality of overlapping regions 615a-c. Rather, the wetting front of the first fluid 720 travels distally into the waste pad 603. However, the target components of the first fluid 720 are selectively retained by the immobilized capture molecules at the second overlapping portion 615b.

Following partial or complete saturation of the waste pad 603 (or at another time deemed appropriate by a user), a second fluid 722 is added to the second porous element 610 via the fluid inlet port 612 (FIG. 7B). The second fluid 722 flows through the body portion 714 and into each leg 716a-c. As described above, the second fluid 722 flows into the first porous element 601 at the overlapping portions 615a-c and substantially stops flow of the first fluid 720 between the first overlapping portion 615a and the third overlapping portion 615c. However, as the second fluid 722 flows through the second leg 716b, the second fluid 722 rehydrates the dehydrated reaction components 630. The rehydrated reaction components 630 flow with the second fluid 722 and into the second overlapping portion 615b (FIG. 7D). Accordingly, the rehydrated reaction components 630 mix with the immobilized target components retained in the overlapping portion 615b. Moreover, because fluid flow is subsequently stopped along the first intermediate region 704a, the second intermediate region 704b, and the overlapping portion 715b, the reaction components 630 remain adjacent the immobilized target components, thereby facilitating interaction between the reaction components 630 and the immobilized target components.

FIGS. 8A-8C illustrate another embodiment of a stopped-flow lateral device 800 configured in accordance with the present technology. Device 800 can operate in a similar manner as devices 400, 600 described above, except device 800 is configured to automate delivery of the second fluid to the second porous element. Device 800 includes a first porous element 801 and a second porous element 810. A proximal end of the first porous element 801 is fluidly coupled to a fluid inlet pad 802, which is configured to receive a first fluid. A distal end of the first porous element 801 is fluidly coupled to a waste pad 803. The second porous element 810 includes a body portion 814 and a plurality of legs 816a-c extending from the body portion 814. The second porous element 810 can also have a fluid port 812 configured to house a second fluid for delivery to the body portion 814.

Device 800 further includes an automated fluid release mechanism 840. Automated fluid release mechanism 840 is configured to automatically control release of the second fluid from the fluid port 812. Automated fluid release mechanism 840 can include a soluble retention element 842 (e.g., a water-soluble paper loop) and pinching members 841a-b. The soluble retention element 842 can retain the pinching members 841a-b in a first position in which the pinching members 841a-b pinch or otherwise restrict the fluid port 812, thereby preventing delivery of the second fluid housed in the fluid port 812 to the second porous element 810.

The soluble retention element 842 can be fluidly coupled to the waste pad 803. Accordingly, as the first fluid flows distally along the first porous element 801 and into the waste pad 803, the first fluid will also flow into the soluble retention element 842. Following sufficient saturation of the soluble retention element 842, the soluble retention element 842 substantially dissolves and/or weakens such that a force exerted on the soluble retention element 842 by the pinching members 841a-b is sufficient to break the soluble retention element 842. The soluble retention element 842 can be designed to rupture based on a predetermined level of saturation to control when the second fluid will be delivered to the second porous element 810. Following rupture of the soluble retention element 842, the pinching members 841a-b transition to a second position (FIG. 8C). In the second position, the pinching members 841a-b do not pinch or otherwise restrict the fluid port 812, thereby allowing delivery of the second fluid housed in the fluid port 812 to the second porous element 810. Following delivery of the second fluid, the device 800 can operate in a similar manner as described above with respect to devices 600, 800.

Accordingly, device 800 enables further automation of processes performed using the lateral flow devices described herein. For example, device 800 removes the need for a user to manually add the second fluid to the second porous element 810. Rather, a user simply adds the first fluid to the first porous element 801. Delivery of the second fluid to the second porous element 810 is automated, as described above. Accordingly, device 800 can be configured to automatically amplify a target component of a biological sample included within the first fluid without a user having to apply a second fluid.

FIG. 9 illustrates another stopped-flow lateral flow device 900 configured in accordance with the present technology. Device 900 can operate in a similar manner as described above with respect to devices 400, 600, 800, except that device 900 is configured to stop flow along a plurality of first porous elements 901, enabling for parallel testing for/amplification of a number of different target components of a first fluid. Device 900 includes a plurality of first porous elements 901 arranged in parallel, a sample input region 902, a rinse buffer well 950 positioned upstream from the plurality of first porous elements 901 and the sample input region 902, a waste pad 903 positioned downstream from the plurality of first porous elements 901, and a second porous element 930. The second porous element 930 extends across the plurality of first porous elements 901 to create a plurality of overlapping regions.

The plurality of first porous elements 901 can include immobilized capture molecules at respective overlapping portions with the second porous element 930. In some embodiments, the immobilized capture molecules can be different for each individual first porous element 901. In other embodiments, the immobilized capture molecules are the same for each individual first porous element 901. Following flow of the first fluid through the plurality of first porous elements 901, the rinse buffer well 950 can release a rinse buffer solution. The rinse buffer solution can flow through the plurality of first porous elements 901, thereby cleansing the capture zones of any unwanted components and preparing the retained target components for subsequent amplification.

The second porous element 930 includes a fluid well 932 configured to retain a rehydration buffer. The second porous element 930 also includes a plurality of legs 934 including dried reaction components (e.g., lyophilized amplification reagents). When the rehydration buffer is released from the fluid well 932, the rehydration buffer flows through the second porous element 930 and into the plurality of legs 934. The rehydration buffer rehydrates the dried reaction components in the plurality of legs 934 and delivers the rehydrated reaction components to the respective overlapping regions of the plurality of first porous elements. Each of the legs 934 can include the same or different reaction components depending on the target components retained in the respective overlapping regions and the desired reaction.

The device 900 can have other components depending on the intended use of the device 900. For example, the device 900 can include electric heaters (not shown) to provide isothermal conditions for amplification. Likewise, the device can be configured to enable real-time fluorescent detection of amplified target components. For example, device 900 can interface with a vacutainer extraction and plasma separation membrane (not pictured) and can use an external reader or cellphone for real-time fluorescent detection. In some embodiments, at least some of the steps performed by device 900 (e.g., release of the rinse buffer solution from the rinse buffer well 950, release of the rehydration buffer solution from the rehydration buffer well 932, etc.) can be automated. Such embodiments can incorporate one or more batteries and electronic control boards to control automated processing (not shown). Device 900 can also be enclosed within an outer casing 960 (cover not shown) configured to protect the device. In some embodiments, device 900 does not include the casing 960.

V. Select Embodiments of Pressure-Based Microfluidic Devices for Automated Dilution

The pressure-based microfluidic devices and systems disclosed herein can include intersecting networks of porous elements. For example, fluidic systems of the present technology may include a first porous network comprising one or more first porous elements, and a second porous network comprising one or more second porous elements that intersect of overlap one or more of the first porous elements. Such a configuration, for example, may be utilized for delivering differing quantities of analyte to assay zones arranged in parallel. FIG. 10, for example, shows one embodiment of a microfluidic device 1000 configured for serial dilutions. As shown in FIG. 10, the device 1000 can include a first porous element 1001 configured to receive a first fluid containing a target analyte. The device 1000 further includes a second porous network 1010. The second porous network 1010 includes a body portion 1012 for receiving a second fluid configured to dilute the first fluid sample. The second porous network 1010 can have five porous elements extending from the body portion 1012, each having a proximal leg 1014a-e and a distal leg 1016a-e. A negative control 1014f can optionally be included which delivers no sample. The second porous elements can be positioned on either side of the porous element 1001 to create overlapping regions with the first porous element 10001. As shown in FIG. 10, one or more of the overlapping regions and/or distal and proximal legs 1014a-e, 1016a-e can have the same or different shapes and/or sizes. The porous element(s) of the second porous network 1010 can have a WRC that is shifted to the left of the WRC of the first porous element 1001 to prevent the first fluid from flowing into the legs 1014a-e. For example, in some embodiments, the first porous element 1001 can be nitrocellulose and each of the second porous network 1010 can be glass fiber. The device 1000 further can include a plurality of nitrocellulose test strips 1018a-f downstream from the distal legs 1016a-e and the optional negative control 1014f. A waste pad 1003 can be positioned downstream from the nitrocellulose test strips 1018a-f.

FIGS. 11A-B are time-lapse views of the microfluidic device 1000 after a first fluid 1120 has been delivered to the first porous element 1001 and a second fluid 1122 has been delivered to the second porous network 1010. As best illustrated by FIGS. 11C-D, the arrangement of porous elements produces five different concentrations of the first fluid 1120. FIG. 11C is a schematic illustration of the diluted first fluid sample analyte in each of the nitrocellulose test strips 1018a-f FIG. 11D is a graph illustrating the normalized pixel intensity corresponding to each of the nitrocellulose test strips 1018a-f. As one skilled in the art will appreciate, the number of legs included on the second porous network 1010 can be adjusted depending on the number of desired dilutions.

EXAMPLES

Certain aspects of the present technology, as well as various applications of the present technology, may be better understood with reference to the following examples. The following examples describe specific embodiments of the present technology but in no way limit the scope of the present technology.

Example 1—Suitable Materials for Isothermal Strand Displacement Amplification Using Stopped-Flow Lateral Flow Devices

Materials capable of supporting isothermal strand displacement amplification (iSDA) using the devices described herein were screened. Suitable materials were selected that (1) have fluidic properties suitable for dual-porous membrane element device design (e.g., materials with suitable flow rates, capillary pressures, volumetric capacity, etc.), (2) are compatible with protein binders, and (3) support nucleic acid amplification. The iSDA test assay was performed with 10 uL porous membrane squares on Fusion 5, Standard 17, and 8950 glass fiber. These materials meet all assay and flow criteria by exhibiting successful amplification of 10{circumflex over ( )}4 input copies of gDNA, as illustrated in FIG. 12.

After demonstrating successful iSDA amplification within a membrane with fresh reagents, the “master-mix” of amplification reagents was lyophilized in Standard 17 (“iSDA Pads”) with fluorescent probes to enable real-time detection using an inverted fluorescent microscope. FIG. 13 shows real-time amplification data from this system where the primary and secondary membranes are modeled by stationary pads rehydrated by pipette with 10{circumflex over ( )}4 gDNA template copies. Amplification occurs in approximately 12 minutes, showing that assay kinetics are not significantly hindered in the porous media format. Work extending from these experiments includes integration of the lyophilized iSDA Pads with the devices described herein.

Example 2—iSDA on Porous Membrane Materials

The present technology enables single template immuno-iSDA, which enables the signal amplification of a traditional lateral flow immunoassay (LFIA) through isothermal amplification of a detection-molecule-bound synthetic oligo template. In one example, the present technology incorporates an influenza nucleoprotein (NP) immunoassay, which is visibly detected with streptavidin-coated AuNPs, with biotinylated synthetic oligonucleotide templates and isothermal strand displacement amplification (iSDA). This assay is called “T1” (for single template) immuno-iSDA. FIG. 14 shows a schematic of one potential assay stack for select embodiments of the present technology. The illustrated assay stack comprises capture antibody 1461 immobilized on the porous element 1404 (e.g., HF120), an analyte 1465 (e.g., influenza A recombinant NP), biotinylated detection antibody 1462, streptavidin-coated AuNPs 1464, and biotinylated single-stranded oligonucleotide template 1463 (e.g., mecA template).

The oligonucleotide template 1463 can be based on the sequence of an existing iSDA assay target (e.g., the mecA gene in methicillin-resistant bacteria) and can be amplified to detectable levels with iSDA reagents (e.g., polymerase, nicking enzyme, target-specific primers, and dNTPs with appropriate salts and buffer) when incubated at 50° C. for about 15 min or more. Comparison of in-tube amplification of biotinylated, synthetic, mecA templates and in-tube amplification of genomic DNA that contained the mecA target confirms amplification. For example, FIG. 15 shows the presence of amplification products when oligo template or genomic DNA were added (columns 2 an 3), and the absence of amplification products when they were not added (column 1), using polyacrylamide gel electrophoresis (PAGE) analysis.

Next, the amplification of the biotinylated oligo template by iSDA within the matrix of a first porous member (HF120) and a second porous membrane (Standard 17) was tested. In this embodiment, the materials were rinsed, allowed to dry, pipetted with an iSDA reaction containing 10⁸copies of biotinylated oligo template, sealed in a hybridization chamber, and incubated at 50° C. for 30 minutes in a clamshell heater. The fluid was then centrifuged out of each material and analyzed by PAGE gel. FIG. 16 shows the PAGE analysis of these iSDA reactions on (a) GF/STD17 blocked by 1% BSA and 0.1% Tween 20 in water (column A), (b) HF120 rinsed by 80 μL of 1% BSA and 0.1% Tween in KiPO₄(column B), and (c) HF120 rinsed with 80 μL of 1% BSA and 0.1% Tween 20 in nuclease-free water (column C). One negative sample (N) and two positive samples (P) were tested for each set up. As illustrated, the incorporation of test with the HF120 and Standard 17 matrices did not substantially affect amplification.

The additional following experimental conditions were used to demonstrate the T1 immuno-iSDA assay. First, the immunoassay capture antibody was hand-spotted on nitrocellulose strips (HF120) at 1 mg/mL and allowed to dry. Next, the nitrocellulose strips were combined with cellulose (C/083) waste pads and placed in successive microplate wells, which contained: (1) nucleoprotein (NP) analyte pre-mixed with detection antibody, (2) streptavidin-coated gold nanoparticle pre-mixed with various amounts of oligonucleotide template, (3) PBS-based wash solution, and (4) KiPO₄-based rinse solution. The rinsing step washes away the PBS, which is required for the immunoassay but is not compatible with iSDA. Next, the cellulose waste pads were removed, and glass fiber source pads (GF/8950), which contained iSDA reagent master mixes, were placed on the nitrocellulose strips over the capture region. This assembly was sealed in a hybridization chamber and incubated on a clamshell heater at 50° C. for 30 minutes. Finally, the fluid was centrifuged out of each material and analyzed by PAGE gel. GF/8950 was used because it also supports iSDA reactions and is another glass fiber material that can be incorporated into the present technology.

FIG. 17 shows the PAGE gel analysis of this experiment. As illustrated, the use of 10⁵copies/μL of oligonucleotide template yielded amplification products in the positive NP condition but did not yield amplification products in the negative NP condition. This demonstrates T1 immuno-iSDA in paper, validating one potential use of the devices described herein. FIG. 17 also indicates that the amount of oligo template relative to the amount of analyte can affect assay performance. Relatively too much template (10⁷copies/μL) appeared to yield false negatives and relatively too little template (10³copies/μL) appeared to yield false negatives. However, PAGE analysis is not a highly sensitive detection method. Accordingly, use of more sensitive detection methods, such as LFT and fluorescence detection, can measure signal amplification levels and assay LOD. Thus, embodiments of the devices herein decouple the dependence of captured oligo template on analyte amount, which can significantly increase the dynamic range of the T1 immuno-iSDA assay.

Additional steps that can be incorporated with the methods described herein include, for example: (1) a rinse with KiPO₄to remove PBS after running the immunoassay and before adding iSDA reagents, (2) use of BSA as a blocking agent for the T1 immuno-iSDA, (3) use of Tween-20 in buffers to maintain the wettability of HF120, and (4) manual addition of glass fiber pads (GF/8950) for introducing iSDA reagents to nitrocellulose (HF120) after running the immunoassay.

Example 3—Preparation of Lateral Flow Device for iSDA

A. Reagent Preparation

Reagents were prepared with sterile molecular biology grade water. Low-molecular-weight chitosan oligosaccharide lactate (average MW 5000), Tris base, 2-(N-morpholino)-ethanesulfonic acid (MES), achromopeptidase (ACP, A3547), ethanol, erioglaucine, and glycogen were used. A working solution of chitosan was prepared at 1 mg/mL in 50 mM MES at pH 5. A working solution of erioglaucine was prepared at 2 mg/mL in sterile water. A working solution of ACP was prepared at 20 U/μL in 10 mM Tris, pH 8. The 50 mM MES DNA wash buffer was prepared in sterile water and the pH was adjusted to 5. The 50 mM Tris DNA elution buffer was prepared in sterile water and the pH was adjusted to 9. De-identified human urine samples were provided by the Global Health STI Laboratory at the University of Washington Harborview Medical Center in Seattle, WA, USA. Urine sample pH and salinity was measured using a pH/conductivity meter. The 40 nm InnovaCoat streptavidin-gold conjugates were purchased from InnovaBiosciences (Cambridge, United Kingdom). Triethyl ammonium bicarbonate (TEAB) at 0.1 M was diluted to 75 μM with sterile water. A biotinylated probe (comprising the sequence: 5′-TTTTTTTTTTTTTTTTTTTT-biotinTEG-3; “T20-biotin”) was added to the TEAB to create a working solution of T20-biotin probe at 200 μM in 75 μM TEAB. A working solution of gold nanoparticles was prepared using 40 nm, streptavidin-coated, gold nanoparticles diluted to OD 0.0625 in phosphate-buffered saline buffer containing Tween-20 and 1% (w/v) bovine serum albumin (PBST+BSA).

B. Material Fabrication and Patterning

Porous and plastic materials were cut to their final shapes using a CO₂laser. For DNA purification/concentration, Fusion 5 membranes were patterned with chitosan and stored in a desiccator. Glass fiber and cellulose membranes were used without modification. Test cards were made with 0.254 mm-thick Melinex backing with adhesive on one side. For parallel dilution, nitrocellulose membranes (HF135) were patterned with T20-biotin probe by a piezoelectric printer. Glass fiber, Fusion 5, and CFSP223000 cellulose membranes were used without modification. The T20-biotin probe solution was filtered using a 0.2-μm nylon membrane at 8000 g for 5 minutes prior to spotting. Test lines were created by placement of 20 spots, spaced 250 μm apart, with 30 droplets per spot. The volume of each droplet was 450-500 pL. After spotting them with T20-biotin probe solution, the nitrocellulose membranes were UV treated for 8 minutes with a UV transilluminator at 300-310 nm and stored under desiccation before use.

C. Device Configuration

Further tests were conducted using devices with Fusion 5 as the primary porous membrane, glass fiber as the secondary porous membrane, and cellulose as the waste pad; and with the membranes held in place by the adhesive layer of the Mylar test card. Device tests were run as previously described, with the following changes. Test samples were prepared as 10 μL of fragmented MRSA gDNA (approximately 104-105 copies/μL) diluted into 990-3,990 μL of either buffer or discarded urine samples for a resulting concentration of approximately 102-103 copies/mL. The full sample volume was then introduced to the upstream end of the first porous membrane of the device and allowed to flow through completely, followed by 250 μL of wash buffer (50 mM MES, pH 5). Next, 250 μL of the elution buffer (50 mM Tris, pH 9) was introduced to the upstream end of the second porous membrane. After the second porous membrane was fully saturated, the chitosan section (e.g., the capture zone) and the elution membrane section were removed from the device. The fluid was recovered from each membrane section by centrifugation at 10,000×g for 3 minutes. The number of copies of target DNA in the fluid recovered from each membrane section was quantified using qPCR, then and adjusted for the volume recovered from each section. FIG. 18 is a graph of the DNA recovery following in-membrane DNA purification and amplification from 1 mL urine samples. Generally, as sample pH increased, recovery of the target DNA decreased (with the exception of patient 10). The non-target DNA in the sample of patient 10 likely overwhelmed the capacity of the DNA purification system leading to poor recovery of target DNA.

Devices were also automated as described above in FIG. 9. The lever arms were set to pinch closed a section of silicone tubing, which was mated to a razor-cut section of a plastic syringe acting as an elution buffer reservoir. A water-soluble membrane looped around laser-cut acrylic lever arms. The entire device was placed over a sample collection well to which the sample was added to initiate the test.

Device automation tests were run with test samples prepared as 10 μL of fragmented MRSA gDNA (approximately 104-105 copies) diluted into 5 mL of buffer (discarded urine samples were not used for these tests) for a resulting concentration of approximately 101-102 copies/mL. In these tests, however, 750 μL of elution buffer (50 mM Tris, pH 9) was loaded into the elution buffer reservoir prior to test initiation. Next, the full volume was added to the sample collection well and allowed to flow through the device without a wash step. The elution buffer automatically released upon sufficient saturation of the waste pads, which enabled sample fluid to traverse the trigger fluid delivery pathway, saturating and dissolving a break in the water-soluble paper loop. After the secondary membrane fully saturated, fluid was recovered from the elution section of the device by centrifugation at 10,000×g for 3 minutes. Recovery of target DNA can be quantified using qPCR results and adjusting for the elution volume.

D. Nucleic Acid Preparation and Quantification

Nucleic acids were purified from 500 μL of discarded urine samples by first harvesting any intact cells by centrifugation at 13,000×g for 3 minutes. The supernatant was saved in a separate tube and cells were resuspended in an equal volume of 10 mM tris at pH 8. Cells were treated with 10 μL of 20 U/μL ACP, incubated at room temperature for 2 minutes, and then heated to 95° C. for 5 minutes. Nucleic acids were purified from both the cell lysate and the supernatant by ethanol precipitation using 1/10 volume of 3 M sodium acetate (pH 5.2), 2 volumes of cold 100% ethanol, and 1/100 volume of 20 mg/mL glycogen. The solutions were mixed by inversion 10-12 times followed by incubation at −20° C. for 15 minutes. After incubation, the samples were centrifuged for 15 minutes at 21,000×g (maximum speed). The glycogen and nucleic acids formed a visible white pellet in the bottom of the tube. The supernatant was removed and the pellet was washed with 1 mL of 70% ethanol. The sample was mixed by inversion 10-12 times followed by centrifugation at maximum speed for 5 minutes. The supernatant was removed and the pellet air-dried for 10 minutes at room temperature. The pellet was then resuspended in 50 μL of sterile water and incubated at 37° C. for 10 minutes. The resulting concentration of nucleic acids was then quantified.

E. MRSA Ldh-1 Gene Quantification

DNA recovery was quantified using a qPCR kit for the ldh-1 gene provided by the ELITechGroup (ELITechGroup Molecular Diagnostics, Bothell, WA, USA). The 20 μL reactions were run on a real-time PCR instrument using the following protocol: 50° C. hold for 2 minutes; 93° C. hold for 2 minutes; 45 cycles of 93° C. for 10 seconds, 56° C. for 30 seconds, and 72° C. for 15 seconds; and 72° C. hold for 5 minutes. Fluorescence data were collected in the Texas Red channel during the 56° C. step of each cycle. The qPCR results were analyzed using the automated threshold cycle (CT) value calculation in the CFX Manager software (Bio-Rad, Hercules, CA, USA), which is sensitive down to approximately 10¹copies of the target sequence.

Examples 4-41

Several additional examples of the present technology are set forth in the following examples.

4. A microfluidic device, the device comprising:

- a first membrane element having a first end region, a second end region, and a flow region extending between the first end region and the second end region, wherein the first end region is configured to receive a first fluid;
- a second membrane element having a fluid input region and one or more legs extending from the fluid input region, wherein each of the one or more legs is fluidly coupled to the fluid input region and the flow region of the first membrane element; and a fluid port in fluid communication with the fluid input region of the second membrane element and configured to receive a second fluid;
- wherein the device is configured such that—
  - delivery of the first fluid to the first end region of the first membrane element causes the first fluid to flow from the first end region of the first membrane element to the second end region of the first membrane element via the flow region without substantially wetting the one or more legs, and
  - delivery of the second fluid to the fluid port causes the second fluid to flow through (a) the fluid input region, (b) the one or more legs, and (c) at least partially into the flow region of the first membrane element, wherein flow of the second fluid into the flow region changes a flow pressure exerted on the first fluid, thereby substantially stopping flow of the first fluid along at least a portion of the flow region.

5. The microfluidic device of example 4 wherein the one or more legs includes a first flow control leg and a second flow control leg, and wherein the first flow control leg is fluidly coupled to the flow region at a first position and the second flow control leg is fluidly coupled to the flow region at a second position downstream from the first position.