Hybrid lateral flow test

Abstract

A hybrid lateral flow test device for detecting a target analyte in a sample includes an enclosed channel with a test area for binding to the target analyte. The channel is a microfluidic channel having an upstream opening and a downstream opening, an upstream wicking area abutting the upstream opening of the channel, a downstream absorbent pad abutting the downstream opening of the channel, a sample receiving area upstream of or encompassing the wicking area, and labeled conjugates for binding with the target analyte upstream of the test area.

Description

HYBRID LATERAL FLOW TESTCROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to US Provisional Application No. 63 / 625,183 filed January 25, 2024 titled “Hybrid Lateral Flow Test” which is hereby incorporated by reference in its entirety.BACKGROUND

[0002] An immunoassay is a biochemical test that detects the presence of a molecule in a solution using an antibody or antigen. Immunoassays are used in a wide variety of rapid diagnostic tests (RDTs) that are single use, inexpensive, and provide results in a matter of minutes. Common formats for RDTs include lateral flow tests and vertical flow tests. RDTs may be used in many industry sectors such as clinical, veterinary, agricultural, food and environmental sectors to confirm the presence or absence of pathogens, biomarkers or contaminants.

[0003] Lateral flow test strips have been widely used for detecting the presence of target analytes, such as the SARS-CoV-2 antigens, in an individual for diagnostic purposes or in other specimen types like wastewater for detection purposes. Often, these test strips are provided as kits for in-home or point-of-care use where all components of the kit are disposable.

[0004] FIG. 1A is a schematic diagram illustrating principles of a prior art lateral flow test strip. In-home test or point-of care kits may instruct an individual or a healthcare worker to collect a biological sample potentially containing an analyte 116 of interest by using a swab and immersing the swab in a fluid. A few drops of the fluid 102 are added to a sample pad 104 at one end of the lateral flow test strip. Through capillary action, fluid 102 is drawn across the lateral flow test strip to conjugate pad 106.

[0005] Conjugate pad 106 stores antibodies 118 or other molecules that bind the analyte 116. Antibodies 118 are conjugated to chemical labels 117 that induce or amplify a signal detectable by the naked eye at sufficiently high concentrations. As sample fluid 102 is drawn into conjugate pad 106, analyte 116 present in sample fluid 102 will bind to the conjugated antibodies and continue to migrate along the test strip.

[0006] From conjugate pad 106, the labeled antibodies with bound analyte 119 (FIG. IB) flow into a nitrocellulose membrane 108. A row of antibodies 118 corresponding to those on conjugate pad 106 are coated on nitrocellulose membrane 108 at test line 112. If target antigens of the analyte 116 are present in the sample fluid and have bound to the labeledantibodies, those antigens will bind to the monoclonal antibodies at the test line 112. This results in an antibody-antigen-antibody-label that shows as a line visible to the naked eye at the test area, as shown in FIG. 1C. The quality control area (C line 114) is coated with antibodies 121 that bind the labeled antibodies (117, 118) that do not have a bound analyte, resulting in a visible C line 114. If the C line 114 does not show any color, it indicates that the result is invalid. Absorbent pad 110 will absorb excess fluid 102.

[0007] Prior art in-home test kits often use colloidal gold nanoparticles as chemical label 117, which generate a colorimetric signal and have low sensitivity. Sample fluids with a small copy number of antigens may not provide enough color in the test area for detection by the naked eye, resulting in doubt on the part of the user that the antigen has been detected. Tests that generate a fluorescent signal can provide much higher sensitivity than colloidal gold colorimetric tests. The fluorescent signal can be generated via excitation of fluorescent labels by ultraviolet (UV) radiation or even visible light, such as near-UV wavelengths in the case of horse radish peroxidase.

[0008] A microfluidic device is another technique used for detecting the presence of target analytes, such as the SARS-CoV-2 antigens, whether in an individual for diagnostic purposes or in other specimen types like wastewater. Microfluidic devices may be used in many industry sectors to confirm the presence or absence of pathogens, biomarkers or contaminants, for example.

[0009] FIGS. ID - IE are schematic diagrams illustrating principles of a prior art microfluidic device 130. Point-of care tests may require the individual or a healthcare worker to collect a biological sample, for example by using a swab, then to immerse the swab in a fluid, then add a few drops of the fluid 102 containing analyte 116 to a loading inlet at one end of a channels 132 of microfluidic device 130. Through capillary action, fluid is drawn across the microfluidic device. In the case of the prior art in FIGS. ID - IE, the microfluidic device is a paper-based chip with microfluidic channels.

[0010] After a period of time, antibody-conjugated fluorescent particles 134 are loaded into each channel 132. They also flow through the paper microfluidic channels passively through capillary action and mix with sample fluid. If target analytes are present, this mixing leads to particle immunoagglutination 136, which is detected with a fluorescence microscope.

[0011] Multiple images must be taken per channel, starting from the particle front (the farthest area that the particles flowed within the channel) and moving the field of view toward the loading inlet, all while refocusing the fluorescence microscope while moving along the channel. The test requires a skilled technician who uses may use a smartphone fluorescence microscope to search for the immunoaggluntinated fluorescent particles.Fluorescent particles cannot be seen by the naked eye. The difficulty in observing fluorescent particles with the naked eye has precluded using them in home tests.SUMMARY OF THE EMBODIMENTS

[0012] A hybrid lateral flow test device for detecting a target analyte in a sample includes an enclosed channel with at least one test area for binding to the target analyte, said channel comprising an upstream opening and a downstream opening; an upstream wicking area abutting the upstream opening of the channel; a downstream absorbent pad abutting the downstream opening of the channel; a sample receiving area upstream of or encompassing the wicking area; and labeled conjugates for binding with the target analyte upstream of the at least one test area.BRIEF DESCRIPTION OF THE FIGURES

[0013] FIGS. 1A - IB illustrate principles of a prior art lateral flow test cartridge.

[0014] FIG. 1C illustrates positive and negative test results of the lateral flow test cartridge of FIGS. 1A - IB.

[0015] FIG. ID - IE illustrate principles of a prior art microfluidic test device.

[0016] FIG. 2A shows a perspective exploded view of a hybrid lateral flow test, in embodiments.

[0017] FIG. 2B shows a perspective detailed view of the hybrid lateral flow test of FIG. 2A.

[0018] FIG. 2C shows a perspective view of the assembled hybrid lateral flow test of FIG. 2A.

[0019] FIGS. 3A - 3C show a perspective views of a hybrid lateral flow test, in embodiments.

[0020] FIGS. 4A is a schematic cross-sectional view of a prior art nitrocellulose paperbased lateral flow test.

[0021] FIGS. 4B and 4C are schematic cross-sectional views of a hybrid lateral flow test device having a microfluidic channel, in embodiments.

[0022] FIG. 4D and 4E are schematic cross-sectional views of a hybrid lateral flow test device where backing material serves as the lower surface of the microfluidic channel, in embodiments.

[0023] FIG. 4F is a schematic cross-sectional view of a hybrid lateral flow test device with a combined sample-conjugate pad, in embodiments.

[0024] FIG. 4G is a schematic cross-sectional view of a hybrid lateral flow test device with wicking pads, in embodiments.

[0025] FIG. 4H is a schematic cross-sectional view of a hybrid lateral flow test device with fluid control features, in embodiments.

[0026] FIG. 41 is a schematic cross-sectional view of a hybrid lateral flow test device with conjugates in the microfluidic channel, in embodiments.

[0027] FIG. 41 is a schematic cross-sectional view of a hybrid lateral flow test device with a sample well, in embodiments.

[0028] FIG. 4K is a schematic cross-sectional view of a hybrid lateral flow test device with a filter pad, in embodiments.

[0029] FIGS. 4L and 4M are schematic cross-sectional views of a hybrid lateral flow test device with a sample well, in embodiments.DETAILED DESCRIPTION OF THE EMBODIMENTS

[0030] Exemplary embodiments will be described in detail herein, with examples thereof represented in the drawings. When the following descriptions involve the drawings, like numerals in different drawings represent like or similar elements unless otherwise indicated. Implementations described in the following exemplary embodiments do not represent all implementations consistent with the present disclosure. Instead, they are merely examples of apparatuses and methods consistent with some aspects of the present disclosure as detailed in the appended claims.

[0031] Rapid diagnostic tests (RDTs) are typically single-use, disposable cartridges that may be used in many industries such as clinical, veterinarian, agricultural, food-processing and environmental sectors to confirm the presence or absence of pathogens, biomarkers, chemicals or contaminants. They may have several form factors, including lateral flow, vertical flow tests and microfluidic devices. Embodiments disclosed herein may be used to detect respiratory viruses such as SARS-CoV-2, influenza viruses, and respiratory syncytial viruses (RSV). In addition, blood borne substances such as antibodies or delta-9- tetrahydrocannabinol (THC) may also be detected using a RDT. Embodiments disclosed herein may be used with environmental contaminants. For purposes of illustration, embodiments will be discussed with regard to the detection of human-borne viruses but would apply equally to any of the detection of any analyte.

[0032] A sample containing an analyte of interest could be acquired from a throat gargle, nasal secretions, saliva, cheek swab, sweat, pus, cerebrospinal fluid, blood, tears, soil, feces, urine, breath, waste water, or drinking water, for example. Samples may be acquired ina liquid form or combined with a liquid solution prior to being used with an RDT.

[0033] FIG. 2A shows a perspective exploded view of hybrid lateral flow test device 200. FIG. 2B shows a perspective detailed view of the hybrid lateral flow test device of FIG. 2A. FIG. 2C shows a perspective view of the assembled hybrid lateral flow test device of FIG. 2 A. FIGS. 2 A - 2C are best viewed together in the following description.

[0034] Backing material 202 provides a base support for all components of device 200. The length and width of backing material 202 may be selected based on the target analytes for detection, for example. Backing material 202 is a material that allows for the smooth and rapid flow of fluid through the microfluidic channel. Materials may include any or a combination of acrylic, glass, PVDC, other plastics. In some embodiments, the backing material is transparent or translucent, allowing illumination from below.

[0035] Backing material 202 supports several components. Sample pad 204 is positioned at one end of backing material 202 to receive a sample fluid that may contain an analyte for testing. Although not shown in FIGS. 2 A - 2C, device 200 may be contained within a housing having a sample well adjacent to sample pad 204 for receiving the sample fluid. Upstream conjugate pad 206 is physically adjacent to and downstream of sample pad 204. Upstream conjugate pad 206 stores antibodies or other molecules that bind the analyte as described above for conjugate pad 106. The antibodies or molecules (such as (i) antisense oligonucleotides for binding targeted stretches of RNA or DNA analytes, (ii) aptamers, or (iii) peptides) are conjugated to chemical labels that induce a signal detectable at sufficiently high concentrations. For clarity of illustration, the conjugated antibodies and labels are not shown in FIGS. 2A - 2C. The chemical labels may be gold or fluorescent nanoparticles, for example. If the target analyte is present, the conjugated antibodies will bind to the target analyte as the sample fluid flows from sample pad 204 through conjugate pad 206.

[0036] Absorbent pad 208 is positioned at an end of backing material 202 opposite sample pad 204. Downstream conjugate pad 210 is upstream of and physically adjacent to absorbent pad 208. Upstream conjugate pad 206 and downstream conjugate pad 210 are separated by microfluidic channel 212. In embodiments, sample pad 204, upstream conjugate pad 206, downstream conjugate pad 210, and absorbent pad 208 may be adhered to backing material 202. In embodiments, backing material 202 may be formed as the base of a test cartridge that forms a housing enclosing the components of FIGS. 2A - 2C.

[0037] Microfluidic channel 212 is an open area between upstream conjugate pad 106 and downstream conjugate pad 210 that is formed by backing material 202, sidewalls 214 and cover plate 216. In embodiments, side walls 214 have a depth D of approximately 10 and 10,000 micrometers. Depth D may be selected based on the target analytes for detection, forexample. As shown by the dotted lines in FIG. 2 A, sidewalls 214 and cover plate 216 extend approximately from sample pad 204 to absorbent pad 208 and cover portions of upstream conjugate pad 206 and downstream conjugate pad 210, in other words, along only a portion of length L of backing material 202. Either or both of sidewalls 214 may extend along the entire length L of backing material 202, or any selected portion in addition to the specific embodiment shown in FIGS. 2 A - 2C.

[0038] In embodiments, sidewalls 214 may be integrally formed with backing material 202, or may be separate elements that are adhered or fused to backing material 202 and cover plate 216, such as double-sided tape. For example, backing material 202 may be widened in the vicinity of microfluidic channel 212 to form flanking regions where double-sided tape may be used onto achieve the elevation difference between backing material 202 and cover plate 216. In embodiments, sidewalls 214 may be made of plastic, glass, double-sided tape, or another material sandwiched between backing material 202 and cover plate 216 and adhered to each. Further, the surface of backing material 202 may be etched down to form a cavity along some or all of its length to form sidewalls 214. Parts of this cavity may be wider than the channel where results are visualized. For example, upstream of the results viewing channel the cavity may widen to accommodate a sample pad and conjugate pad(s) that are wider. Similarly, downstream of the results viewing area the cavity may be wider to accommodate a absorbent pad that is wider than the results viewing area.

[0039] Sample fluid containing an analyte is drawn from upstream conjugate pad 206 into microfluidic channel 212 by capillary action. Absorbent pad 208 will absorb sample fluid after it is drawn through microfluidic channel 212. The size of absorbent pad 208 will impact the quantity of fluid drawn through channel 212 and may be selected based on the target analytes, for example. The flow rate of sample fluid through microfluidic channel 212 may also be controlled by the size and material of upstream conjugate pad 206 and downstream conjugate pad 210, which may be formed of nitrocellulose membrane, for example. Microfluidic channel 212 is initially filled with air, but when a sample is dropped on the sample pad 204, and flows by capillary action through upstream conjugate pad 206 to the upstream end of channel 212, the channel begins to fill with fluid and the fluid moves towards downstream conjugate pad 210. Once the fluid reaches absorbent pad 208, it continues to move through channel 212 until the fluid in sample pad 204 is depleted. Then, as long as absorbent pad 208 still has room for fluid, the remaining fluid flows into absorbent pad 208 and microfluidic channel 212 becomes filled once again by air.

[0040] Results area 218 of microfluidic channel 212 includes test line 220 and control line 222. These lines may be printed on backing material 202. Control line 222 is a qualitycontrol line coated with antibodies that bind the chemical labels in conjugate pad 206, resulting in a visible C line. If the C line does not show any color, it indicates that the result is invalid. Test line 220 includes antibodies or other molecules that will bind with the target analyte. If antigens are present in the sample fluid, those antigens will bind to the antibodies / molecules in the test area. This results in an antibody-antigen-antibody-chemical label sandwich that shows as a line visible to the naked eye at the test area.

[0041] FIGS. 3A - 3C show a perspective views of a hybrid lateral flow test device 300, in embodiments.

[0042] Sample receiving area 302 receives a sample fluid to be tested for a target analyte. In embodiments, sample receiving area 302 may be an absorbent pad or a well, for example. Upstream absorbent pad 304 is physically adjacent to sample receiving area 302. Upstream absorbent pad 304 may be loaded with antibodies or other molecules capable of binding the target analyte. The antibodies or molecules (including (i) antisense oligonucleotides for bind targeted stretches of RNA or DNA analytes, (ii) aptamers, or (iii) peptides) are conjugated to chemical labels that induce a signal detectable at sufficiently high concentrations. The chemical labels may be gold or fluorescent nanoparticles, for example. If the target analyte is present, the conjugated antibodies will bind to the target as the analyte flows from sample receiving area 302 through upstream absorbent pad 304.

[0043] Plate 306 is downstream of upstream absorbent pad 304. Plate 306 forms a bottom of channel 308, which may be a microfluidic channel. In embodiments, upstream absorbent pad 304 may abut channel 308 or protrude into an upstream opening of channel 308. Downstream absorbent pad 311 is downstream of plate 306 and may abut channel 308 or protrude into a downstream opening of channel 308.

[0044] A test area 310 is formed on the surface of plate 306. As described above for FIGS. 2A - 2C, test area includes one or more test lines and control lines located on the surface of plate 306. The test line includes antibodies or other molecules that will bind with the target analyte. If antigens are present in the sample fluid, those antigens will bind to the antibodies / molecules in the test area. This results in an antibody-antigen-antibody-chemical label sandwich that shows as a line visible to the naked eye at the test area.

[0045] In embodiments, sample receiving area 302, upstream absorbent pad 304, plate 306, and downstream absorbent pad 311 may be adhered to a backing material (not shown). In embodiments, backing material 202 may be formed as the base of a test cartridge that forms a housing enclosing the components of FIGS. 3A - 3C. In embodiments, sample receiving area 302, upstream absorbent pad 304, plate 306, and downstream absorbent pad 311 may be adhered to a test cartridge directly.

[0046] Microfluidic channel 308 is formed by plate 306 and sidewalls 312. In embodiments, sidewalls 312 may be formed of adhesive tape secured to plate 306 to provide a channel depth D of approximately 10 and 10,000 micrometers. Depth D may be selected based on the target analytes for detection, for example. Cover plate 314 is positioned on top of sidewalls 312 and either abuts upstream absorbent pad 304 and downstream absorbent pad 311, or partially covers them. Channel 308 is fully enclosed by plate 306, sidewalls 312 and cover 14 except for upstream opening 316 and downstream opening 18.

[0047] FIGS. 4A is a schematic cross-sectional view of a prior art nitrocellulose paperbased lateral flow test cartridge. The prior art test cartridge 400 may be a rapid diagnostic test (RDT) for detecting a virus, for example, and includes a sample pad 404, conjugate pad 406 and absorbent pad 414, as shown. A sample fluid is deposited on sample pad 404, then drawn by capillary action from upstream to downstream starting with the sample pad 404, through the conjugate pad 406, then nitrocellulose membrane 408 including a test line 410 and a control line 412, to the absorbent pad 414. A backing material 402 supports the paper-based pads.

[0048] FIGS. 4B - 4K are schematic cross-sectional views of hybrid lateral flow test devices as disclosed herein. In embodiments, any of the devices of FIGS. 4B - 4K may be used as an RDT by a user or in a point-of-care facility. Nitrocellulose membrane 408 of FIG. 4A, is replaced in the devices of FIGS. 4B - 4K by a microfluidic channel that uses laminar flow instead of capillary action to move a sample fluid to be tested for a target analyte across one or more test and control lines. Laminar flow of a fluid occurs in the direction of the flow line shown. Although several embodiments are discussed below, other variations are contemplated. For example, any of the features discussed herein for a single embodiment may be combined with features discussed for a different embodiment.

[0049] For ease of illustration, the devices and channels are shown as generally rectangular, however, other shapes are possible. In general, a channel is fully enclosed except its upstream opening and downstream opening. It does not need to be rectangular. It can have curved walls and ceiling for example, like many microfluidic devices.

[0050] FIG. 4B shows a hybrid lateral flow test device 420 having a microfluidic channel 430. Sample pad 424, conjugate pad 426 and absorbent pad 428 are mounted to a backing material 422, which may be transparent to visible / UV / infrared light which permits lighting from below, for example, with an LED that excites a fluorescent reporter molecular or nanoparticle. The microfluidic channel 430 is enclosed by top plate 432 and bottom plate 434. Test line 436 and control line 438 are printed on bottom plate 434. In any of the embodiments discussed herein, the test and control lines may be a dot or other symbol insteadof a line, and they may be printed on a cover plate as well as on an inside surface of backing material or any other surface inside of microfluidic channel 430. As shown in FIG. 4B, conjugate pad 426 and absorbent pad 428 protrude slightly into an upstream opening and a downstream opening of the channel, respectively.

[0051] FIG. 4C shows a hybrid lateral flow test device 440 with a microfluidic channel 430 that is similar to FIG. 4B, but conjugate pad 426 and absorbent pad 428 to not protrude into microfluidic channel 430 but instead, abut the upstream and downstream openings of the channel 430.

[0052] FIG. 4D shows a hybrid lateral flow test device 460 where backing material 422 serves as the lower surface of the microfluidic channel 430. FIG. 4D is similar to FIG. 4B wherein conjugate pad 426 and absorbent pad 428 protrude into the channel.

[0053] FIG. 4E show a hybrid lateral flow test device 470 where backing material 422 serves as the lower surface of the microfluidic channel 430. FIG. 4E is similar to FIG. 4C where conjugate pad 426 and absorbent pad 428 abut the upstream and downstream openings of the channel instead of protruding into them.

[0054] FIG. 4F shows a hybrid lateral flow test device 480 where the sample and conjugate pads are combined into a combined sample-conjugate pad 482 which has been printed with molecules conjugate with labels to bind to a target analyte. A sample fluid is added to sample-conjugate pad 482 and flows from the sample-conjugate pad 482 into microfluidic channel 430 where it passes a test area formed by one or more test lines 436 and control lines 438. Sample-conjugate pad 482 may protrude into the channel as shown in FIGS. 4B and 4D or abut it as shown in FIGS. 4C and 4E.

[0055] FIG. 4G shows a hybrid lateral flow test device 490 including upstream wicking pad 492 that is positioned downstream of conjugate pad 426 and upstream of microfluidic channel 430. A wicking material that abuts or tucks slightly into the entrance of the channel can be used to effectively pump the sample through the channel via capillary pressure. In embodiments, a downstream wicking pad 494 may be provided abutting or slightly tucked into the downstream opening of microfluidic channel 430 and connected to absorbent pad 428, which, through capillary action will pull even large volumes through microfluidic channel 430 and leave it dry after all the sample is pulled through, if desired. This combines advantages of conventional paper-based LFTs with those of microchannel microfluidic devices. The material of one or both of upstream wicking pad 492 and downstream wicking pad 494 may be chosen to increase or reduce the flow rate of fluid. For example, wicking pads having different densities or porosities may be chosen. The upstream wicking pad 492 and downstream wicking pad 494 may be made of the same or different materials.

[0056] FIG. 4H shows a hybrid lateral flow test device 500 that includes features to control the flow of fluid through the device. Devices referred to as timers may be provided at various locations in device 500 to control the flow of fluid through the device. Times may be, for example, a laminate film that dissolves at a known rate in the presence of fluid. Timer 502 is provided between the downstream end of sample pad 424 and the upstream end of conjugate pad 426. Timer 504 is positioned between the downstream end of conjugate pad 426 and the upstream end of wicking pad 492. Sample fluid added to sample pad 424 will make contact with the film of timer 502. The transfer of the sample fluid to the conjugate pad will be delayed for the time it takes for the film to dissolve, thus providing a timer function.

[0057] In embodiments, a reagent pad 506 may be provided on top of conjugate pad 426, separated from conjugate pad 426 by a dissolving film, or timer 508. After the sample fluid has entered the conjugate pad, target analyte present in the sample fluid will bind with the conjugated molecules in conjugate pad 426 for a period while the sample fluid is also in contact with timer 508 and causing the film between conjugate pad 426 and reagent pad 506 to dissolve. At that point, fluid in conjugate pad 426 containing conjugated target analyte will combine with reagents in reagent pad 506, thus providing for a two-step process. Device 500 of FIG. 4H provides a more robust analyte processing without requiring a trained technician to complete multiple steps, such as pipetting a series of one or more reagents. An example of a series of reagents used for sample preparation may include primary conjugates followed later by secondary, signal-amplifying conjugates or enzymes. Timer 504 on the downstream end of conjugate pad 426 provides additional time for interaction with a reagent released from reagent pad 506 by delaying the transfer of fluid to wicking pad 492.

[0058] Other arrangements are contemplated. For example, secondary conjugates may be contained in the sample receiving area via lamination around three sides of the perimeter. This lamination will dissolve after the sample fluid flows from sample pad 424 to conjugate pad 426 so that the secondary conjugates encounter the test line later than the primary conjugates.

[0059] FIG. 41 shows a hybrid lateral flow test device 510 that does not have a separate conjugate pad. Instead, conjugates 512 are dried on one or more internal surfaces of the microfluidic channel 430 upstream of test area. Other features may be printed on the inner surface of the microfluidic channel, such as lamination lines, or timers, that dissolve when wetted to control how much time is spent in the sample pad 424 (which may be preloaded with RNase to break down RNA or antibodies to capture non-target molecules) or conjugate pad or conjugate area 512 (to give sufficient time for analyte to bind to the detection conjugate). Another feature that may be included on an inner surface of microfluidic channel430 is columns or other features that extend perpendicularly to the inner surface at test line 436 and / or control line 438 to increase surface area for analyte-reporter complexes to bind to capture molecules.

[0060] FIG. 4J shows a hybrid lateral flow test device 520 that uses a sample well 522 instead of a sample pad. A sample fluid containing a target analyte of interested may be placed into sample well 522. As discussed above with reference to FIG. 4H, timer 524, such a film, may be provided between the downstream end of sample well 522 and the upstream end of conjugate pad 426 to control the flow of sample fluid. As depicted in FIG. 4J, device 520 includes upstreaming wicking pad 492 and downstream wicking pad 494, however, any of the embodiments disclosed herein may use a sample well instead of a sample pad.

[0061] FIG. 4K shows a hybrid lateral flow test device 530 with a filter pad 532 positioned on backing material 422 between sample pad 424 and conjugate pad 426. Filter pad 532 may be used to remove larger molecules and debris from a sample that could impede the laminar flow of fluid through the channel. One or more filter pads may be provided at other any location along backing material 422, both upstream and downstream from microfluidic channel 430.

[0062] FIG. 4L shows a hybrid lateral flow test device 540 where the functions of the sample pad and the conjugate pad are combined in a sample well 542 which is similar to sample well 522 of FIG. 4J. Conjugates 544 or other reagents may be dried on a lower surface of sample well 542. As shown in hybrid lateral flow test device 550 of FIG. 4M, conjugates 544 may also be dried on one or more internal surfaces of the microchannel as described above for FIG. 41.

[0063] In any of the embodiments disclosed herein, the channel may have a width between approximately 0.1 and 10 millimeters and a height between approximately 10 and 10000 micrometers. The upstream absorbent pad and the downstream absorbent pad may be between approximately 0.1 and 200millimeters long, between approximately 0.1 and 100 millimeters wide, and between approximately 0.01 and 50 millimeters thick.

[0064] As disclosed herein, a hybrid lateral flow test strip combines elements of lateral flow test strip using a nitrocellulose membrane (particularly including test lines / dots and control lines / dots, as well as the combination of conjugates stored upstream of the test / control lines and an absorbent pad positioned downstream of the test / control lines) with a microfluidic channel to provide several benefits. A hybrid lateral test flow provides the convenience of a single step test (one application of sample to the sample pad / test well) with the benefits of using a microfluidic channel:

[0065] (1) A large sample volume can traverse the channel, allowing for much moreanalyte to reach test line(s) than with a nitrocellulose membrane-based test trip.

[0066] (2) Immunoassays and other means of analyte detection on microfluidic channels (e.g. aptamers, antisenescence oligonucleotides) may be much more sensitive than ones on nitrocellulose membranes.

[0067] (3) Test results may be received more quickly.

[0068] (4) The number of analytes that can be multiplexed on a single channel is very large because the channel can simply be lengthened to allow for a large number of test lines.

[0069] (5) Multiplexing may also be achieved by running multiple channels off same sample pad or having multiple wells for multiple channels in a single test cassette.

[0070] (6) Multiple different analytes may be detected with a single test. This is often not possible with a single nitrocellulose membrane. For example, it would be highly useful to be able to test for multiple, common respiratory pathogens from a single sample. However, testing for viral pathogens can require nitrocellulose membranes having characteristics such as fiber density that are different than those required to test for bacterial pathogens. Yet the key target for a multiplex respiratory pathogen antigen test would include viral pathogens like influenza virus and SARS-CoV-2 but also a bacterial pathogen, Group A streptococcus (GAS). Testing for both pathogens in the same microfluidic channel is straightforward. Furthermore, single step testing for analytes with divergent molecular structures, such as relatively large nucleic acids and relatively small proteins, is straightforward in a hybrid lateral flow test, whereas it may not be possible using a conventional lateral flow test, where different fiber densities may be required for analytes of such divergent sizes and shapes.

[0071] All of this provides huge benefits for at-home and point-of-care testing. For example, for point of care testing, a test that can be run in 3 minutes versus 15 ties up a provider for fraction of the time, leading to major cost savings. It also saves time for home users, and workers, in workplace settings.

[0072] This would work particularly well with a throat gargle sample collection system to test for multiple respiratory viruses at once in a multiplex antigen test, including influenza viruses (H3N2, H1N1 and influenza B virus) RSV, SARS-CoV-2 and GAS. But a single sample and single test cassette with multiple test strips / microfluidic channels could test for 20 or 30 or more pathogens at the same time.

[0073] As disclosed, the system would be able to test urine, blood, pus, or other samples extremely rapidly for bacterial antigens to ascertain in minutes rather than days (via culturing) what bacteria are infecting a patient. This situation may occur, for example, in sepsis patients. This would lead to rapid decisions about the right antibiotics to deploy.

[0074] For the detection of analytes including pathogen antigens, the use of fluorescentprobes rather than colorimetric probes, such as colloidal gold, offers the opportunity to generate much higher sensitivity tests, with limits of detection (LOD) a tenth the concentration or less compared colloidal gold. This is because some fluorescent probes, when excited by specific wavelengths of UV or visible radiation can generate more intense signals than the colorimetric signals of colloidal gold. Selection of fluorescent probes for binding to sample analytes may be done according to techniques known in the art. Of particular importance is a large Stokes shift, the difference between the emission wavelength and the excitation wavelength of the fluorescent probe. Fluorophores that can exhibit large Stokes shifts include quantum dots (QDs), and monoclonal antibodies that bind to analytes can be conjugated directly to QDs.

[0075] Prior art fluorescent probes may also comprise monoclonal antibodies conjugated to fluorescent QD-nanoparticle complexes that provide a stronger fluorescent band in positive samples and therefore higher sensitivity. Polystyrene microbeads infused with lanthanide chelates, such europium chelate, which exhibits a large Stokes shift (280 nm) and a very long lifetime, are also available (e.g., Fluoro-Max™ Fluorescent Carboxylate-Modified Particles) and can generate high sensitivity tests.

[0076] The devices disclosed herein could be produced at a cost that is feasible for a disposable use and would allow in-home tests and point of care tests for SARS-CoV-2 and other pathogens to be much more sensitive without losing any of the convenience.

[0077] Changes may be made in the above methods and systems without departing from the scope hereof. It should thus be noted that the matter contained in the above description or shown in the accompanying drawings should be interpreted as illustrative and not in a limiting sense. Herein, and unless otherwise indicated: (a) the adjective "exemplary" means serving as an example, instance, or illustration, and (b) the phrase “in embodiments” is equivalent to the phrase “in certain embodiments,” and does not refer to all embodiments. The following claims are intended to cover all generic and specific features described herein, as well as all statements of the scope of the present method and system, which, as a matter of language, might be said to fall therebetween.

[0078] Mucins, found for example in nasal and oral specimens, inhibit lateral flow tests and PCR tests. In an embodiment, a filter that captures mucins and prevents them from reaching downstream parts of the device where the sample encounters detection conjugates and capture molecules on the test line or test spot, will be included. This could be a filter material such a Sepharose matrix that the sample flows through downstream of the sample pad and upstream of the conjugate pad.

[0079] Lysis buffers are used in collection devices to break down cells, virions, or otherstructures within the sample in order to release analytes such as proteins, RNA, or DNA. In embodiments, lateral flow test strips would include filters. However, some components of lysis buffers may interfere with lateral flow test and increase the chances of false negative results. For example, proteinase K, while useful in a lysis buffer, will degrade RNases that are required to break RNA analytes into smaller fragments to increase sensitivity. The lateral flow test strip could include filter materials to capture such unwanted molecules from having. In some embodiments, anti-proteinase K antibodies or aptamers will be used to immobilize proteinase K, which would otherwise degrade the RNase needed after conjugation of RNA to detection complexes. These anti-proteinase K antibodies could be put into the sample pad or into a filter pad between the sample pad and conjugate pads.Combination of Features

[0080] Features described above as well as those claimed below may be combined in various ways without departing from the scope hereof. The following examples illustrate possible, non-limiting combinations of features and embodiments described above. It should be clear that other changes and modifications may be made to the present embodiments without departing from the spirit and scope of this invention:

[0081] Embodiment 1. A hybrid lateral flow test device for detecting a target analyte in a sample, the device comprising: an enclosed channel with at least one test area for binding to the target analyte, said channel comprising an upstream opening and a downstream opening; an upstream wicking area abutting the upstream opening of the channel; a downstream absorbent pad abutting the downstream opening of the channel; a sample receiving area upstream of or encompassing the wicking area; and labeled conjugates for binding with the target analyte upstream of the at least one test area.

[0082] Embodiment 2. The device of embodiment 1 , wherein the upstream wicking area further comprises a conjugate pad loaded with labeled conjugates.

[0083] Embodiment 3. The device of either of embodiments 1 or 2, wherein the upstream wicking area further comprises a conjugate pad loaded with labeled dried conjugates and a wicking pad downstream of the conjugate pad.

[0084] Embodiment 4. The device of any of embodiments 1-3, wherein the upstream wicking area further comprises a conjugate pad loaded with labeled dried conjugates and a filter pad upstream of the conjugate pad.

[0085] Embodiment 5. The device of any of embodiments 1-4, wherein the sample receiving area further comprises a sample pad.

[0086] Embodiment 6. The device of any of embodiments 1-5, wherein the samplereceiving area further comprises a sample well.

[0087] Embodiment 7. The device of any of embodiments 1-6, wherein the upstream wicking area and the sample receiving area are formed from a sample pad loaded with the labeled conjugates.

[0088] Embodiment 8. The device of any of embodiments 1-7, wherein the upstream wicking area further comprises a conjugate pad loaded with the labeled conjugates and a laminate film upstream of the conjugate pad for delaying transfer of a sample from the sample receiving area to the upstream absorbent pad.

[0089] Embodiment 9. The device of any of embodiments 1-8, wherein the upstream wicking area further comprises conjugate pad loaded with the labeled conjugates, a wicking material downstream of the conjugate pad, and a laminate film between the conjugate pad and the wicking pad for delaying transfer of fluid from the conjugate pad to the wicking pad.

[0090] Embodiment 10. The device of any of embodiments 1-9, further comprising a reagent pad on top of the conjugate pad and a laminate film between the reagent pad and the conjugate pad.

[0091] Embodiment 11 . The device of any of embodiments 1-10, wherein the upstream wicking area comprises a wicking material that protrudes into the upstream opening of the channel.

[0092] Embodiment 12. The device of any of embodiments 1-11, wherein the downstream absorbent pad protrudes into the downstream opening of the channel.

[0093] Embodiment 13. The device of any of embodiments 1-12, wherein the downstream absorbent pad further comprises a sample absorbent pad for absorbing excess sample fluid and a wicking pad upstream of the sample absorbent pad.

[0094] Embodiment 14. The device of any of embodiments 1-13, wherein the labeled conjugates are dried onto a surface of the channel.

[0095] Embodiment 15. The device of any of embodiments 1-14, wherein the upstream wicking area and the sample receiving area are combined and comprise a sample well.

[0096] Embodiment 16. The device of any of embodiments 1-15, wherein the test area further comprises a test line or a test spot.

[0097] Embodiment 17. The device of any of embodiments 1-16, further comprising a transparent backing material.

[0098] Embodiment 18. The lateral flow test of any of embodiments 1-17, wherein a width of the channel is between approximately 0.1 and 10 millimeters.

[0099] Embodiment 19. The lateral flow test of any of embodiments 1-18, wherein the channel has side walls having a height between approximately 10 and 10000 micrometers.

[0100] Embodiment 20. The lateral flow test of any of embodiments 1-19, wherein the upstream wicking area and the downstream absorbent pad are between approximately 0.1 and 200 millimeters long, between approximately 0. 1 and 100 millimeters wide, and between approximately 0.01 and 50 millimeters thick.

Claims

CLAIMSWhat is claimed is:

1. A hybrid lateral flow test device for detecting a target analyte in a sample, the device comprising: an enclosed channel with at least one test area for binding to the target analyte, said channel comprising an upstream opening and a downstream opening; an upstream wicking area abutting the upstream opening of the channel; a downstream absorbent pad abutting the downstream opening of the channel; a sample receiving area upstream of or encompassing the wicking area; and labeled conjugates for binding with the target analyte upstream of the at least one test area.

2. The device of claim 1 , wherein the upstream wicking area further comprises a conjugate pad loaded with labeled conjugates.

3. The device of claim 1, wherein the upstream wicking area further comprises a conjugate pad loaded with labeled dried conjugates and a wicking pad downstream of the conjugate pad.

4. The device of claim 1 , wherein the upstream wicking area further comprises a conjugate pad loaded with labeled dried conjugates and a filter pad upstream of the conjugate pad.

5. The device of claim 1, wherein the sample receiving area further comprises a sample pad.

6. The device of claim 1 , wherein the sample receiving area further comprises a sample well.

7. The device of claim 1, wherein the upstream wicking area and the sample receiving area are formed from a sample pad loaded with the labeled conjugates.

8. The device of claim 1 , wherein the upstream wicking area further comprises a conjugate pad loaded with the labeled conjugates and a laminate film upstream of the conjugate pad for delaying transfer of a sample from the sample receiving area to the upstream absorbent pad.

9. The device of claim 1 , wherein the upstream wicking area further comprises conjugate pad loaded with the labeled conjugates, a wicking material downstream of the conjugate pad, and a laminate film between the conjugate pad and the wicking pad for delaying transfer of fluid from the conjugate pad to the wicking pad.

10. The device of claim 1, further comprising a reagent pad on top of the conjugate pad and a laminate film between the reagent pad and the conjugate pad.

11. The device of claim 1 , wherein the upstream wicking area comprises a wicking material that protrudes into the upstream opening of the channel.

12. The device of claim 1, wherein the downstream absorbent pad protrudes into the downstream opening of the channel.

13. The device of claim 1, wherein the downstream absorbent pad further comprises a sample absorbent pad for absorbing excess sample fluid and a wicking pad upstream of the sample absorbent pad.

14. The device of claim 1 , wherein the labeled conjugates are dried onto a surface of the channel.

15. The device of claim 1, wherein the upstream wicking area and the sample receiving area are combined and comprise a sample well.

16. The device of claim 1 , wherein the test area further comprises a test line or a test spot.

17. The device of claim 1 , further comprising a transparent backing material.

18. The lateral flow test of claim 1, wherein a width of the channel is between approximately 0.1 and 10 millimeters.

19. The lateral flow test of claim 1, wherein the channel has sidewalls having a height between approximately 10 and 10000 micrometers.

20. The lateral flow test of claim 1 , wherein the upstream wicking area and the downstream absorbent pad are between approximately 0.1 and 200 millimeters long, between approximately 0.1 and 100 millimeters wide, and between approximately 0.01 and 50 millimeters thick.

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