High-sensitivity diagnostic test devices and methods and apparatus to make and / or process the same

Abstract

Methods, apparatus, systems, and articles of manufacture to make and / or process a highly-sensitive diagnostic test device are disclosed. An example apparatus includes a conjugate pad including an antibody coated molecule corresponding to a target analyte; a microparticle pad including an antibody coated magnetic microparticle corresponding to the target analyte; and a media including a microwell, the media to propagate a flow of a biological sample, the molecule, the magnetic microparticle, and a liquid buffer along the media, the microwell structured to encapsulate the magnetic microparticle.

Description

PATENT15806WOO1HIGH-SENSITIVITY DIAGNOSTIC TEST DEVICES AND METHODS AND APPARATUS TO MAKE AND / OR PROCESS THESAMERELATED APPLICATION

[0001] This patent claims the benefit of U.S. Provisional Patent Application No. 63 / 690.627, which was filed on September 4, 2024. U.S. Provisional Patent Application No. 63 / 690.627 is hereby incorporated herein by reference in its entirety. Priority to U.S. Provisional Patent Application No. 63 / 690,627 is hereby claimed.FIELD OF THE DISCLOSURE

[0002] This disclosure relates generally to biosensors, and, more particularly, to highly- sensitive diagnostic test devices and methods and apparatus to make and / or process the same.BACKGROUND

[0003] A biosensor (e.g.. a lateral flow device, such as a lateral flow assay (LFA)) is a device that is capable of detecting a condition, disease, etc., in a human or animal based on a sample (e.g., a blood sample, a saliva sample, a urine sample, etc.) from the human or animal. LFAs have been used to detect the presence of a target analyte to determine pregnancy, presence of a pathogen, presence of different toxins, etc.BRIEF DESCRIPTION OF THE DRAWINGS

[0004] FIG. 1 A is an example lateral flow immunoassay device disclosed in conjunction with examples disclosed herein.

[0005] FIG. IB is a side view of the example lateral flow immunoassay device of FIG. 1A.

[0006] FIG. 2 illustrates an exploded view of the lateral flow immunoassay device of FIGS. 1A-1B.

[0007] FIG. 3 A illustrates the lateral flow immunoassay device of FIGS. 1A-2 during four example phases of a test.

[0008] FIG. 3B illustrates a cross-sectional view of the lateral flow immunoassay device of FIGS. 1 A-2 during four example phases of a test.PATENT 15806WOO1

[0009] FIG. 3C is a graph that illustrates an example fluorescence intensity during flow of a buffer in media of the lateral flow immunoassay device of FIGS. 1 A-3B with respect to time.

[0010] FIG. 3D includes example images of the example fluorescence intensity of microwells in the lateral flow immunoassay device of FIGS. 1A-3B with respect to time after the completion of the flow of the buffer when a target analyte is present in a sample.

[0011] FIG. 4 illustrates an example lateral flow assay device reader.

[0012] FIG. 5 is a block diagram of an implementation of the controller of FIG. 4.

[0013] FIG. 6 is a flowchart representing a method of manufacturing the lateral flow assay device of FIGS. 1A-2A.

[0014] FIG. 7 illustrates a flowchart representative of machine readable instructions which may be executed to implement the controller of FIG. 5.

[0015] FIG. 8 is a block diagram of an example processing platform structured to execute the instructions of FIG. 7 to implement the controller of FIG. 5.

[0016] The figures are not to scale. Instead, the thickness of the layers or regions may be enlarged in the drawings. In general, the same reference numbers will be used throughout the drawing(s) and accompanying written description to refer to the same or like parts.DETAILED DESCRIPTION

[0017] Example rapid diagnostic tests include a biosensor or test strip device (e.g., lateral flow immunoassay (LFA)), which is a device including a first region to obtain, receive, and / or accept a sample (e.g., blood, urine, saliva, etc.) and a second region that changes (e.g., changes color and / or experiences another change in a physical property) when a target analyte corresponding to a particular disease or condition is present in the sample. For example, a user applies the sample to a sample pad of a test strip device, or simply “test strip” (e.g., an LFA, etc.). Once applied, the sample migrates or flows along the test strip to a conjugate pad that contains conjugates (e.g., detectable labels, tags, linkers, antibodies, antigens, etc.) specific to the target analyte. If the sample includes the target analyte, a reaction (e.g., a chemical reaction, biochemical reaction, physical reaction, etc.) occurs on the conjugate pad to bind the target analyte with the conjugates. The test strip also includes a test line that contains molecules (e.g., immobilized antibodies, antigens, analytes, aptamers, etc., specific to the target analyte), which bind the first set of conjugate molecules (e.g., probe molecules) from the conjugate pad. For example, if the analyte of interest is an antibody, the positive test area includes immobilized antigen. If the analyte of interest is an antigen, the positive test area includes immobilized antibody. The labeled substance or conjugate includes a binding component that is able to bindPATENT 15806WOO1 the analyte of interest and, in some examples, a visualization component. Accordingly, when the sample (e.g., including the bounded target analyte) flows to a test zone (e.g., a reaction zone), the antibodies, analytes, or antigens of the test line bind to the bounded target analyte, thereby immobilizing the target analyte. In some test strips, the immobilized target analytes result in a visual output that identifies that the target analyte is present in the sample. Accordingly, a scanner or user can identify whether the target analyte (e g., corresponding to a condition or disease) is present in the sample based on the visualization (e.g., color) of the test zone.

[0018] “Target analyte,” “analyte” or “analyte of interest” refers to the compound or the composition to be detected or measured from the sample, which has at least one epitope or binding site. The analyte can be any substance for which there exists a naturally occurring analyte-specific binding member or for which an analyte-specific binding member can be prepared. Analytes include, but are not limited to, toxins, organic compounds, proteins, peptides, microorganisms, amino acids, nucleic acids, hormones, steroids, vitamins, drugs (including those administered for therapeutic purposes as well as those administered for illicit purposes), and / or metabolites of or antibodies to any of the above substances. The term "analyte" also includes any antigenic substances, haptens, antibodies, macromolecules, and / or combinations thereof.

[0019] “Label” refers to any substance which is capable of producing a signal that is detectable by visual and / or instrumental means. Various labels suitable for use in examples disclosed herein include labels that produce signals through chemical and / or physical means. Examples include enzymes and substrates, chromagens, fluorescent compounds, chemiluminescent compounds, colored or colorable organic polymer latex particles, liposomes, and / or other vesicles containing directly visible substances. In some examples, radioactive labels, colloidal metallic particles, and / or colloidal non-metallic particles are employed. In some examples, labels include colloidal gold and latex particles.

[0020] “Labeled substance” or “conjugate” refers to a substance that includes a detectable label attached to a specific binding member. The attachment may be covalent or non- covalent binding and may include nucleic acid hybridization. The label allows the labeled substance to produce a detectable signal that is directly or indirectly related to the amount of analyte in a test sample. The specific binding member component of the labeled substance is selected to bind directly or indirectly to the analyte.

[0021] “Specific binding member” or “binding member” refers to a member of a specific binding pair (e.g., two different molecules wherein one of the molecules specifically binds to the second molecule through chemical or physical means). If the specific binding member is anPATENT 15806WOO1 immunoreactant, it can be, for example, an antibody, analyte, antigen, hapten, or complex thereof, and if an antibody is used, it can be a monoclonal or polyclonal antibody, a recombinant protein or antibody, a chimeric antibody, a mixture(s), or fragment(s) thereof, as well as a mixture of an antibody and other specific binding members. Specific examples of specific binding members include biotin and avidin, an antibody and its corresponding antigen (both having no relation to a sample to be assayed), a single stranded nucleic acid and its complement, and the like.

[0022] A “test strip” or “LFA” can include one or more bibulous or non-bibulous materials. If a test strip includes more than one material, the one or more materials are preferably in fluid communication. One material of a test strip may be overlaid on another material of the test strip, such as for example, filter paper overlaid on nitrocellulose. Additionally or alternatively, a test strip may include a region including one or more materials (e.g., media) followed by a region including one or more different materials. In this case, the regions are in fluid communication and may or may not partially overlap one another. Suitable materials for test strips include, but are not limited to, materials derived from cellulose, such as filter paper, chromatographic paper, nitrocellulose, and cellulose acetate, as well as materials made of glass fibers, nylon, dacron, polyvinyl chloride (PVC), polyacry lamide, cross-linked dextran, agarose, poly acrylate, ceramic materials, and the like. The material or materials of the test strip may optionally be treated to modify their capillary flow characteristics or the characteristics of the applied sample. For example, the sample application region of the test strip may be treated with buffers to correct the pH or specific gravity of an applied urine sample, to ensure optimal test conditions.

[0023] The material or materials can be a single structure such as a sheet cut into strips or it can be several strips or particulate material bound to a support or solid surface such as found, for example, in thin-layer chromatography and may have an absorbent pad either as an integral part or in liquid contact. The material can also be a sheet having lanes thereon, capable of spotting to induce lane formation, wherein a separate assay can be conducted in each lane. The material can have a rectangular, circular, oval, triagonal or other shape provided that there is at least one direction of flow or traversal of a test solution by capillary migration. Other directions of traversal may occur such as in an oval or circular piece contacted in the center with the test solution. In the following, disclosed test strips is by way of illustration and not limitation.

[0024] In examples that include a support for the test strip, the support may be water insoluble, non-porous, and / or rigid. In some examples, the support may be elastic, hydrophobic, and / or porous. In some examples, the support is of the same length and width as the strip butPATENT 15806WOO1 may be larger or smaller in other examples. In some examples, the support material is transparent, and, when a test device disclosed herein is assembled, a transparent support material can be on the side of the test strip that can be viewed by the user, such that the transparent support material forms a protective layer over the test strip where it may be exposed to the external environment, such as by an aperture in the front of a test device. A wide variety of non- mobilizable and non-mobilizable materials, both natural and synthetic, and combinations thereof, may be employed provided only that the support does not interfere with the capillary' action of the material or materials, or non-specifically bind assay components, or interfere with the signal producing system. Illustrative polymers include polyethylene, polypropylene, poly (4-methylbutene), polystyrene, poly methacryl ate, poly (ethylene terephthalate), nylon, poly (vinyl butyrate), glass, ceramics, metals, and the like. Elastic supports may be made of polyurethane, neoprene, latex, silicone rubber and the like. Throughout this description, LFAs are disclosed with the understanding that description of the LFAs applies to other types of test strips.

[0025] Point of care testing (PoCT), in some examples, includes the use of lateral flow assay devices and / or digital immunoassay devices to test biological samples for presence of one or more target analytes. Lateral flow assays (LFAs) are devices used as diagnostic testing devices that are low cost and simple to operate. For example, a user can apply a sample (e.g., saliva, blood, urine, etc.) to an opening of an LFA device and the LFA device can perform one or more reactions (e.g., chemical reactions), that can be read (e.g., by a user and / or by a machine) to determine if the sample includes a particular analyte (e g., corresponding to presence of a disease, whether someone is pregnant, etc ).

[0026] LFA devices are structured to include dried reagents that are stored on a pad. When a small amount liquid sample (e.g., 10-60 microliters) is applied to the pad, the sample dissolves and / or resuspends the dried reagents, which causes an immuno-reaction in the pad and / or on a membrane of the LFA. If the sample includes one or more particular analytes, the target analytes bind to the reagents. In some examples, a buffer (e.g.. a liquid) is applied to the pad to cause the sample to flow through a membrane toward a waste pad (also referred to as a wicking pad). In some LFAs, when the target analyte is present in the sample, a particle conjugated antibody (e.g., the target analyte bounded to the reagent) reacts with an antigen bound with an antibody immobilized in a region of the membrane to generate a visible line on the membrane corresponding to a test result. In such LFAs, when the target analyte is not present in the sample, there is no target analyte for the reagent to bind to. Thus, the reagents flow to the waste pad, without binding to the immobilized antibody on the membrane and no visible line is formed.PATENT 15806WOO1

[0027] LFA devices are cost effective, simple, disposable, and user-friendly. Some LFAs are prone to errors due to limited visibility of a positive line and / or human error. Accordingly, some LFAs have limited sensitivity and limited specificity when compared to analysis using laboratory analysis techniques. Sensitivity (also referred to as true positive rate) is the probability of a positive test result, conditioned on the individual truly being positive. Specificity (also referred to as true negative rate) is the probability of a negative test result, conditioned on the individual truly being negative.

[0028] Digital enzyme-linked immunosorbent assay (ELISA) (also referred to as digital immunoassay (Al)) is a laboratory analysis technique developed that provides high sensitivity and specificity. Some digital ELISAs use a single-molecule detection technique with partitioned small water-in-oil droplets and a fluorogenic substrate, allowing the detection of a single enzy me using an optical sy stem. For example, when a target analyte is present in a sample, a reaction occurs to produce a detectable signal by accumulating fluorescent reaction product molecules. However, the digital ELISA structure and process is different than an LFA device. Although digital ELISA devices have better sensitivity and specificity than LFA devices, the digital ELISA is more complicated, requires a large volume of a sample (e.g., over 100 microliters) and may require a trained professional to implement.

[0029] Examples disclosed herein include a PoCT disposable device that combines some of the principles of the digital ELISA device to some of the principles of an LFA device to create a highly sensitive and specific device that has the simplicity of an LFA and removes the complexity of the digital ELISA device. Examples disclosed herein restructure an LFA device to implement components and / or techniques traditionally associated with a digital ELISA device to increase the sensitivity and specificity of the LFA device. Additionally, by implementing digital ELISA techniques in an LFA device, the amount of sample used to perform a test is significantly reduced.

[0030] FIG. 1A includes a portion of an example LFA device 104 (illustrated in an overhead view). The example LFA device 104 includes an example sample pad 106, an example conjugate release pad 108, an example microparticle release pad 109, an example media 110, example microwells 111, and an example wicking pad 112.

[0031] The example LFA device 104 of FIG. 1 A is a device (e.g., a point-of-care device, a rapid diagnostic device, etc.) that obtains a biological sample (e.g., saliva, mucus, blood, urine, etc.) and performs one or more reactions that results in a detectable signal when a target analyte is present in the sample. For example, the LFA device 104 may be a porous membrane device, a media device, a fluid transporting media device, a test strip device, a lateral flow test strip device, and / or any fluid sample device. As further disclosed below, as the sample flows acrossPATENT 15806WOO1 the LFA device, portions of the sample enter the microwells 111. When a target analyte is present in a sample, at least some of the target analyte become trapped in the microwells 111. A fluorescent-based buffer can be applied to the LFA device 104. The fluorescent-based buffer reacts with a conjugate molecule (e.g.. alkaline phosphatase) labeled with a binding member (e.g.. an antigen and / or antibody) bound to the target analyte to generate fluorescence signals that can be identified by an image capturing sensor (e.g., a camera).

[0032] The example LFA device 104 of FIG. 1 A includes the sample pad 106 (e.g., a sample pad, a sample region, a sample area, a sample zone, etc.). The sample pad 106 is structured to act as a sponge to hold a sample of fluid applied to the sample pad 106. In some examples, the sample pad 106 includes buffer components (e.g., salts, surfactants, etc.) to ensure that target analytes that may be in the sample are capable of binding with components of the conjugate release pad 108. When the sample pad 106 is soaked, the fluid held in the sample pad 106 flows to the conjugate release pad 108.

[0033] The conjugate pad 108 of FIG. 1 A includes a reagent (e.g., a labeled substance or conjugate) configured to bind a target analyte. For example, the conjugate release pad 108 includes conjugates or probes (e.g., conjugate molecules) labeled with detectable binding members (e.g., labels, tags, linkers, antibodies, analytes, antigens, etc.) that correspond to a target analyte. The conjugate / probes labeled with a binding member is also referred to as binding member coated molecules. The conjugate release pad 108 may be a fiber pad that has been soaked with alkaline phosphatase labelled antibody (AP -labeled AB) reagent, the antibody corresponding to a particular analyte. The conjugate pad 108 is dried (e.g., using a vacuum dryer at room temperature). The target analyte is a component that corresponds to a particular condition or disease. Accordingly, presence of the target analyte in the sample corresponds to presence of the corresponding condition and / or disease in the person who provided the sample. If the sample includes one or more of the target analytes, the conjugates attaches to the corresponding target analytes. The sample (e.g., including the probes if corresponding target analytes are present in the sample) continues to flow through the example media 110 of the LFA device 104 toward the wi eking pad 112. In some examples, the sample pad 106 may be combined with the conjugate pad 108.

[0034] The microparticle pad 109 of FIG. 1A includes a reagent (e.g., a labeled substance or conjugate) configured to bind a target analyte. For example, the microparticle release pad 109 includes conjugates or probes (e.g., magnetic particles or components) labeled with detectable binding members (e.g., labels, tags, linkers, antibodies, analytes, antigens, etc.) that correspond to the target analyte. The conjugates / probes labeled with a binding member are also referred to as binding member coated magnetic particles. In some examples, the magneticPATENT 15806WOO1 particles may be approximately 3 micrometers in diameter. The microparticle release pad 109 may be a fiber pad that has been soaked with antibody coated superparamagnetic particle (AB- mP) reagent, the antibody corresponding to a particular analyte. The microparticle release pad 109 is then dried (e.g., using a vacuum dryer at room temperature). The target analyte is a component that corresponds to a particular condition or disease. Accordingly, presence of the target analyte in the sample corresponds to presence of the corresponding condition and / or disease in the patient who provided the sample. If the sample includes the one or more of the target analytes, the binding member(s) (e.g., antibodies and / or antigens) coated to the magnetic microparticles attach to the corresponding target analytes. The sample (e.g., including the probes if corresponding target analytes are present in the sample) continues to flow through the example media 110 of the LFA device 104 toward the wicking pad 112. In some examples, any combination of the sample pad 106, the conjugate pad 108, and / or the microparticle pad 109 may be combined.

[0035] While the sample flows across the media 1 10 (also referred to as a flow cell) of FIG. 1A, the sample flows from the pads 108, 109 to the wicking pad 112. The media 110 may be a porous membrane, a nitrocellulose membrane, a polymer (e.g., a cyclo-olefin polymer (COP)), a paper, and / or other substrate including a compartment-free substrate, etc. that propagates the flow of the biological sample and / or liquid buffer. The media 110 includes the example microwells 111. The microwells 1 11 are cavities that are sized large enough to fit at least one magnetic microparticle. The micro wells 111 of FIG. 1 A have a round shape, however, the microwells 111 may have a different geometry. The microwells 111 may be structured in rows and columns and / or in any other pattern. In some examples, there are 100,000 microwells111 in the media 110. However, there may be any number of mi crowells. As further disclosed below, after the sample and / or a buffer is applied to the sample pad 106, the fluid that includes the sample, the buffer, and / or any rehydrated or resuspended reagents flows toward the wicking pad 112. During the flow, the magnetic microparticles can become seeded (e.g., encapsulated) into the microwells 111. In some examples, the LFA device 104 is placed above a magnet to aid in the seeding of the magnetic microparticles into the microwells 1 11 , as further disclosed below. In some examples, the microwells 111 could be replaced and / or implemented with one or more alternative encapsulation structures or methods.

[0036] The example wicking pad 112 of FIG. 1 A (also referred to as a waste pad) is an absorbent material that wicks the liquid through the LFA. In some examples, the wicking pad112 includes cellulose filters. The wicking pad 112 prevents backflow of the liquid. Also, in some examples, the wicking pad 112 is structured as a waste container.PATENT 15806WOO1

[0037] FIG. IB is a side view of the LFA components of the LFA device 104 of FIG.1A. FIG. IB includes the example sample pad 106, the example conjugate pad 108, the example microparticle pad 109, the example media 110, the example micro wells 111, and the example wicking pad 112 of FIG. 1A.

[0038] FIG. 2 illustrates an exploded view of the example lateral flow immunoassay 104 of FIGS. 1A-1B. FIG. 2 includes the example sample pad 106, the example conjugate pad 108, the example microparticle pad 109, the example media 110, and the example wicking pad 112 of FIGS. 1 A-1B. FIG. 2 further includers an example lamination film 202, an example top film 204, an example flow cell pattern layer 206. example adhesive 208. and an example base plate 210.

[0039] The lamination film 202 of FIG. 2 is positioned on top of the sample pad 106, conjugate pad 108, and / or the microparticle pad 109 to protect the respective pads 106, 108, 109 from contamination. Additionally, the lamination film 202 is structured to cause the sample and buffer flow through the respective pads as intended. In some examples, the lamination film 202 may overlap with at least a portion of the top film 204. The lamination film 202 may include a cutout where the sample and / or buffer can be applied to the sample pad 106. In some examples, the lamination film 202 is made of polytetrafluoroethylene (PTFE) or another synthetic film. The top film 204 is positioned on top of the media 110 to protect the media 110 from contamination. In some examples, the top film 204 is made of polytetrafluoroethylene (PTFE) or another synthetic film. In some examples, the lamination film 202 and the top film 204 may be one film (i.e., integral).

[0040] The flow cell pattern 206 of FIG. 2 is structured to set the flow pattern of the sample and / or buffer when applied to the sample pad 106. Although the flow cell pattern 206 of FIG. 2 corresponds to a particular shape, other shapes may be used. The cutout in the flow cell pattern 206 that defines the flow pattern provides a gap between the media 110 and the top film 204. As further disclosed below, the gap or cavity provides an air-sealed layer that creates a physical barrier of air between microwells. In this manner, enzymatic reactions can occur in individual microwells 11 1 without interference from liquid from other microwells.

[0041] The adhesive 208 of FIG. 2 adheres the media 110 to the base plate 210. The adhesive 208 could be tape (e.g., double sided tape), glue, and / or any other adhesive. The base plate 210 is the bottom layer that supports the rest of the LFA device 104.

[0042] FIG. 3A illustrates the LFA 104 of FIGS. 1-2A during four phases of an example test. FIG. 3 A includes a first example phase 300, a second example phase 302, a third example phase 304, a fourth example phase 306, an example sample 310, and an example buffer 312.PATENT 15806WOO1 FIG. 3A further includes the sample pad 106, the conjugate pad 108, the microparticle pad 109, the media 110, the wicking pad 112, and the flow cell pattern layer 206 of FIGS. 1 A-2.

[0043] At the first phase 300 of FIG. 3 A, a user applies the sample 310 to the sample pad 106. The sample 310 is a biological sample that may include saliva, blood, mucus, urine, and / or any other sample. As disclosed above, the sample 310 may be 10-60 microliters of fluid. After the sample 310 is applied to the sample pad 106, the second phase 302 begins. At the second phase 302, the sample 310 flows onto the conjugate pad 108 and / or the microparticle pad 109, thereby resuspending the dried conjugate molecules and / or magnetic molecules. If a target analyte is present in the sample 310. the binding members linked to the molecule in the conjugate pad 108 attach to the target analyte and the binding members of the magnetic microparticles attach to the target analyte in the microparticle pad 109. The second phase 302 has a duration to allow sufficient time for the target analytes to bind to the binding members of the binding member coated molecules and / or the binding member coated magnetic particles of the pads 108. 109. In some examples, the second phase 302 has a duration of about eight minutes.

[0044] At the third phase 304 of FIG. 3A, a user or machine applies the buffer 312 to the sample pad 106. The buffer 312 (also referred to as a chase buffer) may include fluorescence components (e.g., a pyranine-phosphate buffer). The buffer operates as both a wash buffer and a substrate buffer to remove a step from a conventional digital ELISA design. The buffer 312 saturates the pads 106, 108, 109, and causes the sample 310 and the buffer 312 to flow on the media 110 toward the wicking pad 112. When the sample 310 flows over the micro wells 111 of the media 110, the magnetic particles from the microparticle pad 109 become seeded / encapsulated in the microwells 1 11. If a target analyte is present in the sample, at least some of the magnetic particles are attached to the target analyte. Because the target analyte is also coupled to the molecules from the conjugate pad 108, the magnetic particles seeded in the microwells 111 cause at least some of the molecules from the conjugate pad 108 to be immobilized in or near the microwells 111.

[0045] After a duration of time (e g., 1 to 5 minutes), at the fourth phase 306, the buffer and sample have flowed onto the wicking pad 112. At this point, the space between the media 110 and the top fdm 204 defined by the flow- cell pattern layer 206 has been air sealed to allow localized reactions to occur between the molecule labelled with binding members (e.g., antigens and / or antibodies) attached to the target analyte and the fluorescent-based buffer in respective ones of the microw-ells 111. The reactions create fluorescence signals. At the fourth phase 306, a camera or other sensor can capture one or more images, perform image analysis, and / orPATENT 15806WOO1 determine a test result based on the number, quantity, percentage, and / or intensity of the fluorescence signals, if any, as further disclosed below.

[0046] FIG. 3B illustrates a cross-sectional view of the LFA 104 of FIGS. I A w ith respect to the A-A line (FIG. 1A) during four phases of a test. FIG. 3B includes a first example phase 320, a second example phase 322. a third example phase 324, a fourth example phase 326. example magnetic particles 328 (also referred to as magnetic particles, magnetic beads, or magnetic microparticles), and an example magnet 330. The magnet 330 of FIG. 3B is located in or on an external device (e g., a reader), as further disclosed below in conjunction with FIG. 4. However, in some examples, the LFA 104 may include a magnetic layer below the media 110. FIG. 3 A further includes the media 110, the micro wells 111, and the top layer 204 of FIGS. 1A- 2.

[0047] The first phase 320 of FIG. 3 corresponds to prior to the application of the buffer 312 of FIG. 3 A. During the first phase 320, the sample has not yet flowed onto the media 110. Accordingly, the space between the top film 204 and the media 110 and the microwells 111 is empty. At the second phase 322, the sample 310 of FIG. 3 A flows toward the wi eking pad 112 in the space between the top layer 204 and the media 110. As disclosed above, the magnetic particles 328 are microparticles that were dried onto the microparticle pad 109 and then dissolved resuspended, and / or rehydrated by the sample 310. After being dissolved, resuspended and / or rehydrated, the magnetic particles 328 along with the sample flow through the media 110, as shown in the second phase 322. With the assistance of the magnet 330, the magnetic particles 328 are pulled (e.g., seeded) into the microwells 111. As disclosed above, if the target analyte is present in the sample, the target analyte (which is attached to the binding members labelled with a molecule from the conjugate pad 108) attaches to the magnetic particles 328. Accordingly, if the magnetic particles 328 are seeded in the microwells 111 and the target analyte is present in the sample, the molecules of the conjugate also are seeded in the microw ells 111.

[0048] The third phase 324 corresponds to after the buffer 312 has been applied and flows on the media 1 10 toward the wicking pad 1 12. As disclosed above, the buffer 312 includes fluorescent component(s) that can react with the conjugate molecule to create a fluorescent signal. If the target analyte is not present in the sample 310, then the conjugate molecules flow to the wicking pad without being captured. However, if the target analyte is present in the sample 310, the binding members attached to the conjugate molecules, which are then trapped in the microwells 111 with the magnetic particles 328. At the fourth phase 326, after the buffer 312 has flowed tow ard the wicking pad 112, the cavity betw een the top film 204 and the top of the media 110 is filled with an air seal and at least some of the micro wells 111 arePATENT 15806WOO1 filled with the buffer 312 and the magnetic particles 328. As disclosed above, when the target analyte is present in the sample, at least some of the microwells 111 include a conjugate molecule (e.g., alkaline phosphatase) from the conjugate pad 108. The conjugate molecule reacts with the fluorescent buffer to generate a fluorescent signal that can be identified using a reader (e.g., the reader of FIG. 4).

[0049] FIG. 3C is an example graph 331 that illustrates a time trajectory of fluorescent intensity of the media 110 before the microbeads have been encapsulated in the micro wells 111. Within the first 15 seconds, the intensity of the fluorescent signal within on the media 110 is low (e.g., 100 arbitrary units (AU)). However, after approximately 15 seconds, the fluorescence intensity increases to a high intensity (e.g., 200 AU) and the intensity remains high for a duration of time. After about 90 seconds, the fluorescence intensity7decreases signifying the completion of the buffer flow. After the fluorescence intensity7decreases, the microw ells 111 become isolated due to an air seal, as described above. If a target analyte is present, an enzymatic reaction occurs (e.g., between the buffer and the molecule of the of the conjugate) in the isolated microwells 111 to increase the fluorescence to a detectable level that can be determined by a reader. .

[0050] FIG. 3D illustrates example images taken of the LFA device 104 after a sample with a target analyte was applied. The example images 332, 333. 334, 336. 338, 340 correspond to different amounts of time after the buffer 312 was applied. For example, the image 332 is of the media 110 at zero minutes after the completion of the flow of the buffer 312 through the media 110, the image 333 is of the media 110 one minute after the completion of the flow7of the buffer 312 through the media 110, the image 334 is of the media 110 at two minutes after the completion of the flow of the buffer 312 through the media 110, the image 336 is of the media 110 at three minutes after the completion of the flow of the buffer 312 through the media, the image 338 is of the media 110 at four minutes after the completion of the flow of the buffer 312 through the media 110, and the image 340 is of the media 110 at five minutes after the completion of the flow of the buffer 312 through the media. In the images, the white spots correspond to increased fluorescence intensity indicating that the target analyte was present in the sample. As shown in the example images 332, 333, 334, 336, 338, 340, the intensity of the fluorescence spots increases with time. The completion of the flow of the buffer corresponds to the fluorescence drop at phase iv of FIG. 3C.

[0051] FIG. 4 illustrates an example reader device 400 to generate a test result after a sample is applied to the LFA device 104 of FIGS. 1A-3B. The reader device 400 includes an example camera 402, an example lens 404, an example emission (EM) filter 406, an examplePATENT 15806WOO1 LED plate 408, example excitation (EX) filters 410, an example magnet 412, an example controller 414, an example pump 416, and an example buffer reservoir 418.

[0052] The camera 402 of FIG. 4 is a sensor that is capable of capturing one or more images based on one or more control signals from the controller 414. For example, the controller 414 can output a control signal to cause the camera 402 to capture a stream flow of images (e.g., one or more images) of the LFA device 104 and return the image to the controller 414. The camera 402 includes a lens 404. The lens 404 controls an amount of light that enters a camera 402 so that the camera 402 can generate an image based on the light. Additionally, the lens 404 may bend light and refract the bent light into a single sharp focal point. The camera 402 is positioned to image the media 110 to be an image scale of, for example, 1.62 micrometers / pixel upon full zoom of the lens 404. The camera 402 may include a light source to record the images under blue illumination until the decrease in green fluorescence derived from the buffer, thereby corresponding to a depletion of the buffer. However, the buffer may correspond to any color fluorescence. Accordingly, the camera 402 may use a color of illumination that corresponds to the fluorescence.

[0053] The EM fdter 406 of FIG. 4 allows the desirable fluorescence from the sample to reach the camera 402 while blocking unwanted traces of excitation light from the EX filter 410. In some examples, the EM filter 406 is a bandpass filter that allows a narrow band of wavelengths to pass through, around the peak fluorophore emission wavelength. However, the EM filter 406 may be a long-pass filter that cuts excitation light and allows long wavelength light (e.g., including fluorescence of a compound) to pass. The LED plate 408 illuminates the LFA device 104 to provide better lighting conditions for the camera 402 to generate an image. In some examples, the EX filter 410 is a bandpass filter that allows a narrow band of wavelengths to pass through, around the peak fluorophore excitation wavelength, and block wavelengths outside of the narrow band. However, the EM filter 406 may be a short-pass filter that cuts light with a long wavelength and allows short wavelength light to pass. The magnet 412 of FIG. 4 corresponds to the magnet 330 of FIG. 3B and aids in seeding magnetic particles into the microwells 1 11 of the LFA device 104. In some examples, the magnet 412 is a neodymium magnetic that is placed a distance of 2.2 millimeters from the bottom of the media 110.

[0054] The controller 414 of FIG. 4 controls the camera 402 to obtain image(s) of the LFA device 104 and processes the image(s) to determine if the test is positive or negative based on the image(s). Additionally, the controller 414 can cause the pump 416 to pump buffer onto the sample pad 106 of the LFA device 104. The controller 414 tracks the timing of the test to take images based on when the pump 416 was controlled to output the buffer onto the LFAPATENT 15806WOO1 device 104. The controller 414 outputs the results via a user interface. The controller 414 is further disclosed below in conjunction with FIG. 5.

[0055] The pump 416 of FIG. 4 outputs buffer (e.g., 210 microliters of buffer) onto the sample pad 106 of the LFA device 104 based on a signal from the controller 414. For example, when the controller 414 outputs a control signal to the pump 416. the pump 416 pumps buffer from the buffer reservoir 418 toward an outlet that is positioned over an opening of the LFA device 104 so that the sample pad 106 of the LFA device 104 can obtain the appropriate amount of buffer. The buffer reservoir 418 stores the buffer.

[0056] FIG. 5 is a block diagram of an example implementation of the controller 414 of the reader device 400 of FIG. 4. The controller 414 includes a user interface 500, one or more example component interfaces 502, timer circuitry 504 and image analyzer circuitry 506.

[0057] The user interface 500 of FIG. 5 may include one or more components to interact with a user or technician. For example, the user interface 500 can include a button or touch screen to obtain a command from a user and / or technician to initiate a test after the user / technician has applied a sample to the LFA device 104 and placed the LFA device 104 in position for the reader device 400 to run a test. Additionally, the user interface 500 may output a result to the user and / or technician after a test is complete.

[0058] The component interface(s) 502 of FIG. 5 interact(s) with other components of the reader device 400. For example, the component interface(s) 502 can output control signals to the camera 402 to generate an image, obtain generated images from the camera 402, and output control signals to the pump 416 to cause the pump 416 to pump buffer from the buffer reservoir 418 onto the sample pad 106 of the LFA device 104.

[0059] The timer circuitry 504 determines when to output control signals to the camera and / or the pump 416. For example, after the user interface 500 obtains a command to start a test, the timer circuitry' 504 may output a control signal to the camera 402 to capture an initial image of the LFA device 104 before the buffer is applied (e.g., for calibration). In some examples, the user may apply the sample and then enter the LFA device 104 into the reader device 400 and start a diagnostic test. Then the timer circuitry 504 may output a control signal to the pump 416 to cause the pump 416 to output buffer onto the sample pad 106 of the LFA device 104. In such examples, the timer circuitry 504 may wait for a duration of time before outputting the control signal to the pump 416 to allow the sample to properly dissolve and / or bind to components of the conjugate pad 108 and / or microparticle pad 109. After the pump 416 pumps the buffer onto the LFA device 104, the timer circuitry 504 waits for a threshold amount of time before outputting a signal to the camera 402 to cause the camera to generate image(s) of the LFA device 104 at one or more points in time.PATENT 15806WOO1

[0060] The image analyzer circuitry 506 of FIG. 5 analyzes the images obtained from the camera 402 and processes the images to determine a test result. For example, the image analyzer circuitry 506 may align captured images with the original image captured at the start of the test to correct displacement that may be caused by mechanical drift during enzymatic reactions. The image analyzer circuitry 506 may detect the influx of the buffer by thresholding the mean gray (e.g., gray scale image of) a rectangle region of interest at the center of a green channel image. As described above, when the buffer flows across the media 110, the buffer generates a fluorescence signal that will fade with time (e g., approximately 90 seconds). Thus, the image analyzer circuitry 506 processes obtained images (e.g., calculates the mean gray value of the images) to determine the onset of the enzyme reaction based on the time point when the mean gray value is lower than a threshold to begin to analyze images of the microwells 111 to determine if a target analyte is present (e.g., after fluorescence spots appear in the images). The image analyzer circuitry 506 can cause the camera 402 to auto focus to the microwells 111 upon determination of the onset of the enzyme reaction. The contrast between particle-occupied and empty micro wells is maximized by the projection of the minimum value of respective pixels of the different images. The image analyzer circuitry 506 detects fluorescence spots by the slope value of incremental fluorescence intensity at respective pixels of an image. The image analyzer circuitry 506 can perform image registration and object enhancement concurrently with a sequential image capturing during the enzymatic reactions in the microwells. The image analyzer circuitry' 506 may utilize a top-hat filter to mask debris and / or perform background subtraction on consequent particle and fluorescence imaging. In some examples, the image analyzer circuitry’ 506 extracts line profiles along the same region of interest in the multiple images. In some examples, the image analyzer circuitry 506 subtracts the background on a line profile from the first-order differentiation. The max intensity' on background-subtracted line profile around a test base is defined at the intensity' of the signal. In some examples, the image analyzer circuitry' 506 extracts peaks in particle and fluorescence imaging within a region of interest using global thresholding with Gaussian blur and maximum filter. In some examples, the image analyzer circuitry 506 determines a signal percentage which is proportional to the concentration of a target analyte based on a number of extracted peaks in the particle and fluorescence image using the below Equation 1. 100(Equation 1)

[0061] The image analyzer circuitry 506 calculates the number of fluorescent spots matched to particles by (a) dilating binary peak images with 3 by 3 pixel elements and (b)PATENT 15806WOO1 extracting the union of particle and fluorescence peaks using a logical AND operation. The image analyzer circuitry 506 counts the objects on the union image with size greater than 4 pixels as fluorescence spots matched to particle. The image analyzer circuitry 506 determines the number, quantity, percentage, etc. of fluorescence spots spatially matched to particles and determines whether a test is positive or negative based on the number, quantity, percentage, etc. of determined fluorescence spots. In some examples, the controller 414 generates a result approximately 15 minutes after a sample is applied to the LFA device 104.

[0062] FIG. 6 is a flowchart representative of an example method of manufacture 600 that may be performed to create the LFA device 104 of FIGS. 1 A-3B. The example method of manufacture 600 of FIG. 6 begins at block 602, at which the sample pad 106, the first fiber pad108 (e.g., glass, wool, or other substrate), the second fiber pad 109 (e.g., glass, wool, or other substrate), the media 110, and the wi eking pad 112 are assembled and / or generated. For example, a user and / or machine can generate and / or manufacture material into the structure of the LFA 104.

[0063] At block 604, the first fiber pad 108 is soaked with a binding member coated molecule. For example, the first fiber pad 108 can be soaked in with AP -labeled AB reagents to a particular analyte, as further disclosed above in conjunction with FIG. 1A. At block 606, the second fiber pad 109 is soaked with a binding member coated magnetic particle. For example, the second fiber pad 109 can be soaked in antibody coated superparamagnetic particle (AB-mP) reagents to the particular analyte. At block 608, the first fiber pad 108 and the second fiber pad109 are dried. For example, the first fiber pad 108 and the second fiber pad 109 may be dried using a vacuum at room temperature and stored in a location with less than 10% relative humidity until use. At block 610, the lamination film 202, the top film 204, the flow cell pattern layer 206, and the base plate 210 are assembled and / or generated according to the structure of FIGS. 2A-2B. At block 612, the generated components (e.g., the sample pad 106, the first fiber pad 108 after being soaked and dried, the second fiber pad 109 after being soaked and dried, the media 110. the wi eking pad 112. the lamination film 202, the top film 204, the flow cell pattern layer 206, and the base plate 210) are combined according to the structure of FIGS. 2A-2B using an adhesive (e.g., including the adhesive 208 of FIG. 2A).

[0064] While an example manner of implementing the controller 414 of FIG. 4 is illustrated in FIG. 5, one or more of the elements, processes, and / or devices illustrated in FIG. 5 may be combined, divided, re-arranged, omitted, eliminated, and / or implemented in any other way. Further, the user interface 500, the component interface(s) 502, the timer circuitry 504, the image analyzer circuitry 506, and / or, more generally, the example controller 414 of FIG. 5, may be implemented by hardware alone or by hardware in combination with software and / orPATENT 15806WOO1 firmware. Thus, for example, any of the user interface 500, the component interface(s) 502, the timer circuitry 504, the image analyzer circuitry 506, and / or, more generally, the example controller 414, could be implemented by programmable circuitry in combination with machine readable instructions (e.g., firmware or software), processor circuitry, analog circuit(s). digital circuit(s), logic circuit(s), programmable processor(s), programmable microcontroller(s). graphics processing unit(s) (GPU(s)), digital signal processor(s) (DSP(s)), ASIC(s), programmable logic device(s) (PLD(s)), and / or field programmable logic device(s) (FPLD(s)) such as FPGAs. Further still, the example controller 414 of FIG. 5 may include one or more elements, processes, and / or devices in addition to. or instead of. those illustrated in FIG. 5. and / or may include more than one of any or all of the illustrated elements, processes and devices.

[0065] Flowchart(s) representative of example machine readable instructions, which may be executed by programmable circuitry to implement and / or instantiate the controller 414 of FIG. 5 and / or representative of example operations which may be performed by programmable circuitry to implement and / or instantiate the controller 414 of FIG. 5, are shown in FIG. 7. The machine readable instructions may be one or more executable programs or portion(s) of one or more executable programs for execution by programmable circuitry and / or may be one or more function(s) or portion(s) of functions to be performed by the example programmable circuitry (e.g., an FPGA). In some examples, the machine readable instructions cause an operation, a task, etc., to be carried out and / or performed in an automated manner in the real world. As used herein, “automated” means without human involvement.

[0066] The program may be embodied in instructions (e.g., software and / or firmware) stored on one or more non-transitory computer readable and / or machine readable storage medium such as cache memory, a magnetic-storage device or disk (e.g., a floppy disk, a Hard Disk Drive (HDD), etc.), an optical-storage device or disk (e.g., a Blu-ray disk, a Compact Disk (CD), a Digital Versatile Disk (DVD), etc ), a Redundant Array of Independent Disks (RAID), a register, ROM. a solid-state drive (SSD), SSD memory, non-volatile memory (e.g.. electrically erasable programmable read-only memory (EEPROM), flash memory, etc ), volatile memory (e.g., Random Access Memory (RAM) of any type, etc.), and / or any other storage device or storage disk. The instructions of the non-transitory computer readable and / or machine readable medium may program and / or be executed by programmable circuitry located in one or more hardware devices, but the entire program and / or parts thereof could alternatively be executed and / or instantiated by one or more hardware devices other than the programmable circuitry and / or embodied in dedicated hardware. The machine readable instructions may be distributed across multiple hardware devices and / or executed by two or more hardware devices (e.g., aPATENT 15806WOO1 server and a client hardware device). For example, the client hardware device may be implemented by an endpoint client hardware device (e.g., a hardware device associated with a human and / or machine user) or an intermediate client hardware device gateway (e.g., a radio access network (RAN)) that may facilitate communication between a server and an endpoint client hardware device. Similarly, the non-transitory computer readable storage medium may include one or more mediums. Further, although the example program is described with reference to the flowchart(s) illustrated in FIG. 7, many other methods of implementing the example controller 414 may alternatively be used. For example, the order of execution of the blocks of the flowchart(s) may be changed, and / or some of the blocks described may be changed, eliminated, or combined. Additionally or alternatively, any or all of the blocks of the flow chart may be implemented by one or more hardware circuits (e.g., processor circuitry, discrete and / or integrated analog and / or digital circuitry, an FPGA, an ASIC, a comparator, an operational-amplifier (op-amp), a logic circuit, etc.) structured to perform the corresponding operation without executing software or firmware. The programmable circuitry may be distributed in different network locations and / or local to one or more hardware devices (e.g., a single-core processor (e.g., a single core CPU), a multi-core processor (e.g., a multi-core CPU, an XPU, etc.)). For example, the programmable circuitry may be a CPU and / or an FPGA located in the same package (e.g., the same integrated circuit (IC) package or in two or more separate housings), one or more processors in a single machine, multiple processors distributed across multiple servers of a server rack, multiple processors distributed across one or more server racks, etc., and / or any combination(s) thereof.

[0067] The machine readable instructions described herein may be stored in one or more of a compressed format, an encrypted format, a fragmented format, a compiled format, an executable format, a packaged format, etc. Machine readable instructions as described herein may be stored as data (e.g., computer-readable data, machine-readable data, one or more bits (e.g., one or more computer-readable bits, one or more machine-readable bits, etc.), a bitstream (e.g., a computer-readable bitstream, a machine-readable bitstream, etc.), etc.) or a data structure (e.g., as portion(s) of instructions, code, representations of code, etc.) that may be utilized to create, manufacture, and / or produce machine executable instructions. For example, the machine readable instructions may be fragmented and stored on one or more storage devices, disks and / or computing devices (e.g., servers) located at the same or different locations of a network or collection of networks (e.g., in the cloud, in edge devices, etc.). The machine readable instructions may require one or more of installation, modification, adaptation, updating, combining, supplementing, configuring, decry ption, decompression, unpacking, distribution, reassignment, compilation, etc., in order to make them directly readable, interpretable, and / orPATENT 15806WOO1 executable by a computing device and / or other machine. For example, the machine readable instructions may be stored in multiple parts, which are individually compressed, encrypted, and / or stored on separate computing devices, wherein the parts when decry' pted, decompressed, and / or combined form a set of computer-executable and / or machine executable instructions that implement one or more functions and / or operations that may together form a program such as that described herein.

[0068] In another example, the machine readable instructions may be stored in a state in which they may be read by programmable circuitry, but require addition of a library (e.g., a dynamic link library (DLL)), a software development kit (SDK), an application programming interface (API), etc., in order to execute the machine-readable instructions on a particular computing device or other device. In another example, the machine readable instructions may need to be configured (e.g., settings stored, data input, network addresses recorded, etc.) before the machine readable instructions and / or the corresponding program(s) can be executed in whole or in part. Thus, machine readable, computer readable and / or machine readable media, as used herein, may include instructions and / or program(s) regardless of the particular format or state of the machine readable instructions and / or program(s).

[0069] The machine readable instructions described herein can be represented by any past, present, or future instruction language, scripting language, programming language, etc. For example, the machine readable instructions may be represented using any of the following languages: C, C++, Java, C#, Perl, Python, JavaScript, HyperText Markup Language (HTML), Structured Query Language (SQL), Swift, etc.

[0070] As mentioned above, the example operations of FIG. 7 may be implemented using executable instructions (e.g.. computer readable and / or machine readable instructions) stored on one or more non-transitory computer readable and / or machine readable media. As used herein, the terms non-transitory' computer readable medium, non-transitory computer readable storage medium, non-transitory machine readable medium, and / or non-transitory machine readable storage medium are expressly defined to include any type of computer readable storage device and / or storage disk and to exclude propagating signals and to exclude transmission media. Examples of such non-transitory' computer readable medium, non- transitory computer readable storage medium, non-transitory' machine readable medium, and / or non-transitory machine readable storage medium include optical storage devices, magnetic storage devices, an HDD, a flash memory, a read-only memory (ROM), a CD, a DVD, a cache, a RAM of any t pe, a register, and / or any other storage device or storage disk in which information is stored for any duration (e.g., for extended time periods, permanently, for brief instances, for temporarily buffering, and / or for caching of the information). As used herein, thePATENT 15806WOO1 terms “non-transitory computer readable storage device” and “non-transitory machine readable storage device” are defined to include any physical (mechanical, magnetic and / or electrical) hardware to retain information for a time period, but to exclude propagating signals and to exclude transmission media. Examples of non-transitory computer readable storage devices and / or non-transitory machine readable storage devices include random access memory of any type, read only memory of any type, solid state memory, flash memory, optical discs, magnetic disks, disk drives, and / or redundant array of independent disks (RAID) systems. As used herein, the term “device” refers to physical structure such as mechanical and / or electrical equipment, hardware, and / or circuitry that may or may not be configured by computer readable instructions, machine readable instructions, etc., and / or manufactured to execute computer-readable instructions, machine-readable instructions, etc.

[0071] FIG. 7 is a flowchart representative of example machine readable instructions and / or example operations 700 that may be executed, instantiated, and / or performed by programmable circuitry to run a test to determine if a sample includes a target analyte. The example machine-readable instructions and / or the example operations 700 of FIG. 7 begin at block 702, at which the timer circuitry 504 determines if a test has started. For example, a user and / or technician may interact with the user interface 500 to indicate that the LFA device 104 is ready to be tested. In some examples, the user and / or technician may indicate the start of a test after applying the buffer 312 to the LFA device 104. In some examples, the user and / or technician may indicate the start of a test after applying the buffer 312 to the LFA device 104 and waiting for a threshold amount of time before initiating a test (e.g., 8 minutes) to give the sample time to react with the molecule of the reagent (e.g., conjugate) in the conjugate pad 108 and / or the microparticle pad 109.

[0072] If the timer circuitry 504 determines that a test has not started (block 702: NO), control returns to block 702. If the timer circuitry' 504 determines that a test has been started (block 702: YES), the timer circuitry' 504 causes the camera 402 to take an initial image of the LFA device 104 (e.g., a region of interest on the LFA device 104) by sending a control signal via the component interface 502 (block 704). Additionally, after a test starts, the timer circuitry 504 can initiate a timer. At block 706, the example timer determines if a threshold amount of time has occurred. The threshold amount of time corresponds to the amount of time for the sample 310 to react with the reagents (e.g., alkaline phosphatase in the reagent) on the LFA device 104, if a target analyte is present in the sample 310. If the user waits for the duration of time before starting the test, this step may be skipped. If the user applies the sample 310 and then starts the test, the threshold amount of time may be 8 minutes, for example. The thresholdPATENT 15806WOO1 amount of time may be different based on the type of sample, the structure of the LFA 104, the analyte of interest, the device testing, etc.

[0073] If the timer circuitry7504 determines that the threshold amount of time has not occurred (block 706: NO), the timer circuitry 504 waits until the threshold amount of time has occurred. If the timer circuitry 504 determines that the threshold amount of time has occurred (block 706: YES), the timer circuitry drives (e.g., using a control signal sent via the component interface(s) 502) the pump 416 of FIG. 4 to pump the buffer 312 onto the LFA device 104 (block 708). At block 710, the timer circuitry 504 determines if a second threshold amount of time (e.g., 1-5 minutes) has occurred. The second threshold amount of time corresponds to an amount of time for the buffer 312 to flow toward the wi eking pad 112 and for the solution in the microwells 111 to react if a target analyte is present in the sample 310. The second threshold amount of time may be based on the structure of the LFA 104, the structure of the reagents, the structure of the buffer, the analyte of interest, etc. If the timer circuitry 504 determines that the second threshold amount of time has not occurred (block 710: NO), control returns to block 710.

[0074] If the timer circuitry 504 determines that the second threshold amount of time has occurred (block 710: YES), the timer circuitry' 504 transmits a control signal to the camera 402 via the component interface 502 to cause the camera 402 to take one or more images of the LFA device 104 (e.g., the region of interest of the LFA device 104) (block 712). At block 714, the component interface 502 obtains the generated image(s) and the image analyzer circuitry 506 processes the image(s) from the camera 402 to determine a number, quantity, percentage, intensity7, and / or density7of fluorescence spots based on the image(s), as further disclosed above in conjunction with FIG. 5. The fluorescence spots are spots with fluorescence intensity above a threshold.

[0075] At block 714, the image analyzer circuitry 506 determines if the number, quantity7, percentage, etc. of identified fluorescence spots is above a threshold number of spots. If the image analyzer circuitry 506 determines that the number, quantity7, percentage, etc. of identified fluorescence spots is above a threshold (block 716: YES), the user interface 500 indicates a positive result to the user and / or technician (block 718). After block 718, the instructions end. If the image analyzer circuitry7506 determines that the number, quantity7, percentage, etc. of identified fluorescence spots is not above a threshold (block 716: NO), the user interface 500 indicates a negative result to the user and / or technician (block 720). After block 720, the instructions end.

[0076] FIG. 8 is a block diagram of an example programmable circuitry7platform 800 structured to execute and / or instantiate the example machine-readable instructions and / or the example operations of FIG. 7 to implement the controller 414 of FIG. 5. The programmablePATENT 15806WOO1 circuitry platform 800 can be, for example, a server, a personal computer, a workstation, a mobile device (e.g., a cell phone, a smart phone, a tablet such as an iPad™), or any other type of computing and / or electronic device.

[0077] The programmable circuitry platform 800 of the illustrated example includes programmable circuitry 812. The programmable circuitry 812 of the illustrated example is hardware. For example, the programmable circuitry 812 can be implemented by one or more integrated circuits, logic circuits, FPGAs, microprocessors, CPUs, GPUs, DSPs, and / or microcontrollers from any desired family or manufacturer. The programmable circuitry 812 may be implemented by one or more semiconductor based (e.g., silicon based) devices. In this example, the programmable circuitry 812 implements the user interface 500, the component interface(s) 502, the timer circuitry' 504, and the image analyzer circuitry 506.

[0078] The programmable circuitry7812 of the illustrated example includes a local memory 813 (e.g., a cache, registers, etc.). The programmable circuitry7812 of the illustrated example is in communication with main memory 814. 816, which includes a volatile memory 814 and a non-volatile memory 816, by a bus 818. The volatile memory 814 may be implemented by Synchronous Dynamic Random Access Memory7(SDRAM), Dynamic Random Access Memory (DRAM), RAMBUS® Dynamic Random Access Memory (RDRAM®), and / or any other type of RAM device. The non-volatile memory 816 may be implemented by flash memory and / or any other desired type of memory device. Access to the main memory 814, 816 of the illustrated example is controlled by a memory controller 817. In some examples, the memory7controller 817 may be implemented by one or more integrated circuits, logic circuits, microcontrollers from any desired family or manufacturer, or any other ty pe of circuitry to manage the flow of data going to and from the main memory 814, 816.

[0079] The programmable circuitry platform 800 of the illustrated example also includes interface circuitry7820. The interface circuitry7820 may be implemented by hardware in accordance w ith any type of interface standard, such as an Ethernet interface, a universal serial bus (USB) interface, a Bluetooth® interface, a near field communication (NFC) interface, a Peripheral Component Interconnect (PCI) interface, and / or a Peripheral Component Interconnect Express (PCIe) interface.

[0080] In the illustrated example, one or more input devices 822 are connected to the interface circuitry 820. The input device(s) 822 permit(s) a user (e.g., a human user, a machine user, etc.) to enter data and / or commands into the programmable circuitry7812. The input device(s) 822 can be implemented by, for example, an audio sensor, a microphone, a camera (still or video), a keyboard, a button, a mouse, a touchscreen, a trackpad, and / or a voice recognition system.PATENT 15806WOO1

[0081] One or more output devices 824 are also connected to the interface circuitry 820 of the illustrated example. The output device(s) 824 can be implemented, for example, by display devices (e.g., a light emitting diode (LED), an organic light emitting diode (OLED), a liquid crystal display (LCD), a cathode ray tube (CRT) display, an in-place switching (IPS) display, a touchscreen, etc.), and / or speaker. The interface circuitry 820 of the illustrated example, thus, typically includes a graphics driver card, a graphics driver chip, and / or graphics processor circuitry such as a GPU.

[0082] The interface circuitry' 820 of the illustrated example also includes a communication device such as a transmitter, a receiver, a transceiver, a modem, a residential gateway, a wireless access point, and / or a network interface to facilitate exchange of data with external machines (e.g., computing devices of any kind) by a network 826. The communication can be by, for example, an Ethernet connection, a digital subscriber line (DSL) connection, a telephone line connection, a coaxial cable system, a satellite system, a beyond-line-of-sight wireless system, a line-of-sight wireless system, a cellular telephone system, an optical connection, etc.

[0083] The programmable circuitry platform 800 of the illustrated example also includes one or more mass storage discs or devices 828 to store firmware, software, and / or data. Examples of such mass storage discs or devices 828 include magnetic storage devices (e.g., floppy disk, drives, HDDs, etc.), optical storage devices (e.g., Blu-ray disks, CDs, DVDs, etc.), RAID systems, and / or solid-state storage discs or devices such as flash memory devices and / or SSDs.

[0084] The machine readable instructions 832, which may be implemented by the machine readable instructions of FIG. 7. may be stored in the mass storage device 828. in the volatile memory 814, in the non-volatile memory 816, and / or on at least one non-transitory computer readable storage medium such as a CD or DVD which may be removable.

[0085] Example methods, apparatus, systems, and articles of manufacture to make and / or process a highly-sensitive diagnostic test device are disclosed herein. Further examples and combinations thereof include the following: Example 1 includes a lateral flow assay device comprising a conjugate pad including an antibody coated molecule corresponding to a target analyte, a microparticle pad including an antibody coated magnetic microparticle corresponding to the target analyte, and a media including a microwell, the media to propagate a flow of a biological sample, the antibody coated molecule, the antibody coated magnetic microparticle, and a liquid buffer along the media, the microwell structured to encapsulate the antibody coated magnetic microparticle.PATENT 15806WOO1

[0086] Example 2 includes the lateral flow assay device of example 1, wherein the molecule is alkaline phosphatase.

[0087] Example 3 includes the lateral flow assay device of any one of examples 1 -2, wherein the antibody coated molecule is dried onto the conjugate pad and the antibody coated magnetic microparticle is dried onto the microparticle pad.

[0088] Example 4 includes the lateral flow assay device of example 3, the biological sample, when applied, resuspends the dried molecule and the dried antibody coated magnetic microparticles.

[0089] Example 5 includes the lateral flow assay device of any one of examples 1-4, wherein, when the target analyte is present in the biological sample, the target analyte attaches to (a) the antibody coated molecule and (b) the antibody coated magnetic microparticle.

[0090] Example 6 includes the lateral flow assay device of example 5, wherein an enzymatic reaction occurs between the molecule and the liquid buffer in the microwell.

[0091] Example 7 includes the lateral flow assay device of example 6, wherein the enzymatic reaction generates a fluorescence signal indicative of the target analyte being present in the biological sample.

[0092] Example 8 includes the lateral flow assay device of any one of examples 1-7, further including a flow cell pattern layer in contact with the media, the flow cell pattern layer to define the flow of the biological sample, the antibody coated molecule, the antibody coated magnetic microparticle, and the liquid buffer along the media.

[0093] Example 9 includes the lateral flow assay device of example 8, further including a top layer in contact with the flow cell pattern layer, the top layer and the flow cell pattern layer defining a cavity between the top layer and the media.

[0094] Example 10 includes the lateral flow assay device of example 9, wherein, after the liquid buffer has flowed across the media, the microwell encapsulates a portion of the buffer.

[0095] Example 11 includes the lateral flow assay device of example 10, wherein the portion of the buffer is a first portion and the microwell is a first microwell, the cavity providing an air seal between the first portion of the buffer and a second portion of the buffer encapsulated in a second microwell of the media.

[0096] Example 12 includes the lateral flow assay device of any one of examples 1-11, further including a wicking pad attached to the media, the wicking pad to facilitate the flow of the biological sample, the antibody coated molecule, the antibody coated magnetic microparticle, and the liquid buffer along the media.

[0097] Example 13 includes a lateral flow assay device reader comprising a camera, a pump, machine readable instructions, and a controller to execute the machine readablePATENT 15806WOO1 instructions to at least responsive to determining that a test to read a lateral flow assay device has initiated, cause the pump to pump buffer onto a sample pad of a lateral flow assay device, after a threshold amount of time, cause the camera to capture an image of the lateral flow assay device, process the image to determine a number of fluorescence spots in the image, and output a test result based on the number of the fluorescence spots.

[0098] Example 14 includes the lateral flow assay device reader of example 13, further including a buffer reservoir to store the buffer, the pump to pump the buffer out of the buffer reservoir based on a control signal from the controller.

[0099] Example 15 includes the lateral flow assay device of any one of examples 13-14, further including a magnet to cause a magnetic particle in a sample to become encapsulated in a microwell of the lateral flow assay device.

[0100] Example 16 includes the lateral flow assay device of any one of examples 13-15, wherein the controller is to output a test result corresponding to a positive test when the number of the fluorescence spots is above a threshold.

[0101] Example 17 includes the lateral flow assay device of any one of examples 13-16, wherein a fluorescence spot is a spot in the image that corresponds to an intensity above a threshold.

[0102] Example 18 includes the lateral flow assay device of any one of examples 13-17, wherein the buffer is a fluorescence buffer.

[0103] Example 19 includes the lateral flow assay device of any one of examples 13-18, further including a filter to block wavelengths outside of a narrow band from reaching the camera, the narrow band corresponding to fluorophore excitation wavelengths.

[0104] Example 20 includes a method for generating a lateral flow assay device, the method comprising soaking a first pad with an antibody coated molecule, the antibody coated molecule corresponding to a target analyte, the antibody coated molecule to, when mixed with a buffer, react with the buffer to generate a fluorescence signal, soaking a second pad with an antibody coated magnetic microparticle, the antibody coated magnetic microparticle corresponding to the target analyte, drying the first and second pads, placing the first pad in contact with the second pad, and placing the second pad in contact with a media that includes microwells.

[0105] Example 21 includes a machine-readable storage medium that includes machine- readable instructions, that when executed, realize a controller as recited in any of examples 13- 19 or implements a method as recited in any preceding example.

[0106] The disclosed methods, apparatus and articles of manufacture improve the diagnostic testing systems using a highly sensitive diagnostic device that includes techniquesPATENT 15806WOO1 from a lateral flow assay device and a digital IA device. As disclosed above, digital IA devices utilize magnetic microparticles that bind to a target analyte and utilize fluorescence to react with a molecule that is labelled with a binding member corresponding to the target analyte to generate fluorescence signals that correspond to a test result with high sensitivity and / or high specificity. However, digital IA devices have complicated setups and may require a professional to perform the test. LFA devices have a simple process that can be done by a user that has not been trained. However, the sensitivity and / or specificity of LFA devices tends to be lower than digital IA devices. Accordingly, examples disclosed herein restructure the LFA device to leverage digital I A techniques to increase sensitivity and / or specificity with a simpler LFA device structure. Accordingly, examples disclosed herein provide a diagnostic testing device that is simple to operate and is highly sensitive and highly specific.

[0107] Descriptors "first," "second," "third," etc. are used herein when identifying multiple elements or components which may be referred to separately. Unless otherwise specified or understood based on their context of use, such descriptors are not intended to impute any meaning of priority, physical order or arrangement in a list, or ordering in time but are merely used as labels for referring to multiple elements or components separately for ease of understanding the disclosed examples. In some examples, the descriptor "first" may be used to refer to an element in the detailed description, while the same element may be referred to in a claim with a different descriptor such as "second" or "third." In such instances, it should be understood that such descriptors are used merely for ease of referencing multiple elements or components.

[0108] ’Including" and ‘"comprising” (and all forms and tenses thereof) are used herein to be open ended terms. Thus, whenever a claim employs any form of “include” or “comprise” (e.g., comprises, includes, comprising, including, having, etc.) as a preamble or within a claim recitation of any kind, it is to be understood that additional elements, terms, etc. may be present without falling outside the scope of the corresponding claim or recitation. As used herein, when the phrase "at least" is used as the transition term in, for example, a preamble of a claim, it is open-ended in the same manner as the term "comprising" and “including” are open ended. The term “and / or” when used, for example, in a form such as A, B, and / or C refers to any combination or subset of A, B, C such as (1) A alone, (2) B alone, (3) C alone, (4) A with B, (5) A with C, (6) B with C, and (7) A with B and with C. As used herein in the context of describing structures, components, items, objects and / or things, the phrase "at least one of A and B" is intended to refer to implementations including any of (1) at least one A, (2) at least one B, and (3) at least one A and at least one B. Similarly, as used herein in the context of describing structures, components, items, objects and / or things, the phrase "at least one of A or B" isPATENT 15806WOO1 intended to refer to implementations including any of (1) at least one A, (2) at least one B, and (3) at least one A and at least one B. As used herein in the context of describing the performance or execution of processes, instructions, actions, activities and / or steps, the phrase "at least one of A and B" is intended to refer to implementations including any of (1) at least one A, (2) at least one B. and (3) at least one A and at least one B. Similarly, as used herein in the context of describing the performance or execution of processes, instructions, actions, activities and / or steps, the phrase "at least one of A or B" is intended to refer to implementations including any of (1) at least one A, (2) at least one B, and (3) at least one A and at least one B.

[0109] As used herein, singular references (e.g.. “a,” "an.” "first." "second." etc.) do not exclude a plurality. The term “a” or “an” entity, as used herein, refers to one or more of that entity. The terms “a” (or “an”), “one or more,” and “at least one” can be used interchangeably herein. Furthermore, although individually listed, a plurality of means, elements or method actions may be implemented by, e.g., a single unit or processor. Additionally, although individual features may be included in different examples or claims, these may possibly be combined, and the inclusion in different examples or claims does not imply that a combination of features is not feasible and / or advantageous.

[0110] The following claims are hereby incorporated into this Detailed Description by this reference, with each claim standing on its own as a separate embodiment of the present disclosure.

Claims

PATENT15806WOO1What Is Claimed Is:

1. A lateral flow assay device comprising: a conj ugate pad including an antibody coated molecule corresponding to a target analyte; a microparticle pad including an antibody coated magnetic microparticle correspondingo the target analyte; and a media including a microwell, the media to propagate a flow of a biological sample, the antibody coated molecule, the antibody coated magnetic microparticle, and a liquid buffer along the media, the microwell structured to encapsulate the antibody coated magnetic microparticle.

2. The lateral flow assay device of claim 1, wherein the molecule is alkaline phosphatase.

3. The lateral flow assay device of any of claims 1-2, wherein the antibody coated molecule is dried onto the conjugate pad and the antibody coated magnetic microparticle is dried onto the microparticle pad.

4. The lateral flow assay device of claim 3, the biological sample, when applied, resuspends the dried molecule and the dried antibody coated magnetic microparticles.

5. The lateral flow assay device of any of claims 1-4, wherein, when the target analyte is present in the biological sample, the target analyte attaches to (a) the antibody coated molecule and (b) the antibody coated magnetic microparticle.

6. The lateral flow assay device of claim 5, wherein an enzymatic reaction occurs between the molecule and the liquid buffer in the microwell.

7. The lateral flow assay device of claim 6, wherein the enzymatic reaction generates a fluorescence signal indicative of the target analyte being present in the biological sample.

8. The lateral flow assay device of any of claims 1-7, further including a flow cell pattern layer in contact with the media, the flow cell pattern layer to define the flow of the biological sample, the antibody coated molecule, the antibody coated magnetic microparticle, and the liquid buffer along the media.

9. The lateral flow assay device of claim 8, further including a top layer in contact with the flow cell pattern layer, the top layer and the flow cell pattern layer defining a cavity between the top layer and the media.

10. The lateral flow assay device of any of claims 1-9, wherein, after the liquid buffer has flowed across the media, the microwell encapsulates a portion of the buffer.

11. The lateral flow assay device of claim 10, wherein the portion of the buffer is a first portion and the microwell is a first microwell, the cavity providing an air seal between thePATENT 15806WOO1 first portion of the buffer and a second portion of the buffer encapsulated in a second microwell of the media.

12. The lateral flow assay device of any of claims 1-11, further including a wicking pad attached to the media, the wicking pad to facilitate the flow of the biological sample, the antibody coated molecule, the antibody coated magnetic microparticle, and the liquid buffer along the media.

13. A lateral flow assay device reader comprising: a camera; a pump; machine readable instructions; and a controller to execute the machine readable instructions to at least: responsive to determining that a test to read a lateral flow7assay device has initiated, cause the pump to pump buffer onto a sample pad of a lateral flow assay device; after a threshold amount of time, cause the camera to capture an image of the lateral flow assay device; process the image to determine a number of fluorescence spots in the image; and output a test result based on the number of the fluorescence spots.

14. The lateral flow7assay device reader of claim 13, further including a buffer reservoir to store the buffer, the pump to pump the buffer out of the buffer reservoir based on a control signal from the controller.

15. The lateral flow assay device of any of claims 13-14, further including a magnet to cause a magnetic particle in a sample to become encapsulated in a microwell of the lateral flow assay device.

16. The lateral flow7assay device of any of claims 13-15, wherein the controller is to output a test result corresponding to a positive test when the number of the fluorescence spots is above a threshold.

17. The lateral flow7assay device of any of claims 13-1 , wherein a fluorescence spot is a spot in the image that corresponds to an intensity above a threshold.

18. The lateral flow7assay device of any of claims 13-17, wherein the buffer is a fluorescence buffer.

19. The lateral flow7assay device of any of claims 13-18, further including a filter to block wavelengths outside of a narrow7band from reaching the camera, the narrow band corresponding to fluorophore excitation wavelengths.

20. A method for generating a lateral flow assay device, the method comprising:PATENT 15806WOO1 soaking a first pad with an antibody coated molecule, the antibody coated molecule corresponding to a target analyte, the antibody coated molecule to, when mixed with a buffer, react with the buffer to generate a fluorescence signal; soaking a second pad with an antibody coated magnetic microparticle, the antibody coated magnetic microparticle corresponding to the target analyte; drying the first and second pads; placing the first pad in contact with the second pad; and placing the second pad in contact with a media that includes microwells.

21. A machine-readable storage medium comprising machine-readable instructions, that when executed, realize a controller as recited in any of claims 13-19 or implements a method as recited in any preceding claim.