Lateral flow assay system and method of reading and quantifying analyte concentration based on visual width of bound analyte

Abstract

The disclosure relates to lateral flow assay (LFA) systems and methods of reading and quantifying analyte concentration in a test sample. The LFA is configured to bind analyte within a test band whereby a visual width of bound analyte within the test band is proportional to the concentration of analyte.

Description

LATERAL FLOW ASSAY SYSTEM AND METHOD OF READING AND QUANTIFYING ANALYTE CONCENTRATION BASED ON VISUAL WIDTH OF BOUND ANALYTE CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to and the benefit of United States Provisional Patent Application Number 63 / 775,650 filed on March 21, 2025, entitled “LATERAL FLOW ASSAY SYSTEM AND METHOD OF READING AND QUANTIFYING ANALYTE CONCENTRATION BASED ON VISUAL WIDTH OF BOUND ANALYTE,” the entire contents of which are incorporated by reference.FIELD

[0002] The disclosure relates to lateral flow assay (LFA) systems and methods of reading and quantifying analyte concentration in a test sample. The LFA is configured to bind analyte within a test band whereby a visual width of bound analyte within the test band is proportional to the concentration of analyte.BACKGROUND

[0003] Lateral flow assays are widely used for point-of-care diagnostics and wellness due to their simplicity, cost-effectiveness, and rapid results. Traditional LFAs provide qualitative or semi-quantitative results often relying on the visual intensity of test and control lines. Generally, LFAs designed for qualitative analysis for the presence of an analyte return a positive, negative or inconclusive result. LFAs have been designed for semi-quantitative analysis via test line intensity wherein the color intensity of a test line provides an estimation of the quantity of analyte in a sample. However, intensity-based measurements are subject to variability due to environmental factors, user interpretation, and inconsistencies in manufacturing. Moreover, intensity-based measurement requires color quantification and the use of a universal reader, which is subject to error due to background light and shadows. As a result, a more reliable method for quantitative analysis is needed to improve accuracy and reproducibility.SUMMARY

[0004] In accordance with the disclosure, a Lateral flow assay (LFA) system is described comprising: a body having: a sample pad configured to receive a sample containing an analyte; a conjugate release pad configured to the sample pad and configured with a conjugate / anti-analyte detector antibody complex (OY); a membrane configured to the conjugate release pad, the membrane having: a test band configured with an anti-analyte capture antibody (Y1); a control line configured with an anti-OY detector antibody (Y2); an absorbent pad configured to the membrane, wherein each of the sample pad, conjugate release pad, membrane and absorbent pad are mounted to a backing; wherein the quantity of OY is greater than a predetermined amount of analyte in the sample and the quantity of Y1 configured to the test band is greater than a pre-determined amount of analyte in the sample; wherein analyte, OY and Y1 may form a visual analyte-OY-Y1 complex in the test band; and, wherein the test band has a length parallel to a direction of flow sufficient to discern variations in length of the visual analyte-0Y-Y1 complex in the test band proportional to a concentration of analyte in the sample.

[0005] In various embodiments, any of the following elements may be configured to form a useful combination of elements including intermediate generalizations thereof.

[0006] In various embodiments:• The test band includes two or more sub-bands, each sub-band being configured with Y1.• Each sub-band includes a sub-band zone between adjacent sub-bands without Y1.• Each sub-band is configured with an equal amount of Y1 and the total amount of Y1 in the test band corresponds to an anticipated maximum amount of analyte in the sample.• The test band is configured with at least one additional downstream sub-band configured with additional Y1 over and above the upstream amount of Y1.• The test band includes two or more parallel test bands each configured with Y 1 , wherein Y1 in each parallel test band is specific to a different analyte.• The test band is configured with 10 sub-bands each sub-band configured to correspond to a 10% increment of a range of concentration of analyte in the sample.• The test band is configured with an additional downstream 11thsub-band configured with additional Y1 over and above the upstream amount of Y1.

[0007] In another aspect, the disclosure describes a system for obtaining and analyzing images from a biomarker test comprising: a smartphone having a camera and a smartphone processor configured with smartphone application software and having non-transitory memory including instructions configured to: prompt a user to initiate image acquisition from the biomarker test; activate the camera; prompt the user to position the biomarker test on a surface and position the camera over the biomarker test; obtain at least one captured image; and deliver the at least one captured image to a back-end computer system over the internet; wherein the back-end computer system includes a back-end processor configured with back-end application software having non-transitory memory including instructions configured to: receive the at least one captured images; conduct a test band width analysis of the captured images; and, compare the test band width analysis against a calibration curve and obtain a diagnostic test result.

[0008] In various embodiments:• The back-end processor is configured with instructions to report the diagnostic test result to one or more stakeholders over the internet.• The smartphone processor is configured with instructions to obtain first and second captured images in succession.• The first and second captured images are RAW data images.• The smartphone processor is configured with instructions to activate a camera flash during acquisition of the first or second captured image.• The back-end computer system includes a back-end database storing biomarker test parameters and the back-end processor is configured with instructions to search the back-end database to access a testing protocol specific to a biomarker test kit.• The back-end computer system is configured with instructions to analyze a relative test band line length and correlate the test band line length to a quantitative level of analyte from a biomarker test.

[0009] In another aspect, a method of obtaining and analyzing images from a biomarker test is described, the method comprising the steps of: from a smartphone having a camera and a smartphone processor configured with application software and having non-transitory memory including instructions: prompting a user to initiate image acquisition; activating the smartphone camera; prompting the user to position a biomarker test on a surface and position the camera over the completed test; obtaining at least one capture image; and, delivering captured images to a back-end system over the internet.

[0010] In various embodiments:• The method includes the step of reporting the diagnostic test result to one or more stakeholders over the internet.• The method includes the step of obtaining first and second captured images in succession.• The method includes the step of activating a camera flash during acquisition of the first or second captured image.• The method includes the step of searching a back-end database to access a testing protocol specific to a biomarker test kit.BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Various objects, features and advantages of the disclosure will be apparent from the following description of particular embodiments, as illustrated in the accompanying drawings. The drawings are not necessarily to scale, emphasis instead being placed upon illustrating the principles of various embodiments of the disclosure. Similar reference numerals indicate similar components.Figure 1 is a schematic plan view of a typical lateral flow assay (LFA) test device in accordance with the prior art showing the visual results of diagnostic test including a positive (A), a negative (B) and an inconclusive (C) test.Figure 2 is a schematic side view of a typical mechanism of operation of an LFA in accordance with the prior art showing the configuration of a test kit before use (A) and the binding and migration of analytes across a test kit during a test (B).Figure 3 is a schematic side view of an LFA in accordance with the disclosure showing the configuration of a test kit before use (A).Figures 3A, 3B and 3C are schematic plan views of a test band having sub-bands in accordance with one embodiment of the disclosure. Figure 3B shows binding across a number of sub-bands of the test band and Figure 3C shows a test band configured to capture different analytes within a test sample showing different concentrations of captured analytes.Figure 4 is a flowchart showing representative steps of a testing procedure including different sample preparation steps.Figures 5 and 5A are schematic views of an LFA in accordance with the disclosure showing how different amounts of analyte in a sample result in different quantities of bound analyte within a test band and corresponding differences in measured signal width via signal intensity and signal area.Figures 6 and 7 are schematic diagrams showing a communication system configured to obtain, analyze and report test results to stakeholders in accordance with the disclosure. Figure 6 shows collecting an image from an LFA test and Figure 7 shows how data from the test may be processed and delivered to stakeholders.Figure 8 is a flowchart showing steps of obtaining, analyzing and reporting test results to stakeholders in accordance with the disclosure.Figure 9 is a graph showing analyte concentration as a function of test band width as measured in pixels.Figure 10 is a flowchart showing steps of identifying a bioassay test kit and activating a test protocol based on known or generic test protocols in accordance with the disclosure.DETAILED DESCRIPTION

[0012] With reference to the figures, lateral flow assay (LFA) systems and methods of reading and quantifying analyte concentration based on a visually determined width of bound analyte within a lateral flow assay test kit are described.Terminology

[0013] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the disclosure. As used herein, the singular forms “a”, “an” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise. It will be understood that the terms “comprises” and / or “comprising,” when used in this specification, specify the presence of stated features, steps, operations, elements, and / or components, but do not preclude the presence or addition of one or more other features, steps, operations, elements, components, and / or groups thereof. As used herein, the term “and / or” includes any and all useful combinations of one or more of the associated listed items. Moreover, the specification includes appended claims, that at the time of writing utilizes specific words, chosen to distinguish the subject matter herein over prior art. The specification also introduces elements which in combination with other elements constitutes a patentable combination. It is understood and expressly stated that combinations may not be, in all cases, directly described or claimed utilizing words as they exist in the specification when written; however, as this specification is written for interpretation by one of ordinary skill in the art, various combinations and the specific language used to interpret those combinations may be reasonably inferable and / or unambiguously derived from the collective language and drawings of this description as understood by one of ordinary skill in the art.

[0014] It is understood that the language used in this description is intended to provide both breadth and depth to the scope of the appended claims and that any language utilized to express breadth and depth is intended to cover any element that may be considered to be intermediate between a broad form of that element and a more specific form of that element.

[0015] Spatially relative terms may be used herein for ease of description to describe one element or feature's relationship to another element(s) or feature(s) as illustrated in the figures. It will be understood that the spatially relative terms are intended to encompass different orientations of the device in use or operation in addition to the orientation depicted in the figures. For example, if a feature in the figures is inverted, elements described as “under” or “beneath” other elements or features would then be oriented “over” the other elements or features. Thus, the exemplary term “under” can encompass both an orientation of over and under. A feature may be otherwise oriented (rotated 90 degrees or at other orientations) and the spatially relative descriptors used herein interpreted accordingly. Similarly, the terms “upwardly”, “downwardly”, “vertical”, “horizontal” and the like are used herein for the purpose of explanation only unless specifically indicated otherwise.

[0016] It will be understood that when an element is referred to as being “on”, “attached” to, “connected” to, “coupled” with, “contacting”, etc., another element, it can be directly on, attached to, connected to, coupled with or contacting the other element or intervening elements may also be present. In contrast, when an element is referred to as being, for example, “directly on”, “directly attached” to, “directly connected” to, “directly coupled” with or “directly contacting” another element, there are no intervening elements present.

[0017] It will be understood that, although the terms “first”, “second”, etc. may be used herein to describe various elements, components, etc., these elements, components, etc. should not be limited by these terms. These terms are only used to distinguish one element, component, etc. from another element, component. Thus, a “first” element, or component discussed herein could also be termed a “second” element or component without departing from the teachings of the present disclosure. In addition, the sequence of operations (or steps) is not limited to the order presented in the claims or figures unless specifically indicated otherwise.

[0018] Other than described herein, or unless otherwise expressly specified, all of the numerical ranges, amounts, values and percentages, such as those for amounts of materials, elemental contents, times and temperatures, ratios of amounts, and others, in the following portion of the specification and attached claims may be read as if prefaced by the word “about” even though the term “about” may not expressly appear with the value, amount, or range. Accordingly, unless indicated to the contrary, the numerical parameters set forth in the following specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained by the present disclosure. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.

[0019] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

[0020] Various aspects of the disclosure will now be described with reference to the figures. The disclosure may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein; rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scopeof the disclosure to those skilled in the art. Moreover, the drawings are not necessarily drawn to scale and are intended to emphasize principles of operation rather than precise dimensions.Introduction

[0021] Test kits for assaying various biomarkers are known. For example, during the COVID-19 pandemic, the world became particularly familiar with conducting lateral flow assay (LFA) tests.

[0022] As is known, an LFA test is typically conducted by accessing a test kit, obtaining a biological sample, introducing the biological sample potentially containing an analyte of interest to a lateral flow medium, introducing buffer and waiting a period of time for the biological sample to react with a test line and a control line to produce a test result.

[0023] By way of example, as shown in Figures 1 and 2, an LFA test kit (or simply LFA) 10 generally includes a sample pad 10b, a conjugate release pad 10c, a membrane 10d, a test line 10e, a control line 10f and an absorbent pad 10g mounted to a backing 10a. In some embodiments, additional components will be readily appreciated by a person of ordinary skill in the art as being required for the particular test, for example a plasma separator membrane. An LFA may also include a casing for stability and handling, which casing may be referred to as a body, housing, or other related term. Such casing may be formed of one or more components joined together and may permanently retain the other components of LFA 10 or may be adapted so that other components of LFA 10 may be removable or semi-removable from the casing, for example having two halves that are separable (i.e. a clamshell design). As will be appreciated, the casing may have one or more apertures extending therethrough adapted to receive the sample and allow the sample to flow into sample pad 10b. The casing may also have one or more apertures extending therethrough and / or substantially transparent portions through which test line 10e and control line 10f may be observed. Embodiments without such apertures or transparent portions may be adapted to have removable or semi-removable cards or cassettes comprising at least test line 10e and control line 10f to permit observation of test results. Except as expressly stated in the claims, the configuration of an operable LFA for a particular test, including necessary components and their arrangement, attachment, and design will be readily apparent to a person of ordinary skill in the art and are not intended to be limiting.

[0024] As shown in Figure 2, a liquid sample containing the analyte of interest Z is applied to the sample pad 10a which can then diffuse / move via capillary action through the various zonesof the membrane where the analyte can interact with various capture reagents contained in different zones of the LFA.

[0025] For example, the test kit may be configured with antibodies Y (anti-analyte detector antibody) that are specific to the analyte Z which bind the analyte as the sample migrates across the strip. The antibodies Y are typically conjugated to one or more reporter groups such as colored or fluorescent particles-most commonly colloidal gold or latex microspheres (marker particles) O shown as a Y-0 complex (referred to as a “conjugate complex”) within the conjugate release pad 10c. Other types of reporter groups may be conjugated to antibodies Y provided that such reporter groups are adapted to produce a detectable line across the membrane, as will be readily apparent to a person of ordinary skill in the art. It is anticipated that reporter groups that produce a “visible line” via a colorimetric signal visible to the human eye (i.e. within the visual light spectrum of about 380 nm to 750 nm) or a grayscale signal with an intensity that is visible to the human eye may be preferred; however, it is also anticipated that other reporter groups may be used provided that they produce a signal that is detectable by known means, including reporter groups that produce colorimetric and / or grayscale signals that are not visible to the human eye, radioactive reporter groups, isotopic reporter groups, and any combination of known reporter groups that may be detected by known readers and instruments. The type of reporter group is not intended to be limiting. In the present example, as the analyte Z migrates across the membrane 10d, it binds to the Y-0 complex to form a Y-O-Z complex which migrates to the test line 10e. The test line is conjugated with an anti-analyte antibody Y1 (anti-analyte capture antibody) which can form a Y1-Z-Y-0 complex. Binding at the test line results in a visible line.

[0026] The control line 10f is conjugated with antibodies Y2 (anti-conjugate detector antibody) specific to Y such that mobilized Y-0 complexes will bind to the control line as Y2-Y-0 complexes. Binding at the control line results in a visible line.

[0027] If the analyte of interest is present in the sample, the Y-Z-0 complex forms which can then be captured at the test line as a Y1 -Y-Z-0 complex. Y-0 will also be bound at the control line as Y2-Y-O. The combination of these two visible lines indicates a positive test (Figures 1A and 2B).

[0028] If the analyte is not present in the sample, the Y-Z-0 complex does not form at the test line while Y-0 binding occurs at the control line as Y2-Y-0 which indicates a negative test (Figure 1B).

[0029] Weakly appearing lines can indicate an inconclusive test (Figure 1C).

[0030] In accordance with the disclosure, and with reference to Figure 3, a lateral flow assay device 30 having a wide test band 30e is shown having a width in the range of 5-15 mm. As above, the LFA comprises a sample pad 30b, a conjugate pad 30c, a nitrocellulose membrane 30d, a control line 30f and an absorbent pad 30g mounted to a backing (or membrane card) 30a. The test band 30e is preferably multiple sub-bands, each sub-band having a width of approximately 0.5-1 mm. A gap may exist between each sub-band and may be 0-1 mm wide (preferably about 0.1-0.5 mm wide) or the test band may be a single, continuous band having a width of about 5-15 mm as noted above.

[0031] As above, the test band includes an immobilized capture antibody Y1 that binds to the analyte / conjugate (Y-Z-0 complex). The Y1-Y-Z-0 produces a visual signal (e.g. red color) obtained on the test band from binding of the Y-Z-0 complex with the test band antibody Y1.

[0032] The relative amount of analyte within the volume of the sample applied to the LFA, and by extension, the concentration of analyte within the analyte source (e.g. the blood of a test subject) can be determined by configuring the LFA such that the number of O-Y complexes and Y1 binding sites are proportional to analyte concentration in the test sample.

[0033] In order to provide a quantifiable determination of the concentration of analyte in a test sample, substantially all analyte molecules Z that are added to the LFA must be fully bound to O-Y complexes and resulting Y-Z-0 complexes must be fewer in number than available Y1 binding sites in the test band.

[0034] That is, as Y-Z-0 complexes migrate over the test band, they become fully bound to Y1 binding sites such that, at a point of their linear progression, substantially all Y-Z-0 complexes are bound to Y1. As such, no additional Y1-Y-Z-0 complexes are formed past a linear position in the test band.

[0035] Accordingly, the linear distance over which binding occurs is a proportional measure of the total number of analyte molecules in the test sample with a furthest visible lateral edge or sub-band of the test band proportional to the total number of analyte molecules in the test sample.

[0036] The number of available Y-0 complexes and Y1 antibodies in the test band is determined by an expected relative range of analyte concentration in the analyte source (e.g. the blood of a test subject) or a diluted test sample as described below.

[0037] By way of example, and as shown in Figure 3A, a test band may be configured with 11 separate bands across the test band length, progressively numbered as bands 1-11. The analyte is known to exist in the analyte source in a concentration between 0 and 10 mg / ml and is applied as test sample directly to the LFA. An appropriate and corresponding number of Y-O complexes are available for binding up to 10 mg / ml of analyte Z. As such, as Z is applied to the LFA, all analyte Z binds with Y-0 as Z migrates across the band and the Y-Z-0 will bind with Yl in the test band. If the test sample contains 3 mg / ml of analyte, subsequent binding will occur across the first 3 sub-bands of the test band as shown in Figure 3B as a visual signal. Different analyte concentrations would result in proportionally greater or fewer sub-bands becoming visible.

[0038] The test band may be configured with additional bands beyond the expected 100% range of analyte as shown in Figures 3A and 3B. While the appearance of the 11thband may be an accurate indicator of a positive test result, its appearance may also indicate an inconclusive quantification measurement and / or may be an indicator of an improperly prepared test sample.

[0039] In various embodiments, the test band may also be configured to capture different analytes as shown in Figure 3C. In this case, different antibody / analyte chemistries may be configured to the test band as a grid where each row shown in Figure 3C is configured for a different analyte. Upon completion of the test different concentrations of the different analytes may be observed. Each row of the test band may be configured for different analyte concentrations and hence not utilize the same scale across the grid.

[0040] As shown in Figure 4, depending on the test being performed, sample preparation steps may be performed prior to introducing a sample volume to the LFA in order to ensure the relative number of molecules required for a quantification measurement to align. Such steps may include collecting the sample and dilution of the sample before applying a sample volume to the LFA and conducting a subsequent analysis. Such steps, including other steps such as denaturing the sample with a denaturing agent, may be required to ensure that the total number of Z, O-Y and Y1 molecules / complexes are within the required ranges for a specific test.

[0041] In various embodiments, LFA 10 and / or its casing may display printed labels corresponding with the location of the test band and control band. Having regard for Figure 1 , for example, the location of the test band may be marked with a label T and the location of the control band may be marked with a label ‘C’. In another example, printed labels may be displayed corresponding with the location of one or more of the test band sub-bands and / or additional bands shown in Figures 3A, 3B, and 3C, the labels reporting the number of the band (i.e. sub-band 1 , sub-band 2, sub-band 3, additional band 1 , etc.) or the apparent concentration of the analyte source (i.e. 0 mg / ml, 1 mg / ml, 2 mg / ml, 3 mg / ml etc.). In other embodiments utilizing readers such as smartphones, as will be described in more detail below, printed labels may report information readable by the reader via register marks, bar codes, QR codes, or other known methods. As will be appreciated, the printed labels allow for convenient and accurate reading of the LFA results by a user, who may compare the resulting visible band or bands with the printed labels corresponding with the location of each band.Analysis

[0042] Test results can be interpreted by eye (i.e. visible lines) or by using dedicated readers (i.e. visible lines or lines detectable by other known means). While test results can often be readily understood, particularly if the stakeholder is skilled in reading the LFA, there can be an element of subjectivity, particularly in the case of interpretation by eye by unskilled stakeholders (e.g. the test subject). Furthermore, dedicated readers may not be readily available and / or may be prohibitively expensive. Moreover, the reporting of test results to stakeholders can be nonexistent and / or subject to significant delays.

[0043] In accordance with this description, the inventors have recognized that smartphones can be utilized to address these challenges which can improve the efficiency and accuracy of conducting tests, interpreting results and delivering those results to stakeholders.

[0044] As shown in Figures 5 and 5A, different examples of conjugate / antibody binding at the test band are shown for different analyte concentrations and methodologies for determining the measured width in pixels across the test band. Figure 5 shows an example where changes in the intensity of the signal as a function of pixel distance is used to determine the relative width and, hence, concentration of analyte in the sample. Figure 5A shows an example where the concentration of analyte is determined by calculating the area of the intensity / pixel distance signal to determine relative analyte concentration.

[0045] Figure 6 shows a smartphone 30 having a camera (with camera application software) in a position to obtain images of test results from a test kit 10 for automatic determination of test band width. In this example, a stakeholder such as a test subject / patient and / or a caregiver obtains a photo of the LFA for analysis as explained below.

[0046] Figure 7 shows stakeholders in a multi-user system together with a back-end computer system 40 (with back-end application software) that can be used to collect and analyze test data and deliver that analyzed data to multiple stakeholders via the cloud.

[0047] Data collection and analysis is described via a representative example, where a patient and / or their caregiver (a “user”) wishes to complete a test and have those results delivered back to the user and / or to a medical team.

[0048] A LFA test is completed in the usual manner as described and a result is obtained where 3 test lines are visible (see Figure 3B) as well as a control line (e.g. a positive test).

[0049] The user accesses a smartphone 30 having application software configured to obtain images of the test kit and automatically deliver those images to a back-end system having back-end software to receive and interpret image results.

[0050] In one embodiment, the smart phone application software instructs the user to place the completed test on a surface in a manner that enables one or more images of the test kit to be obtained to enable accurate interpretation of image(s) of the test kit.

[0051] In a typical system, the user will be prompted through a series of instructions to position the smartphone camera over the completed test, frame the completed test and obtain one or more images of the test kit (the user may also be instructed on how to conduct the test including any required steps to prepare the sample). The images may be automatically delivered via the internet / cloud to the back-end system 40 where the images are interpreted and a result obtained. Such results may be automatically reported back to any one of or a combination of the patient 40a, a caregiver 40b, a health care provider 40c and / or a health authority 40d as shown in Figure 7.

[0052] In various embodiments as shown in Figure 8, upon activation of the smartphone application the smartphone application may be configured to:• Prompt the user to complete a test including preparing the test sample.• Prompt the user to confirm that the test is completed.• Automatically activate the smartphone camera.• Prompt the user to position the completed test on a surface and position the camera in a particular manner over the completed test.• Automatically obtain images and / or prompt a user to obtain images.• Automatically deliver captured images to a back-end computer system over the internet.

[0053] In various embodiments, the smartphone application software may also be configured to:• Activate a smartphone flash 30a and deliver a known flash intensity as images are being collected.• Prompt the user to locate the test kit at a specific distance and orientation to the camera and / or frame the test kit.

[0054] Importantly, as the location and lighting conditions that may be present after completion of a test will be highly variable, the smartphone may be configured to capture at least two RAW images of one or more LFA test lines. In one embodiment, a first image is taken without a flash and a second image is taken with a flash resulting in an image pair. The images of the image pair are taken in quick succession to eliminate movement variation between the images.

[0055] If the images are satisfactory, the selected images are uploaded to a back-end computer system for analysis.

[0056] In various embodiments, where the test kit is configured to determine analyte concentration, the relative number of visible sub-bands and / or width of the test band may be correlated to an analyte concentration. For example, as shown in Figures 5 and 9, the number / width of test sub-bands / test band can be correlated to analyte concentration via predetermined calibration curves. In the case of multiple analytes, each analyte may have a separate calibration curve. It is understood that other forms of the test bands could be incorporated on the membrane such as a linear array of dots.

[0057] The positive / negative and / or quantitative measurement can be sent back to the user’s smartphone, and / or to other stakeholders if permissions have been granted.

[0058] In various embodiments, the smartphone application software is configured to automatically recognize the type of test being conducted which may be determined by ID markings / codes 60a on the test kit, such as QR codes (Figure 6).

[0059] By recognizing a specific test kit (as shown in Figure 10), which may be specific to a manufacturer and / or may be a test kit for a specific analyte or multiple analytes, the smartphone application software may deliver prompts to the user specific to that test kit, follow different image capture protocols and / or mark the image data as specific to a type of test, enabling the back-end to then recognize the images and analyze the image(s) according to a preferred analysis protocol for that specific test kit. If an ID code is not recognized, a generic test protocol may be activated that may simply assess basic test results such as standard T and C lines on an LFA.

[0060] As described above, the smartphone application software may also be configured to automatically recognize other information about the test kit or its results by corresponding labels printed on the test kit.

[0061] Non-limiting examples of analytes that can be analyzed using the system may include Ferritin(iron), Progesterone, Estradiol, HcG (Human Gonadotropin), Luteinizing Hormone (LH), ApoB, Total Cholesterol, LDL, HDL, Haemoglobin, Vitamin D, etc.

[0062] Advantages of the system include:• Objectivity given that subjective interpretation of line intensity is not required.• Enhanced precision as distance is a fixed physical parameter that is unaffected by ambient light or visual perception.• A reproducible signal can generally be obtained by most smartphones.• The system enables a wide dynamic range by incorporating multiple test lines allowing measurement of a broader range of analyte concentrations.• The system can be integrated with existing LFA formats and optical readers for compatibility and enhanced functionality.• An LFA may be configured to quantitatively measure different analytes within the same sample.

[0063] Although the present disclosure has been described and illustrated with respect to preferred embodiments and preferred uses thereof, it is not to be so limited since modifications and changes can be made therein which are within the full, intended scope of the disclosure as understood by those skilled in the art.

Claims

CLAIMS1. A lateral flow assay (LFA) system comprising:a body having:a sample pad configured to receive a sample containing an analyte;a conjugate release pad configured to the sample pad and configured with a conjugate / anti-analyte detector antibody complex (OY);a membrane configured to the conjugate release pad, the membrane having:a test band configured with an anti-analyte capture antibody (Y1);a control line 10f configured with an anti-OY detector antibody (Y2);an absorbent pad configured to the membrane,wherein each of the sample pad, conjugate release pad, membrane and absorbent pad are mounted to a backing;wherein the quantity of OY is greater than a pre-determined amount of analyte in the sample and the quantity of Y1 configured to the test band is greater than a pre-determined amount of analyte in the sample;wherein analyte, OY and Y1 may form a visual analyte-OY-Y1 complex in the test band; and,wherein the test band has a length parallel to a direction of flow sufficient to discern variations in length of the visual analyte-OY-Y1 complex in the test band proportional to a concentration of analyte in the sample.

2. The LFA as in claim 1 wherein the test band includes two or more sub-bands, each sub-band being configured with Y1.

3. The LFA as in claim 2 wherein each sub-band includes a sub-band zone between adjacent sub-bands without Y 1.

4. The LFA as in claim 2 or 3 wherein each sub-band is configured with an equal amount of Y1 and the total amount of Y1 in the test band corresponds to an anticipated maximum amount of analyte in the sample.

5. The LFA as in claim 4 wherein the test band is configured with at least one additional downstream sub-band configured with additional Y1 over and above the amount of Y1 of claim 4.

6. The LFA as in any one of claims 1-5 wherein the test band includes two or more parallel test bands each configured with Y1, wherein Y1 in each parallel test band is specific to a different analyte.

7. The LFA as in any one of claims 1-5 wherein the test band is configured with 10 sub-bands each sub-band configured to correspond to a 10% increment of a range of concentration of analyte in the sample.

8. The LFA as in any one of claims 1-4 wherein the test band is configured with an additional downstream 11thsub-band configured with additional Y1 over and above the amount of Y1 defined in claim 4.

9. A system for obtaining and analyzing images from a biomarker test comprising:a smartphone having a camera and a smartphone processor configured with smartphone application software and having non-transitory memory comprising instructions configured to:prompt a user to initiate image acquisition from the biomarker test;activate the camera;prompt the user to position the biomarker test on a surface and position the camera over the biomarker test;obtain at least one captured image; and,deliver the at least one captured image to a back-end computer system over the internet;i) wherein the back-end computer system includes a back-end processor configured with back-end application software having non-transitory memory comprising instructions configured to:receive the at least one captured images;conduct a test band width analysis of the captured images; and,compare the test band width analysis against a calibration curve and obtain a diagnostic test result.

10. The system as in claim 11 wherein the back-end processor is configured with instructions to report the diagnostic test result to one or more stakeholders over the internet.

11. The system as in claim 9 or 10 wherein the smartphone processor is configured with instructions to obtain first and second captured images in succession.

12. The system as in claim 11 where the first and second captured images are RAW data images.

13. The system as in any one of claims 10-12 where the smartphone processor is configured with instructions to activate a camera flash during acquisition of the first or second captured image.

14. The system as in any one of claims 10-13 wherein the back-end computer system includes a back-end database storing biomarker test parameters and the back-end processor is configured with instructions to search the back-end database to access a testing protocol specific to a biomarker test kit.

15. The system as in any one of claims 10-14 wherein the back-end computer system is configured with instructions to analyze a relative test band line length and correlate the test band line length to a quantitative level of analyte from a biomarker test.

16. A method of obtaining and analyzing images from a biomarker test comprising the steps of:from a smartphone having a camera and a smartphone processor configured with application software and having non-transitory memory comprising instructions configured to:prompting a user to initiate image acquisition;activating the smartphone camera;prompting the user to position a biomarker test on a surface and position the camera over the completed test;obtaining at least one capture image; and,delivering captured images to a back-end system over the internet.

17. The method as in claim 16 further comprising the step of reporting the diagnostic test result to one or more stakeholders over the internet.

18. The method as in claim 16 or 17 further comprising the step of obtaining first and second captured images in succession.

19. The method as in claim 18 further comprising the step of activating a camera flash during acquisition of the first or second captured image.

20. The method as in any one of claims 16-19 further comprising the step of searching a back-end database to access a testing protocol specific to a biomarker test kit.

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