LATERAL FLOW DEVICE FOR DETECTING COLORECTAL CANCER

FIELD

[0001] Aspects provided herein are directed to diagnostic and analytical systems for the detection of an analyte in a sample. More specifically, embodiments described herein concern devices and methods for identifying the presence or absence of an intestinal disease or condition including colorectal cancer and its precursor lesions.

BACKGROUND

[0002] Fecal immunochemical Tests (FITs) typically detect hemoglobin (Hb) in the stool as an indicator of bleeding, which may be caused by lower intestinal pathologies such as colorectal cancer and its precursor lesions (polyps). Most FITs require sampling a stool collected in a sling suspended from the toilet seat. This means that only the exposed stool surface is available for sampling, typically by a spatula or spear from several points on the stool surface. As any blood is likely to be spotted or striped by lesions on the inside wall of the intestine these are hit-or-miss methods of sampling (except in the unlikely cases when the blood is uniformly distributed throughout the stool). After sampling, the collected sample is typically added to a tube or card for transporting the sample to a lab for analysis. Accordingly, there is a delay between the time point of sample collection and analysis, which contributes to the degradation of the analyte (Hb). The need for more diagnostic devices and methods for analyzing lower intestinal pathologies, such as colorectal cancer and its precursor lesions (polyps), is manifested.

SUMMARY

[0003] Embodiments provided herein relate to devices and methods of use thereof, which allow for the detection or identification of lower intestinal pathologies such as colorectal cancer and its precursor lesions (polyps) in a subject, such as an animal, preferably a human.

[0004] In general, the device comprises a housing having at least one sample receiving port configured to receive a sample, wherein the housing further comprises a detection window configured to permit identification of a test result, a sample receiving matrix, which preferably comprises a color catcher or dye trapping reagent and, optionally a stabilizer, such as an albumin e.g., bovine serum albumin, a buffer, surfactant, and/or solubilizing reagent, an immunochromatographic test strip having a test line, a control line and, optionally an interceptor line (e.g., comprising an immobilized immunoglobulin G (IgG) of the same species as the test line), in liquid communication with the sample receiving matrix, wherein the immunochromatographic test strip further comprises a m obi liz able antibody conjugate or antibody binding fragment conjugate, such as an anti-human hemoglobin antibody or a binding fragment thereof conjugated to a label (e.g., latex or nano-gold) and, optionally an absorbent pad configured to wick sample through the test strip. In preferred embodiments, the device further comprises an obscuring arrangement or cover, which hides or obscures the detection window. Greater detail on the components of these devices and use of these devices is provided below.

[0005] In some embodiments, the devices include a sample receiving matrix positioned behind the at least one sample receiving port and having a defined saturation capacity.

[0006] In some embodiments, the sample receiving matrix comprises a positively charged hydrophilic material.

[0007] In some embodiments, the color catcher or dye trapping aspect comprises a cellulosic substrate activated with a positively charged compound. In some embodiments, the cellulosic substrate is paper, and the positively charged compound comprises a sodium carbonate, an ammonium, a polyamine, an N-trisubstituted ammonium-2-hydroxy-3-halopropyl, glycidyltrimethylammonium chloride (GMAC), 3- chloro-2-hydroxypropyltrimethylammonium chloride, an epoxy propyl ammonium, or a derivative or salt thereof.

[0008] In some embodiments, the sample receiving matrix further comprises a stabilizer configured to stabilize the analyte and prevent or reduce non-specific binding and loss of the analyte. In some embodiments, the stabilizer comprises an amount of an albumin or fragment thereof such as a bovine serum albumin (BSA) or a fragment thereof in an amount of less than 1% w/v but not zero. In some embodiments, the sample receiving matrix further comprises at least one reagent for solubilizing and buffering the analyte in the sample before transfer to the test strip. In some embodiments, at least one reagent comprises a borate buffer. In some embodiments, the sample receiving matrix further comprises one or more lytic agents for lysing and releasing the contents of cells present in the sample. In some embodiments, the sample receiving matrix comprises one or more surfactants to prevent or reduce non-specific binding and loss of the analyte to the test strip.

[0009] In some embodiments, the sample receiving matrix is dimensioned to provide a saturation capacity equal to or greater than a volume of the sample added to the device. In some embodiments, the sample receiving matrix is configured to filter solids, stool pigments, and negatively charged contaminants from the sample before transfer to the test strip.

[0010] In some embodiments, the device comprises an immunochromatographic test strip located within the housing and in liquid communication with the sample receiving matrix, wherein the immunochromatographic test strip comprises a mobilizable conjugate, wherein the test strip comprises a test line and a control line, wherein the control line is downstream of the test line, and wherein the conjugate is upstream of the test line.

[0011] In some embodiments, the mobilizable conjugate comprises a detectable label and a binding agent that specifically binds an analyte of interest in the sample, such as hemoglobin (Hb). In some embodiments, the detectable label is a metal nanoparticle, a colored latex particle, a magnetic particle, a carbon nanoparticle, a quantum dot, an up-converting phosphor, an organic fluorophore, a textile dye, an enzyme, or a liposome. In some embodiments, the detectable label is a gold nanoparticle. In some embodiments, the binding agent is an anti-analyte antibody or binding fragment thereof. In some embodiments, the conjugate comprises an anti-hemoglobin antibody or binding fragment thereof conjugated to a gold nanoparticle, preferably a polyclonal antibody conjugated to a gold nanoparticle. Exemplary anti-hemoglobin antibodies, which may be incorporated with any one or more of the devices or methods or both described herein as conjugate include for example, commercially available antibodies such as monoclonal antibodies ab92492, ab214049, ab 137096, ab 109444, ab217074, ab211589, abl 16628, ab215919, ab214379, ab240106, ab240390, ab401, ab248384, ab 128940, abl56584, ab227671, ab269881, ab77125, ab240408, abl31225, or abl74536, available from Abeam or rabbit polyclonal antibodies ab82871, ab93825, abl 53898, ab202399, ab238597, ab231803, ab231584, ab231732, or ab227552, goat polyclonal antibodies abl9362, abl93950, abl9363, or abl9361, mouse polyclonal antibodies abl72019, sheep polyclonal antibodies abl9364, ab33615, abl9366, or abl9365, or chicken polyclonal antibodies abl7542 or ab28961 also available from Abeam. The aforementioned exemplary anti-hemoglobin antibodies are provided to demonstrate that the skilled person is well aware of many different options for both monoclonal and polyclonal antibodies, which are readily available commercially and, which can be used with one or more devices or methods described herein but the skilled person also recognizes that that are many more commercially available anti-hemoglobin antibodies available, which can be used with one or more of the devices or methods described herein. Accordingly, as used herein, the term anti-hemoglobin antibodies refers generically to any commercially available anti-hemoglobin antibodies whether monoclonal or polyclonal unless further specified as such, as well as, anti-hemoglobin antibodies whether monoclonal or polyclonal unless further specified as such, which can be generated using techniques available to the skilled immunologist.

[0012] In some embodiments, the test line comprises an immobilized antibody that specifically binds the analyte of interest, such as hemoglobin (Hb). In some embodiments, the test line comprises anti-human hemoglobin antibody or a binding fragment thereof. Again, exemplary anti-hemoglobin antibodies, which may be incorporated with any one or more of the devices or methods or both described herein at the test line include for example, commercially available antibodies such as monoclonal antibodies ab92492, ab214049, abl37096, abl09444, ab217074, ab211589, abl 16628, ab215919, ab214379, ab240106, ab240390, ab401, ab248384, abl28940, abl56584, ab227671, ab269881, ab77125, ab240408, abl31225, or abl74536, available from Abeam or rabbit polyclonal antibodies ab82871, ab93825, abl53898, ab202399, ab238597, ab231803, ab231584, ab231732, or ab227552, goat polyclonal antibodies abl9362, abl93950, abl9363, or abl9361, mouse polyclonal antibodies abl72019, sheep polyclonal antibodies abl 9364, ab33615, abl 9366, or abl 9365, or chicken polyclonal antibodies abl7542 or ab28961 also available from Abeam. Additional examples of antibodies can be monoclonal or polyclonal antibodies from chickens, goats, guinea pigs, hamsters, horses, mice, rabbits, rats, or sheep. The aforementioned exemplary anti hemoglobin antibodies are provided to demonstrate that the skilled person is well aware of many different options for both monoclonal and polyclonal antibodies, which are readily available commercially and, which can be used with one or more devices or methods described herein but the skilled person also recognizes that that are many more commercially available anti-hemoglobin antibodies available, which can be used with one or more of the devices or methods described herein. Accordingly, as used herein, the term anti-hemoglobin antibodies refers generically to any commercially available anti hemoglobin antibodies whether monoclonal or polyclonal unless further specified as such, as well as, anti-hemoglobin antibodies whether monoclonal or polyclonal unless further specified as such, which can be generated using techniques available to the skilled immunologist.

[0013] In some embodiments, the control line comprises an immobilized analyte, such as hemoglobin, preferably human hemoglobin. Some embodiments include a plurality of control lines, wherein each of the plurality of control lines has a different amount of immobilized analyte. In some embodiments, the amount of immobilized analyte ranges from 0.1 mg/ml to 1 mg/ml, such as 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1 mg/ml or a range defined by any two of the aforementioned concentrations.

[0014] In some embodiments, the test strip further comprises an interceptor line, wherein the interceptor line is upstream of the test line and downstream of the mobilizable conjugate. In some embodiments, the interceptor line comprises an immobilized immunoglobulin G (IgG). In some embodiments, the immobilized IgG is an IgG that is from the same species that the immobilized antibody at the test line is from. In some embodiments, the immobilized IgG is from mouse, goat, sheep, chicken, rabbit, or horse.

[0015] In some embodiments, the test strip is formed from an elongate strip of hydrophilic material, one end of the test strip providing a first location for sample reception, and opposing end of the test strip providing a second location for the indication showing the test result. Some embodiments of the device also comprise an absorbent pad configured to wick sample through the test strip.

[0016] In some embodiments, the devices further include an obscuring arrangement or cover, which hides or obscures the detection window. In some embodiments, the obscuring arrangement or cover comprises a first hinged flap positioned over the detection window, wherein said hinged flap is configured to permit subsequent detection of the indication. In some embodiments, the obscuring arrangement or cover further comprises a second hinged flap positioned over the at least one sample receiving port wherein said second hinged flap covers said at least one sample receiving port. In some embodiments, the device further comprises a seal, which at least partially or completely seals the second hinged flap over the at least one sample receiving port after sample collection. In some embodiments, the seal also comprises a bar code label or a unique identifier. [0017] In some embodiments, the housing further includes an inspection window, which permits a visual indication confirming the flow of the sample through the test strip and/or the result of the analysis.

[0018] Additional embodiments provided herein relate to a kit comprising one or more of the above devices and, optionally a sample transfer apparatus. In some embodiments, the sample transfer apparatus is a brush or a brush-like apparatus having flexible or semi-flexible bristles. In some embodiments, the sample transfer apparatus has a sample volume that is less than, equal to, or greater than the defined saturation capacity of the sample receiving matrix.

[0019] More embodiments relate to methods for measuring an analyte, such as hemoglobin in a sample utilizing one or more of the devices described above. In some embodiments, the methods include contacting any one of the above devices with a sample desired to be tested or analyzed. In some embodiments, the method further includes detecting a signal generated by the detectable label at the test line. In some embodiments, the method further includes measuring a presence and/or quantity of the analyte in the sample.

[0020] In some embodiments, the analyte detected by the device is hemoglobin. In some embodiments, the sample analyzed is stool or blood or a medium comprising fecal matter and/or blood such as water in the vicinity of a stool or blood. In preferred embodiments, the method comprises collecting the sample from toilet water. In some embodiments, collecting the sample comprises mixing stool into toilet water using a sample transfer apparatus. In some embodiments, the sample is collected from toilet water and delivered to the at least one sample receiving port of the device using a sample transfer apparatus. In some embodiments, the sample transfer apparatus is a brush or a brush-like apparatus having flexible or semi-flexible bristles. In some embodiments, the sample transfer apparatus has a sample volume that is less than, equal to, or greater than the defined saturation capacity of the sample receiving matrix.

[0021] In some embodiments, contacting the device with the sample solubilizes the conjugate in the sample receiving matrix. In some embodiments, when the analyte is present in the sample, the conjugate binds to the analyte, flows through the test strip through capillary action to the test line, and binds to immobilized antibody at the test line. In some embodiments, when the analyte is not present in the sample, the conjugate flows through the test strip through capillary action to the control line, and binds to immobilized analyte at the control line. [0022] In some embodiments, detecting the signal comprises visual measurement or identification or non-visual image capture and analysis of detectable signal at the control line and/or at the test line. In some embodiments, the signal is non visual and is detected using spectrometry, fluorimetry, or magnetometry. In some embodiments, measuring is qualitative, quantitative, or semi-quantitative. In some embodiments, the measuring comprises comparing the signal at the test line to the signal at the control line, wherein the signal at the control line is indicative of the amount of analyte found in a healthy or diseased subject (e.g., the amount of analyte present in a subject lacking an intestinal pathology such as colon cancer or polyps and/or a subject having an intestinal pathology such as colon cancer or polyps).

[0023] Preferred embodiments are exemplified by the following alternatives.

[0024] 1. A device comprising: a housing having at least one sample receiving port configured to receive a sample, wherein the housing further comprises a detection window configured to permit identification of a test result; a sample receiving matrix positioned behind the at least one sample receiving port and having a defined saturation capacity, wherein the sample receiving matrix comprises a color catcher or dye trapping reagent; and an immunochromatographic test strip located within the housing and in liquid communication with the sample receiving matrix, wherein the test strip comprises a mobilizable conjugate, and wherein the test strip further comprises a test line and a control line, wherein the control line is downstream of the test line, and wherein the conjugate is upstream of the test line.

[0025] 2. The device of alternative 1, wherein the sample receiving matrix further comprises a positively charged hydrophilic material.

[0026] 3. The device of any one of alternatives 1-2, wherein the color catcher or dye trapping reagent comprises a cellulosic substrate activated with a positively charged compound.

[0027] 4. The device of alternative 3, wherein the cellulosic substrate is a paper, and wherein the positively charged compound comprises a sodium carbonate, an ammonium, a polyamine, an N-trisubstituted ammonium-2-hydroxy-3-halopropyl, an epoxy propyl ammonium, or a derivative or salt thereof.

[0028] 5. The device of any one of alternatives 3 or 4, wherein the positively charged compound is glycidyltrimethylammonium chloride (GMAC) or 3-chloro-2- hydroxypropyltrimethylammonium chloride. [0029] 6. The device of any one of alternatives 1-5, wherein the sample receiving matrix further comprises a stabilizer configured to stabilize the analyte and prevent non-specific binding and loss of the analyte to the test strip.

[0030] 7. The device of alternative 6, wherein the stabilizer comprises an albumin, such as a bovine serum albumin (BSA), preferably in an amount of less than 1% w/v but not zero.

[0031] 8. The device of any one of alternatives 1-7, wherein the sample receiving matrix further comprises at least one reagent for solubilizing and buffering the analyte in the sample before transfer to the test strip.

[0032] 9. The device of alternative 8, wherein the at least one reagent comprises a borate buffer.

[0033] 10. The device of any one of alternatives 1-9, wherein the sample receiving matrix further comprises one or more lytic agents for lysing and releasing the contents of cells present in the sample.

[0034] 11. The device of any one of alternatives 1-10, wherein the sample receiving matrix further comprises one or more surfactants capable of reducing non specific binding or loss of the analyte to the test strip or both.

[0035] 12. The device of any one of alternatives 1-11, wherein the sample receiving matrix is dimensioned to provide a saturation capacity equal to or greater than a volume of the sample added to the device.

[0036] 13. The device of any one of alternatives 1-12, wherein the sample receiving matrix is configured to filter solids, stool pigments, or negatively charged contaminants from the sample before transfer to the test strip.

[0037] 14. The device of any one of alternatives 1-13, wherein the mobilizable conjugate comprises a detectable label and a binding agent that specifically binds an analyte of interest in the sample

[0038] 15. The device of alternative 14, wherein the detectable label is a metal nanoparticle, a colored latex particle, a magnetic particle, a carbon nanoparticle, a quantum dot, an up-converting phosphor, an organic fluorophore, a textile dye, an enzyme, or a liposome.

[0039] 16. The device of any one of alternatives 14-15, wherein the detectable label is a gold nanoparticle.

[0040] 17. The device of any one of alternatives 14-16, wherein the binding agent is an anti-analyte antibody or binding fragment thereof. [0041] 18. The device of any one of alternatives 14-17, wherein the mobilizable conjugate comprises an anti-hemoglobin antibody or binding fragment thereof conjugated to a gold nanoparticle, preferably a polyclonal antibody conjugated to a gold nanoparticle.

[0042] 19. The device of any one of alternatives 1-18, wherein the test line comprises an immobilized antibody or binding fragment thereof, which specifically binds the analyte of interest.

[0043] 20. The device of any one of alternatives 1-19, wherein the test line comprises anti-human hemoglobin antibody or binding fragment thereof, which may be a polyclonal antibody or a monoclonal antibody or a binding fragment thereof, such as AbD05988, abl91183, A80-134A, A80-134P, GTX77484, at>92492, ab214049, abl74536, abl91183, ab82871, abl9362, abl37096, abl9364, abl09444, ab24048 , ab240390 , ab77125, at>227552, ab227671, abl7542, ab231584, ab401, abl56584, ab28961, abl72019, ab248384, abl28940, ab238597, ab240106,abl 16628, ab214379, ab248234, abl53898, ab33615, ab93825, abl9366, LS-B7869, LS-B 12169, LS-B13233, LS-B7869, LS-C211255, LS-C294442, LS-B4914, LS-B11162, , LS- C195465, LS-C195444, LS-C294440, LS-B7868, LS-C681796, or LS-C681795 or a binding fragment thereof.

[0044] 21. The device of any one of alternatives 1-20, wherein the control line comprises an immobilized analyte, such as hemoglobin or a fragment thereof.

[0045] 22. The device of any one of alternatives 1-21, comprising a plurality of control lines, wherein each of the plurality of control lines has a different amount of immobilized analyte, such as hemoglobin or a fragment thereof.

[0046] 23. The device of alternative 22, wherein the amount of immobilized analyte ranges from 0.1 mg/ml to 1 mg/ml, such as 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1 mg/ml or an amount within a range defined by any two of the aforementioned concentrations.

[0047] 24. The device of any one of alternatives 1-23, wherein the test strip further comprises an interceptor line, wherein the interceptor line is upstream of the test line and downstream of the conjugate.

[0048] 25. The device of alternatives 24, wherein the interceptor line comprises an immobilized immunoglobulin G (IgG).

[0049] 26. The device of alternative 25, wherein the interceptor line comprises an immobilized mouse IgG, chicken IgG, sheep IgG, goat IgG, rabbit IgG, or horse IgG. [0050] 27. The device of any one of alternatives 24-26, wherein the interceptor line comprises mouse IgG.

[0051] 28. The device of any one of alternatives 1-27, wherein the test strip is formed from an elongate strip of hydrophilic material, one end of the test strip providing a first location for sample reception, and opposing end of the test strip providing a second location for the indication showing the test result.

[0052] 29. The device of any one of alternatives 1-28, further comprising an absorbent pad configured to wick sample through the test strip.

[0053] 30. The device of any one of alternatives 1-29, wherein the device further comprises an obscuring arrangement or cover, which hides or obscures the detection window.

[0054] 31. The device of alternative 30, wherein the obscuring arrangement or cover comprises a first hinged flap positioned over the detection window, wherein said hinged flap is configured to permit subsequent detection of the indication.

[0055] 32. The device of any one of alternatives 30-31, wherein the obscuring arrangement or cover further comprises a second hinged flap positioned over the at least one sample receiving port wherein said second hinged flap covers said at least one sample receiving port.

[0056] 33. The device of alternative 32, wherein the device further comprises a seal, which seals or partially seals the second hinged flap over the at least one sample receiving port after sample collection.

[0057] 34. The device of alternative 33, wherein the seal comprises a bar code label or an identifier.

[0058] 35. The device of any one of alternatives 1-34, wherein the housing further comprises an inspection window, which permits a visual indication confirming the flow of the sample through the test strip.

[0059] 36. A kit comprising: the device of any one of alternatives 1-35; and a sample transfer apparatus.

[0060] 37. The kit of alternative 36, wherein the sample transfer apparatus is a brush or a brush-like apparatus having flexible or semi-flexible bristles.

[0061] 38. The kit of any one of alternatives 36-37, wherein the sample transfer apparatus has a sample volume that is less than, equal to, or greater than the defined saturation capacity of the sample receiving matrix. [0062] 39. A method for measuring an analyte in a sample, the method comprising: contacting a device of any one of alternatives 1-35 with a sample; detecting a signal generated by the detectable label at the test line; and measuring a presence and/or quantity of the analyte in the sample.

[0063] 40. The method of alternative 39, wherein the analyte is hemoglobin.

[0064] 41. The method of any one of alternatives 39-40, wherein the sample is stool or blood or water in the vicinity of stool or blood.

[0065] 42. The method of any one of alternatives 39-41, further comprising collecting the sample from toilet water.

[0066] 43. The method of alternative 42, wherein collecting the sample comprises mixing stool into toilet water using a sample transfer apparatus.

[0067] 44. The method of alternative 43, wherein the sample is collected from toilet water and delivered to the at least one sample receiving port of the test device using a sample transfer apparatus.

[0068] 45. The method of any one of alternatives 43-44, wherein the sample transfer apparatus is a brush or a brush-like apparatus having flexible or semi-flexible bristles.

[0069] 46. The method of any one of alternatives 43-45, wherein the sample transfer apparatus has a sample volume that is less than, equal to, or greater than the defined saturation capacity of the sample receiving matrix.

[0070] 47. The method of any one of alternatives 39-46, wherein contacting the device with the sample solubilizes the conjugate in the sample receiving matrix.

[0071] 48. The method of alternative 47, wherein when analyte is present in the sample, the conjugate binds to analyte, flows through the test strip through capillary action to the test line, and binds to immobilized antibody at the test line.

[0072] 49. The method of alternative 47, wherein when analyte is not present in the sample, the conjugate flows through the test strip through capillary action to the control line, and binds to immobilized analyte at the control line.

[0073] 50. The method of any one of alternatives 39-49, wherein detecting the signal comprises visual measurement or non-visual image capture and analysis of detectable signal at the control line and/or at the test line.

[0074] 51. The method of any one of alternatives 39-50, wherein said signal is non- visual and is detected using spectrometry, fluorimetry, or magnetometry. [0075] 52. The method of any one of alternatives 39-51, wherein measuring is qualitative, quantitative, or semi-quantitative.

[0076] 53. The method of any one of alternatives 39-52, wherein measuring comprises comparing the signal at the test line to the signal at the control line, wherein the signal at the control line is indicative of a healthy or disease state.

[0077] 54. The method of alternative 53, wherein the disease state is colorectal cancer or precursor lesions, such as polyps.

[0078] Certain embodiments are described further in the following description, examples, claims, and drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

[0079] The drawings illustrate certain embodiments of the technology and are not limiting. For clarity and ease of illustration, the drawings are not made to scale and in some instances, various aspects may be shown exaggerated or enlarged to facilitate an understanding of particular embodiments.

[0080] FIG. 1A depicts an example device shown assembled. FIG. IB depicts an example device shown assembled with the sample flap in an open position.

[0081] FIG. 2 depicts an example device unassembled, showing the inner components of the device, including a sample receiving matrix and a test strip in a center panel.

[0082] FIG. 3 depicts an example test strip having a sample receiving matrix for application of a sample, an interceptor line, a test line, and a control line. Conjugate is placed in the sample receiving area, and upon application of a sample, the sample, together with the conjugate, flows through the test strip.

[0083] FIG. 4 depicts exemplary test strips after flow of a sample through the test strips. In (a) and (b), a negative stool sample was tested; in (c), hemoglobin was added to a negative stool sample, which was then tested. In test strip (a), the test strip lacks an interceptor line and lacks a color catcher or dye trapping agent. In test strip (b), the test strip has an interceptor line but no color catcher or dye catching material. In test strip (c), the test strip includes an interceptor line and a color catcher or dye catching material.

[0084] FIG. 5 depicts exemplary test results showing sensitivity of the device, from 0 to 50 pF of blood per liter of water. DET AILED DESCRIPTION

[0085] The following detailed description is merely exemplary in nature and is not intended to limit embodiments or the application and uses of embodiments. Furthermore, there is no intention to be bound by any theory presented in the preceding background or the following detailed description.

[0086] Some embodiments provided herein related to a device for detection of an analyte in a sample. The device includes a housing having at least one sample receiving port configured to receive a sample, wherein the housing further comprises a detection window configured to permit identification of a test result; a sample receiving matrix positioned behind the at least one sample receiving port and having a defined saturation capacity, wherein the sample receiving matrix, which preferably comprises a color catcher or dye trapping reagent and, optionally a stabilizer; and an immunochromatographic test strip located within the housing and in liquid communication with the sample receiving matrix, wherein the test strip comprises a mobilizable conjugate, and wherein the test strip further comprises a test line and a control line, wherein the control line is downstream of the test line, and wherein the conjugate is upstream of the test line, and optionally an absorbent pad configured to wick sample through the test strip. In some embodiments of the device, the test strip comprises an interceptor line, wherein the interceptor line is upstream of the test line and downstream of the mobilizable conjugate. In some embodiments, the device further comprises an obscuring arrangement or cover, which hides or obscures the detection window.

[0087] According to some embodiments, the test strip may include, for example, an area with conjugate comprises dried down gold conjugated polyclonal anti human hemoglobin (Hb) antibody downstream from the sample receiving matrix. A test line of immobilized monoclonal anti-Hb antibody may be located further downstream on the test strip. The sample receiving matrix receives a water sample, which then passes through the area having the conjugate; if Hb is present in the sample, gold conjugated Ab- Hb complexes will form; the test line captures and accumulates any such gold conjugated Ab-Hb complexes to thereby produce a visible line. This line therefore provides visual confirmation of the presence of Hb in the water sample. Alternatively, other disclosing agents may be used which enable the indication to be detected, for example fluorescent dyes or particles, or magnetic particles. In these cases, the detection of bound aggregates of Hb and the disclosing agent may be by other means, e.g. spectrometry, fluorimetry or magnetometry. This can be a convenient means of preventing a patient from being able to read their own test results. Accordingly, reference to an “indication” should be understood as a reference to both an indication which is visible to the naked eye and one which requires additional means to enable its detection, such as via spectrometry, fluorimetry or magnetometry.

[0088] Other embodiments provided are kits comprising the device and a sample transfer apparatus. Still other embodiments provided are methods of using the device for detection of an analyte in a sample.

[0089] Greater detail on the components of these devices and use of these devices is provided below.

Sample Receiving Port

[0090] In Fecal immunochemical Tests (FIT) applications, the liquid containing the sample may be the toilet water within which the person being tested has deposited feces. The toilet water can therefore act as both the sample and the test developer.

[0091] The sample may be collected by dipping at least a portion of the device into a liquid containing the sample such that the sample receiving matrix comes into contact with the liquid to thereby collect said sample. In this arrangement, the sample receiving port would be located on the portion of the device being dipped. The device may be dipped one or more times into the liquid for collecting one or more samples. The defined saturation capacity of the sample receiving matrix enables the appropriate volume of liquid to be retained by the test strip.

[0092] The liquid containing the sample may alternatively be collected using a sample collection apparatus for collecting the sample and delivering the sample to the sample receiving port. In some embodiments, collecting the sample comprises mixing stool into toilet water using a sample transfer apparatus. The mixing makes sure that the device detects blood from the whole stool surface and, to the extent the stool disintegrates on stirring, also internal blood.

[0093] The sample collection apparatus may collect one or more samples and may deliver one or more samples to the sample receiving port. Further discussion of sample collection apparatus can be found in the later paragraphs.

Detection window [0094] The detection arrangement for allowing detection of the indication of the test strip may include at least one detection window provided in the housing and configured to permit identification of a test result. A visible indication could be viewed through this window or an indication not visible to the naked eye could be detected through this window, such as using fluorimetry. Alternative viewing aspects are also envisaged, for example, a transparent section may be provided for the housing to allow for the viewing a visual indication.

[0095] The visual indication may be, for example, in the form of one or more lines extending across the exposed portion of the test strip. The position of the lines and/or the color density of each line may provide the visual indication of the test result. A general assessment of the test results may be obtained by viewing the visual indication with the naked eye. Alternatively, a quantitative result may be obtained by using a viewing apparatus for measuring the color density and distribution of the one or more lines of the visual indication.

Sample receiving matrix

[0096] Stool water testing provides several challenges. These include variability in the water quality (e.g. pH, ionic concentration, contamination by rust, toilet cleaners etc.), stool heterogeneity (e.g. in consistency, dietary and microbial content, pigmentation etc.) and potentially cross-reacting stool contaminants that may interfere with the test reagents.

[0097] A sample receiving matrix is positioned behind the sample receiving port. The purpose of the sample receiving matrix is to receive a sample and facilitate the transfer of the sample delivered to the sample receiving port to the test strip.

[0098] In some embodiments, the sample receiving matrix comprises a positively charged hydrophilic material to retain sufficient sample to complete a test.

[0099] In some embodiments, the color catcher or dye trapping reagent of the sample receiving matrix are a cellulosic substrate activated with a positively charged compound, for example as detailed in U.S. Patent No. 9,845,570, the disclosure of which is expressly incorporated herein by reference in its entirety. It will be appreciated by a skilled person that the disclosure is not limited to use with a cellulosic substrate, and that any other suitable absorbent substrate, for example glass wool or non-woven polyester may be used. The cellulosic substrate may be paper, and the positively charged compound may comprise an ammonium, a polyamine, an N-trisubstituted ammonium-2-hydroxy-3- halopropyl, an epoxy propyl ammonium, a derivative thereof, or a combination thereof. In a preferred embodiment, the disclosed positively charged compounds herein are dye scavenging compounds. It will be appreciated by a skilled person that the disclosure is not limited to use with those compounds, and that any other suitable dye scavenging compounds may be used.

[0100] In some embodiments, the sample receiving matrix further contain a stabilizer configured to stabilize the analyte and prevent non-specific binding and loss of the analyte to the test strip. In some embodiments, the stabilizer comprises an amount of an albumin or fragment thereof such as a bovine serum albumin (BSA) or a fragment thereof in an amount of less than 1% w/v but not zero. In some embodiments, the stabilizer is whole milk or milk powder. In some embodiments, the sample receiving matrix further comprises at least one reagent for solubilizing and buffering the analyte in the sample before transfer to the test strip. In some embodiments, at least one reagent comprises a borate buffer.

[0101] In some embodiments, the sample receiving matrix further comprises one or more lytic agents for lysing and releasing the contents of cells present in the sample. In some embodiments, the sample receiving matrix comprises one or more surfactants (e.g. TritonXIOO) to prevent or reduce non-specific binding and loss of the analyte to the test strip.

[0102] In some embodiments, the sample receiving matrix is dimensioned to provide a saturation capacity equal to or greater than a volume of the sample added to the device. In some embodiments, the sample receiving matrix is configured to filter solids, stool pigments, and negatively charged contaminants from the sample before transfer to the test strip.

[0103] The above embodiments of the sample receiving matrix, alone or in combination with others, filter particulates and remove various dyes or pigmentation from the sample that may obscure the test result, and precondition the sample so that its pH and ionic strength is compatible with the conjugate before its addition to the test strip enclosed within the device.

Test strip

[0104] In some embodiments, the device comprises an immunochromatographic test strip located within the housing and in liquid communication with the sample receiving matrix, wherein the immunochromatographic test strip comprises a mobilizable conjugate, wherein the test strip comprises a test line and a control line, wherein the control line is downstream of the test line, and wherein the conjugate is upstream of the test line.

[0105] As used herein, the term “liquid communication” shall be taken to mean that a sample collected within a solvent such as water, when applied via the sample receiving port to the sample receiving matrix is capable of being in liquid communication with the test strip under sufficient conditions of hydration to enable transfer of at least part of the sample, or a component thereof, to the test strip.

[0106] After the sample is delivered to the sample receiving port, any fecal solids within the sample are then filtered by the sample receiving matrix before the remaining part of the sample is transferred to the test strip by virtue of the liquid communication with the test strip. The collected sample then migrates along the test strip through capillary action to areas within the strip containing the reagents for detecting the analyte within the sample. The test strip furthermore provides a visual indication of the test result that can be viewed through the detection window. In some embodiments, the test device contains an immunochromatographic test strip so that on water sample addition the test develops immediately, thereby providing an accurate assay of the sample at the time of collection. The test results may then be read immediately, or at any time thereafter without risk of Hb loss through degradation.

[0107] The test strip may comprise a mobilizable conjugate, an interceptor, a test line, and a control line, wherein the control line is downstream of the test line, and wherein the conjugate is upstream of the test line.

Conjugate

[0108] In some embodiments, the mobilizable conjugate comprises a detectable label and a binding agent that specifically binds an analyte of interest in the sample, such as hemoglobin (Hb). Other analytes of interest could include, for example, stool pathogens, such as Clostridium difficile, or other indicators of gastrointestinal pathologies. In some embodiments, the detectable label could be a metal nanoparticle, a colored latex particle, a magnetic particle, a carbon nanoparticle, a quantum dot, an up- converting phosphor, an organic fluorophore, a textile dye, an enzyme, or a liposome. In one non-limiting example, gold nanoparticles are used to label the binding agent because they are relatively inexpensive, stable, and provide easily observable color indications based on the surface plasmon resonance properties of gold nanoparticles. In some embodiments, the binding agent is an anti-analyte antibody or binding fragment thereof. In some embodiments, the antibody that binds to the analyte is any commercially available antibody, such as a monoclonal or polyclonal antibody, or an aptamer, or any fragment or derivative thereof. In some embodiments, the m obi 1i /able conjugate comprises an anti-hemoglobin antibody or binding fragment thereof conjugated to a gold nanoparticle, preferably a polyclonal antibody conjugated to a gold nanoparticle.

[0109] In some embodiments, this signal provides qualitative information, such as to show whether the analyte is present in the sample. In some embodiments, this signal provides quantitative information, such as a measurement of the quantity of analyte in the sample. More detail about qualitative or quantitative signal can be found in later paragraphs.

Test line and control line

[0110] In some embodiments of the device, the test line comprises an immobilized antibody that specifically binds the analyte of interest, such as hemoglobin (Hb). The antibody may be an anti-human hemoglobin antibody or a binding fragment thereof, which may be a polyclonal antibody or a monoclonal antibody or a binding fragment thereof, such as AbD05988, abl91183, A80-134A, A80-134P, GTX77484, ab92492, ab214049, abl74536, abl91183, ab82871, abl9362, abl37096, abl9364, ab 109444, ab24048,ab240390, ab77125, ab227552, ab227671, abl7542, ab231584, ab401, abl56584, ab28961, abl72019, ab248384, abl28940, ab238597, ab240106,abl 16628, ab214379, ab248234, abl53898, ab33615, ab93825, abl9366, LS- B7869, LS-B 12169, LS-B 13233, LS-B7869, LS-C211255, LS-C294442, LS-B4914, LS- B11162, , LS-C195465, LS-C195444, LS-C294440, LS-B7868, LS-C681796, or LS- C681795 or a binding fragment thereof. Depending on the analyte to be detected, a skilled person may use other types of agents that specifically bind the analyte of interest in the test line. When the analyte is present in the sample, the conjugate binds to the analyte to form a conjugate-analyte complex. The complex then binds to immobilized antibody at the test line.

[0111] In some embodiments of the device, the control line comprises an immobilized analyte. Some of the free conjugate not bound to the analyte in the sample will bind to the immobilized analyte at the control line. Depending on the detectable label used in the conjugate, the signal at the test line and control line can be detected and analyzed by e.g. visual measurement, spectrometry, fluorimetry, or magnetometry.

[0112] The control line serves at least two purposes: one, because the control line is located downstream of the test line, detecting signal from the control line indicates that the sample has passed through the test line and reached the control line, which suggests sufficient sample has been added to complete the test.

[0113] Two, the signal at the control line could indicate whether a high dose hook effect (prozone) from excessive analyte present in the sample. Most immunochemical tests suffer from a prozone phenomenon, which causes a diminished signal at very high analyte concentrations. Specifically, in some embodiments, diminished signal occurs when a sample contains elevated levels of analyte that exceed the concentration of labeled conjugate deposited on the test strip. In this scenario, when the sample is placed on the test strip, the labeled conjugate becomes saturated by the analyte, and not all of the analyte becomes labeled. The unlabeled analyte flows through the test strip and binds at the test line, out-competing the labeled conjugate-analyte complex, and thereby reducing the detectable signal. At the same time, because almost all labeled conjugate has become saturated, not much labeled conjugate binds to the control line, also resulting in diminished signal. Thus, diminution of the intensities of both the test and control lines indicates a high dose hook effect. When embodiments apply to detect hemoglobin, including a control line could eliminate the risk of missing an advanced cancer due to hook effect caused false negative signal, which can occur where high concentrations of blood are present in the patient sample.

[0114] In some embodiments, the device could comprise a plurality of control lines, wherein each of the plurality of control lines has a different amount of immobilized analyte. The amount of immobilized analyte can range from 0.1 mg/ml to 1 mg/ml, such as 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or lmg/ml or a range defined by any two of the aforementioned concentrations. The signal intensity at each control line is proportionate to the amount of immobilized analyte. Thus, comparing the signal intensity of the test line to those of the multiple control lines can measure the analyte concentration in the sample qualitatively, semi-quantitatively, or quantitatively.

Interceptor line

[0115] In some embodiments of the device, the test strip further comprises an interceptor line, wherein the interceptor line is upstream of the test line and downstream of the mobilizable conjugate. The interceptor line may comprise an immobilized immunoglobulin G (IgG). In some embodiments, the immobilized IgG at the interceptor line is an IgG that is from the same species that the immobilized antibody at the test line is from. In some embodiments, the immobilized IgG is from mouse, goat, sheep, chicken, rabbit, or horse. The interceptor line can arrest non- specific ally binding stool contaminants or conjugated complexes. Depending on the source of the antibody used in the test line, a skilled person can use IgG from other suitable sources to arrest the non- specifically binding stool contaminants· Thus, for example, where the immobilized IgG is from mouse, any molecules in the sample that non-specifically bind to the mouse IgG are intercepted and removed.

Elongate strip

[0116] The test strip may be formed from an elongate strip of hydrophilic material, one end of the strip may provide a first location for sample reception, and the opposing end of the strip providing a second location for the indication, such as a visual indication, showing the test result. The first location of the test strip may therefore be located in liquid communication with the sample receiving port. The transferred sample may then migrate along the test strip to a downstream area of the test strip where the presence of the analyte may be indicated.

[0117] The sample receiving matrix may be added onto the end of the test strip and laminated as a single web, ensuring direct contact between the sample receiving matrix and the test strip. This web may then be impregnated with the same solutions used to prepare the sample receiving matrix and test strip. In addition, the distance between the sample receiving matrix and the indication zone, for example a visual indication zone such as a gold conjugate zone, may be varied, to ensure adequate mixing and/or rehydration of the collected sample with the dried buffer/reagents on the sample receiving matrix.

[0118] Some embodiments of the above device further comprise an absorbent pad configured to wick sample through the test strip and accept and retain spent sample and reactants through the test strip.

Cover

[0119] In some embodiments, an obscuring arrangement or cover is provided to hide the detection window. This is likely to be of particular significance, for example, where the indication is a visual indication and it is desired that the patient not be able to read the test result. It may also be useful if the detection means, even if not visible to the naked eye, is preferably not exposed to daylight, such as a fluorescent marker. Some embodiments include at least one hinged flap for hiding the detection window. The provision of a said flap over the detection window limits any anxiety that may arise from the test subject observing the visual indication provided by the test. [0120] In other embodiments, an obscuring arrangement or cover is provided to hide the at least one sample receiving port. The obscuring arrangement or cover over the sample receiving port provides sample containment and therefore also saves the test subject from the embarrassment of providing an exposed sample. Furthermore, the obscuring arrangement or cover over the sample receiving port provides for hygiene as it seals the port and prevents fecal contamination·

[0121] Other embodiments include both types of obscuring arrangements or covers: one being locatable over the detection window, the other being locatable over the sample receiving port. The obscuring arrangements or covers can be hinged flaps, which may be formed from the same sheet of material as the rest of the housing.

Seal

[0122] In some embodiments, the device further comprises a seal, which at least partially or completely seals the second hinged flap over the at least one sample receiving port after sample collection. In some embodiments, the seal also comprises a bar code label or a unique identifier.

[0123] In some embodiments, an identifier means is provided on the housing that links the test subject to the test results. In some embodiments, the identifier is in the form of a coded label that may be used to seal the flap over the sample port. In some embodiments, the test result and the coded ID are captured in a digital image linking the test subject to the test result.

Inspection window

[0124] In some embodiments, the housing further comprises an inspection window downstream of the sample receiving port; the window permits a visual indication confirming the flow of the sample through the test strip. For example, a colored dye may be used to indicate the mobilization and flow of the collected sample through the test strip. The provision of the inspection port will allow the test subject to determine whether or not the collected sample has been properly transferred into the test strip, with a further sample addition being required if no flow is observed. In one embodiment, the inspection window is located at the distal end of the test strip, between the control line and the absorbent pad. A band of water-soluble dye (such as food dye) is dried onto this region of the test strip during its manufacture. The sample is applied to the receiving port (for example using a brush or by dipping the device into a liquid sample). The liquid sample is contacted with the sample port for a period of time until the band of dye is seen to be mobilized through the inspection window. Thereafter the contact can be ended. Where a brush is used, it may take 15-20 seconds of contacting the brush with the receiving port. If no flow is observed within 30 seconds, then another delivery of sample is required.

The kit

[0125] In some embodiments, a kit comprises a device of any of the above embodiments and a sample transfer apparatus. The sample transfer apparatus could be a brush or a brush-like apparatus have flexible or semi-flexible bristles. In some embodiments, the sample transfer apparatus has a sample volume that is less than, equal to, or greater than the defined saturation capacity of the sample receiving matrix.

[0126] The advantage of using a brush or brush like apparatus is that it allows the collection of liquid samples within the bristles of the brush or brush- like apparatus. Such a sample collection apparatus is particularly applicable for use in FITs because it allows the collection of fecal material released from a stool located within the water of a toilet bowl. In addition, the bristles allow brushing around the stool to disperse any blood into the surrounding water. Furthermore, the water collected within the bristles of the brush or brush-like apparatus can act as a solvent to facilitate the liquid communication of the collected sample within the test strip. The brush or brush-like apparatus can be specified to collect a sufficient volume of liquid to complete the test. The brush or brush like apparatus may have a liquid holding capacity equal to or greater than the defined saturation capacity of the sample receiving matrix. This ensures that the sample delivered by the brush or brush-like apparatus saturates the sample receiving matrix. In an alternative embodiment, and as discussed further below, the brush may have a liquid holding capacity less than the defined saturation capacity of the sample receiving matrix.

[0127] The brush may be of any suitable size. Where a large brush with capacity to deliver larger volumes of liquid is used, only one application of the liquid sample may be required. However, in one embodiment, in order to enable the use and packaging of a smaller brush, it may be desirable to deliver two or more sequential liquid samples to the receiving port. This may also be desirable from the point of view that fecal contamination may cause an undesirable level of background discoloration to the test strip that may possibly obscure a borderline positive result. In order to minimize the possibility of this occurring, one may elect to use the following protocol:

(a) Use the brush to add a toilet water sample to the sample port;

(b) Flush the toilet; and

(c) Use the same brush to add a sample of clean water from the toilet bowl to the sample port. [0128] In this embodiment the smaller brush or brush-like apparatus may have a liquid holding capacity less than the defined saturation capacity of the sample receiving matrix. It would be appreciated by the skilled person that the total volume of liquid delivered by the smaller brushes or brush-like apparatuses is sufficient to saturate the sample receiving matrix.

[0129] The term “brush” herein is used to denote an apparatus comprising a stem or handle, usually elongate, and a clump, bunch or group of bristles, hair or other similar flexible or semi-flexible elongate strands, laminar flaps or the like attached to the stem or handles. The term “brush-like apparatus” is used herein to denote an apparatus which is similar to a brush in that it includes a bunch, clump or group of bristles, hair or other similar flexible or semi-flexible elongate strands, laminar flaps or the like. Whilst reference is made throughout the present specification to the collection of sample within the bristles of a brush or brush-like apparatus, it is to be understood that the reference to “bristles” is used to include the hairs or other similar flexible or semi-flexible elongate strands, laminar flaps or the like of a brush or brush-like apparatus.

Methods

[0130] More embodiments relate to methods for measuring an analyte, such as hemoglobin in a sample utilizing one or more of the devices described above. In some embodiments, the method comprises contacting any one of the above devices with a sample desired to be tested or analyzed. In some embodiments, the method further includes detecting a signal generated by the detectable label at the test line. In some embodiments, the method further includes measuring a presence and/or quantity of the analyte in the sample.

[0131] In some embodiments, the analyte detected by the device is hemoglobin. In some embodiments, the sample analyzed is stool or blood or a medium comprising fecal matter and/or blood such as water in the vicinity of a stool or blood.

[0132] In preferred embodiments, the method comprises collecting the sample from toilet water. The sample could be collected from toilet water by dipping the device directly into the toilet water. The portion of the device having a sample receiving port is dipped into the toilet water to thereby saturate the underlying sample receiving matrix with the toilet water sample. The test results can then be obtained by viewing the visual indication provided by the test strip through the detection window.

[0133] In some embodiments, the sample is collected from toilet water and delivered to the at least one sample receiving port of the device using a sample transfer apparatus. The sample collecting may include mixing stool into toilet water using the sample transfer apparatus. The sample transfer apparatus could be a brush or a brush-like apparatus having flexible or semi-flexible bristles.

[0134] The apparatus may be sized to collect a quantity of the toilet water sample to fully saturate the sample receiving matrix. In some embodiments, a smaller brush may be used to deliver more than one sample to fully saturate the sample receiving matrix. It is also possible that the fecal contamination may cause an undesirable level of background discoloration to the test strip that may obscure a borderline positive test result. The above test method can therefore be altered by using a small brush to add a toilet water sample to the sample receiving port, flushing the toilet, and using the same brush to add clean water from the toilet bowl to the sample receiving port.

[0135] In some embodiments, contacting the device with the sample solubilizes the conjugate in the sample receiving matrix. In some embodiments, when the analyte is present in the sample, the conjugate binds to the analyte to form a conjugate- analyte complex; the complex flows through the test strip through capillary action to the test line and binds to immobilized antibody at the test line. In some embodiments, when the analyte is not present in the sample, some of the free conjugate flows through the test strip to the control line and binds to immobilized analyte at the control line. Depending on the detectable label used in the conjugate, the signal at the test line and control line can be detected and analyzed by e.g. visual measurement, spectrometry, fluorimetry, or magnetometry.

[0136] The device could comprise one or multiple control lines, wherein each of the multiple control lines has a different amount of immobilized analyte. Since the signal intensity at each control line is proportionate to the amount of immobilized analyte, comparing the signal intensity of the test line to that of the control lines can measure the analyte concentration in the sample qualitatively, semi-quantitatively, or quantitatively. Depending on the signal at the control line is indicative of a healthy or disease state, comparing the signal at the test line to that at the control line can tell whether the test subject is in a healthy or disease state.

[0137] Some embodiments of the method are to detect disease state of colorectal cancer or precursor lesions, such as polyps.

[0138] The device, kit, or method according to the present disclosure therefore allows for the immediate testing of collected samples on site without the need to use the facilities of an offsite pathology lab. This self-testing aspect of the device also makes it suitable for use in the home or in the field where no laboratory facilities are available.

[0139] While the present disclosure has been described with respect to its use in FITs, it is to be appreciated that the present disclosure can be used in other applications such as the sampling and analysis of other biological fluids such as blood, urine, semen and saliva, or may be adapted to analyze the presence of contaminants in ground water, or bacteria such as E.coli in food.

[0140] Persons skilled in the art will recognize that many modifications or variations may be made to the device described in detail herein in order to suit other testing purposes or by way of adaption for optimal function, without departing from the spirit and scope of the present disclosure as broadly described above.

EXAMPLE 1

[0141] Some aspects of the embodiments discussed above are disclosed in further detail in the following examples, which are not in any way intended to limit the scope of the present disclosure. Those in the art will appreciate that many other embodiments also fall within the scope of the disclosure, as it is described herein above and in the claims.

[0142] The following example describes a device to detect an analyte of interest as described herein. In this non-limiting example, the analyte of interest is the protein globin moiety of the hemoglobin (Hb) molecule, present in a stool sample in toilet water. Detection of Hb in the stool is typically used as an indicator of bleeding potentially caused by lower intestinal pathologies such as colorectal cancer and its precursor lesions (polyps).

[0143] The sample receiving matrix of the device is made from a commercially available laundry dye-catching material (e.g. Color Catcher®, Henkel), cut to a size e.g. 30 x 20mm capable of accepting sufficient water to complete the test reaction, such as a material disclosed in U.S. Patent No. 9,845,570, the disclosure of which is expressly incorporated by reference herein.

[0144] The immunochromatographic test strip has the following reagents in order from the point of sample application:

• Dried borate buffer.

• Dried conjugate (anti-Hb polyclonal goat antibody conjugated to colloidal gold).

• An Interceptor line of immobilized mouse immunoglobulin (IgG). • A Test line of monoclonal mouse anti-Hb antibody (IgG).

• A Control line of immobilized Hb.

[0145] After the person being tested has deposited feces into toilet water, a brush or other means is used to add toilet water to the sample-receiving matrix. The matrix filters and traps stool particulates and pigments, so that only clear sample is applied to the test strip. The applied water solubilizes and mobilizes the borate buffer so that its pH and ionic strength is compatible with the conjugate, which is in turn mobilized by the advancing waterfront.

[0146] Any cross-reacting stool components or conjugate complexes that may otherwise be non- specifically captured by the Test line are arrested by the Interceptor line, so only Hb labelled with conjugate is specifically captured by the Test line. Conjugate solution not complexed with Hb and arrested at the Test line then binds with the Hb immobilized at the Control line. Visual measurement can then be performed to detect and analyze the signal at the Test and Control lines.

[0147] If Hb is not present in the sample, the Test line should not have any signal, and the Control line show a strong signal. This result indicates no blood is present in the stool.

[0148] If the Hb concentration in the sample is low to medium, the Test line should have a low to medium signal, and the Control line show a strong signal as most of the labeled conjugate is unreacted with Hb and binds to the Control line. This result indicates that blood is present in the stool sample. The person being tested may be asked to perform other tests to confirm whether colorectal cancer or its precursor lesions are present.

[0149] If the Hb concentration in the sample is high, both the Test and Control lines will display signals with diminution of intensity due to high dose hook or prozone effect. These results indicate that heavy bleeding, which may be caused by advanced cancers, is present. The person being tested may be asked to perform other tests to confirm.

EXAMPLE 2

[0150] The following example describes a device to detect an analyte of interest as described herein. Preparation of the device

[0151] The components of the test strip were prepared as shown in FIG. 3, wherein the test strip includes a sample receiving matrix having conjugate provided thereon. Some test strips were prepared with a color catcher or dye trapping reagent therein, and some test strips were prepared with an interceptor line, as described below.

[0152] Hemoglobin antibody: BiosPacific Hemoglobin mab (Cat# 7204- 100080), diluted to 2.0 mg/ml with PBS.

[0153] Control line: Freshly collected human red blood cells lysed in distilled water and diluted to 500ug/ml with PBS.

[0154] Interceptor line: Sigma mouse IgG from mouse serum (Cat# 18765 - 5xl0mg), diluted to 2.5mg/ml with PBS.

[0155] Conjugate: BiosPacific Goat anti-Haemoglobin antibody (Cat# G-134- C), conjugated to colloidal gold (EY or Innova3) standardized to OD520 = 4 with 40mM borate just before use. The conjugate was diluted 1:1 with 5mM borate buffer, pH 8.5 containing 50% sucrose, 6% BSA, and 2% Triton X100.

[0156] The Sample Receiving Matrix (Sample Pad) included commercial store-bought Color Catcher® laundry dye catcher manufactured by Henkel (Ireland), which was saturated and dried with BSA in the concentration ranging from 0.1 to 1%, depending on the sensitivity required. It was observed that the sensitivity of the test could be improved by drying low concentrations of bovine serum albumin (BSA) into the Color Catcher® matrix, presumably by blocking non-specific binding of hemoglobin to the matrix. At BSA concentrations >1% false positive results with some stools were observed, possibly by the BSA blocking the positively charged sites in the matrix that would otherwise bind the stool contaminants responsible for false positivity. The optimal BSA concentration was determined to be between 0.1% and 0.5%.

[0157] The Color Catcher® paper sheets were cut into pads 20x30mm and mounted onto the device as shown in FIG. 2, after which PVA glue was applied at the glue lines shown in FIG. 2. The right panel was folded over the center panel, and then the left panel over the right panel to obtain an assembled device as illustrated in FIG. 1A. In some embodiments, staples may be used instead of glue.

[0158] As shown in FIG. 4, various test strips were prepared to test the ability of the interceptor line and the color catcher material to improve test results at the test line. In (a) and (b), a negative stool sample was tested; in (c), hemoglobin was added to a negative stool sample, which was then tested. In (a), the test strip lacks an interceptor line and lacks a color catcher or dye trapping agent. With no interceptor line or color catcher, non-specific binding material accumulated at the test line, causing a false positive result. In (b), the test strip has an interceptor line but no color catcher material. The interceptor line was successful at removing some non-specific complexes, but due to excess non specific binding material, the interceptor line was overwhelmed, and a false positive appeared at the test and control lines, though in lesser amounts than in test strip (a). In (c), the test strip has both an interceptor line and a color catcher. The result shows that the color catcher completely removed non-specific binding material, leaving no signal at the interceptor line and a clear signal at the test line.

Use of the device

[0159] Fresh human blood of assumed Hb concentration of 150 mg/mL was diluted 1/200 in water and then further diluted to a concentration of 1 mg/mL in a stabilizing solution comprised of 3% BSA, 10% ethanol and 0.1% sodium azide in 40mM sodium borate buffer at pH 8.5 and stored for use within 3 hours.

[0160] Human stool from a normal defecation was dispersed into the toilet bowl water by mixing for 5 seconds with a brush. 40 mL aliquots of the water were withdrawn using a 50 mL syringe and used within 3 hours.

[0161] Diluted blood was added to 10 mL aliquots of the toilet water samples to a blood concentration equivalent to 0, 5, 10, 20, 40, 80, 160 and 320 pL of blood per liter of water.

Results and Discussion.

[0162] A typical result is shown in FIG. 5, indicating a sensitivity for blood is approximately 5 pL blood/liter of water. As blood is assumed to have a Hb concentration of 150 mg/mL, 5 pL blood has about 750 pg Hb; diluted 5 pL blood in 1 liter water and the concentration of Hb was about 750 ng/ml.

[0163] If a normal stool is taken as 150 g, then 5ul of blood per 150 g stool = 5 pg Hb/g stool. This detection limit is comparable to the claimed detection limit of commercial quantitative FITs, e.g., OC Sensor Diana claims a lower limit of detection of 10 pg Hb/g of stool. It is notable that the commercial tests in the report sample from only 2 to 10 mg of the stool, which therefore may not be representative of the whole stool. The FITs discussed in the above report collect stool into a liquid medium for transport to the lab during which the protein globin moiety of the Hb may be degraded. However, sample stability is not an issue with the current disclosure as the test develops immediately on sample addition and the result is stable for several weeks thereafter.

[0164] Toilet water samples containing dispersed stool caused an undesirable background color and false positive test results with some stools. As the latter were assumed to be caused by entities in the stool with an affinity for the mouse IgG of the test line, an interceptor line of non-immune mouse IgG was added as an interceptor line upstream of the test line. This innovation reduced the false positives, but not the background color, which was undesirable for both visual and instrumented result reading.

[0165] Various filter materials were tested as sample pads to filter fine particulates that may cause background color, but bile pigments (e.g. biliverdin and bilirubin) continued to provide an undesirable yellow background in the test strip with samples with a heavy stool load. To remove these pigments various commercial dye catcher matrices were evaluated and one in particular (Color Catcher®) found to be particularly effective in removing both pigments and colored fines. It was unexpected, however, that Color Catcher® also proved effective in removing the molecules responsible for causing false positive results, suggesting that these entities were negatively charged and therefore arrested by the positively charged matrix.

[0147] Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present disclosure belongs. (All patents, applications, published applications and other publications referenced herein are expressly incorporated by reference in their entireties unless stated otherwise. For purposes of the present disclosure, the following terms are defined below.)

[0166] Throughout this specification, unless the context requires otherwise, the words “comprise,” “comprises,” and “comprising” will be understood to imply the inclusion of a stated step or element or group of steps or elements but not the exclusion of any other step or element or group of steps or elements.

[0167] It will be readily understood that the aspects of the present disclosure, as generally described herein, can interchangeably be used in another embodiment unless such a replacement is not technically feasible. It will be appreciated by those skilled in the art that various other omissions, additions and modifications may be made to the methods and structures described above without departing from the scope of the claimed subject matter. All such modifications and changes are intended to fall within the scope of the subject matter, as defined by the appended claims.

[0168] With respect to the use of substantially any plural and/or singular terms herein, those having skill in the art can translate from the plural to the singular and/or from the singular to the plural as is appropriate to the context and/or application. The various singular/plural permutations may be expressly set forth herein for sake of clarity.

[0169] It will be understood by those within the art that, in general, terms used herein, and especially in the appended claims (for example, bodies of the appended claims) are generally intended as “open” terms (for example, the term “including” should be interpreted as “including but not limited to,” the term “having” should be interpreted as “having at least,” the term “includes” should be interpreted as “includes but is not limited to,” etc.). It will be further understood by those within the art that if a specific number of an introduced claim recitation is intended, such an intent will be explicitly recited in the claim, and in the absence of such recitation no such intent is present. For example, as an aid to understanding, the following appended claims may contain usage of the introductory phrases “at least one” and “one or more” to introduce claim recitations. However, the use of such phrases should not be construed to imply that the introduction of a claim recitation by the indefinite articles “a” or “an” limits any particular claim containing such introduced claim recitation to embodiments containing only one such recitation, even when the same claim includes the introductory phrases “one or more” or “at least one” and indefinite articles such as “a” or “an” (for example, “a” and/or “an” should be interpreted to mean “at least one” or “one or more”); the same holds true for the use of definite articles used to introduce claim recitations. In addition, even if a specific number of an introduced claim recitation is explicitly recited, those skilled in the art will recognize that such recitation should be interpreted to mean at least the recited number (for example, the bare recitation of “two recitations,” without other modifiers, means at least two recitations, or two or more recitations). It will be further understood by those within the art that virtually any disjunctive word and/or phrase presenting two or more alternative terms, whether in the description or claims, should be understood to contemplate the possibilities of including one of the terms, either of the terms, or both terms. For example, the phrase “A or B” will be understood to include the possibilities of “A” or “B” or “A and B.”

[0170] As will be understood by one skilled in the art, for any and all purposes, such as in terms of providing a written description, all ranges disclosed herein also encompass any and all possible sub-ranges and combinations of sub-ranges thereof. Any listed range can be easily recognized as sufficiently describing and enabling the same range being broken down into at least equal halves, thirds, quarters, fifths, tenths, etc. As a non-limiting example, each range discussed herein can be readily broken down into a lower third, middle third and upper third, etc. As will also be understood by one skilled in the art all language such as “up to,” “at least,” “greater than,” “less than,” and the like include the number recited and refer to ranges which can be subsequently broken down into sub-ranges as discussed above. Finally, as will be understood by one skilled in the art, a range includes each individual member. Thus, for example, a group having 1-3 articles refers to groups having 1, 2, or 3 articles. Similarly, a group having 1-5 articles refers to groups having 1, 2, 3, 4, or 5 articles, and so forth.

[0171] While various aspects and embodiments have been disclosed herein, other aspects and embodiments will be apparent to those skilled in the art. The various aspects and embodiments disclosed herein are for purposes of illustration and are not intended to be limiting, with the true scope and spirit being indicated by the following claims.