Dual lateral flow device for simultaneous determining the ratio of glycated to non-glycated biomarker

Abstract

The present invention relates to a lateral flow device for measuring the amount of a glycated biomarker in a sample. In particular, the device comprises a first test strip for measuring the amount of a glycated biomarker, and a second test strip for measuring the level of the non- glycated biomarker. The present invention also relates to a test strip for a lateral flow device, the use of the lateral flow device for measuring the amount of glycated biomarker in a sample that, and a method of measuring the amount of glycated biomarker in a sample using the lateral flow device. The devices, strips and methods of the present invention are particularly suitable for measuring the amount of HbA1c in a sample.

Description

[0001] Biomarker detection device

[0002] Field of the Invention

[0003] The present invention provides devices and test strips for biomarker detection, and methods for detecting biomarkers. In particular, the invention provides devices, test strips and methods that may be used for detecting and measuring glycated biomarkers such as glycated haemoglobin (HbA1c).

[0004] Background to the Invention

[0005] Diabetes is a disorder characterised by elevated levels of blood sugar. Such elevated levels can result in a corresponding increase in glycated biomarkers, the levels of which in a sample can be interrogated to provide information about the progression of diabetes.

[0006] When exposed to sugar molecules, for instance by permeation of the sugar into the red blood cells (RBCs) from the bloodstream, a glycated form of haemoglobin (HbA1c) can form. This results from the spontaneous formation of a chemical bond between the sugar molecule (typically galactose, fructose or glucose) and e.g. the N-terminal end of a monomeric unit of the haemoglobin. Since the binding of the sugar molecule to haemoglobin is permanent, the level of HbA1 c can be used as an indicator of average blood sugar levels over the previous 3- month period ( / .e. the average lifespan of an RBC). Elevated HbA1c concentrations also correlate well with the risk of long-term diabetes complications, and can be regarded as independent risk factors for coronary heart disease and stroke.

[0007] Blood sugar levels are an important indicator in the endocrine disease diabetes. Recent estimates show that diabetes affects nearly 425 million people globally, and accounts for a significant proportion of healthcare spending; in the UK, for instance, treatment of Type 2 diabetes (characterised by the development of insulin resistance, rather than loss of insulin productive ability) accounts for just under 9% of the annual NHS budget. To monitor the blood sugar levels of such patients, biomarkers such as HbA1c can be measured from whole blood samples - in fact, both the American Diabetes Association (ADA) and the UK’s National Institute for Health and Care Excellence (NICE) suggest using HbA1 c tests as the sole tool for diagnosing diabetes.

[0008] A key stage in the progression of diabetes in patients is the prediabetic stage, which is defined as a state where blood sugar levels are higher than normal, but not high enough to be diagnosed with diabetes. The diagnostic criteria for prediabetes are, according to NICE, HbA1c levels of 42-47 mmol / mol ( / .e. 6.0-6.4% of the haemoglobin), while ADA defines prediabetic as having HbA1c levels of 39-47 mmol / mol ( / .e. 5.7-6.4%). At the prediabetic stage, lifestyle changes can be used to prevent progression into full diabetes, and therefore the ability to accurately identify the level of HbA1c in a patient blood sample is important in order to identify patients whose condition can still be readily reversed without pharmaceutical intervention.

[0009] Blood samples are often sent off to be tested in clinical laboratories; however, such an approach is unsuitable for larger scale population screening, and is not useful for reaching marginalised communities who are more difficult to reach through typical out-reach programmes. Therefore, “point-of-care” (POC) devices - which are able to run tests more locally - represent a promising tool for diabetes management, not least because they have the potential to be used as screening devices for large populations.

[0010] Many of the POC devices that are currently on the market are based on sandwich immunoassays, wherein the analyte to be detected is captured between two layers of antibodies: a capture antibody, which immobilises the analyte; and a detection antibody which binds to the antigen. Reporter molecules (e.g. enzyme, fluorophore, or biotin) can be directly attached to the detection antibody or to a secondary antibody which binds the detection antibody (e.g. goat, anti-mouse IgG - HRP) in order to quantify the

[0011] However, POC devices that are currently on the market lack the accuracy and precision required to provide reliable information for patients in the prediabetic range, and are often focused on providing qualitative results. Thus, there is a need for improved means for measuring diabetes biomarkers.

[0012] Summary of the Invention

[0013] The present invention relates to discoveries that may improve the accuracy of diabetes tests. Firstly, a device comprising both (i) a competitive lateral flow test strip for measuring glycated biomarker levels and (ii) a sandwich lateral flow test strip for measuring non-glycated biomarker levels can more precisely and efficiently calibrate the glycated biomarker results to improve their reliability. Secondly, by utilising a competitive immunoassay lateral flow test strip having a particular ratio of binding molecule in the glycated biomarker conjugate:antigen to measure the level of glycated biomarker in a sample, a higher degree of accuracy can be achieved which can better distinguish results in the prediabetic range from those in the non-diabetic and diabetic ranges.

[0014] The present invention therefore provides a lateral flow device for measuring the amount of a biomarker that is in a glycated form in a sample, the device comprising: a first test strip for measuring the level of a glycated biomarker in a competitive assay; a second test strip for measuring the level of the non-glycated biomarker in a sandwich assay; and a sample inlet for receiving a sample and passing it to the first and second test strips.

[0015] Also provided is a test strip for a lateral flow device, wherein the test strip is for measuring the level of a glycated biomarker in a sample in a competitive assay and comprises: a conjugate region comprising a glycated biomarker conjugate, the glycated biomarker conjugate comprising a glycated biomarker binding molecule linked to a reporter; and one or more detection regions comprising an antigen, wherein the ratio of the number of glycated biomarker binding molecules in the conjugate region to the number of antigen molecules in the detection region is at least 1 : 1000.

[0016] The present invention further provides the use of a lateral flow device of the present invention for measuring the amount of biomarker that is in glycated form in a sample, wherein the sample is preferably a blood sample, and preferably wherein the lateral flow device is for measuring the amount of haemoglobin (Hb) that is in the form of HbA1c.

[0017] A method for measuring the amount of biomarker that is in glycated form in a sample is also provided, said method comprising: adding a sample to a lateral flow device as defined herein; waiting for the sample to flow along the first test strip and the second test strip; and detecting an output from the first and second test strips.

[0018] Description of the Figures

[0019] Figure 1 is an exploded view of a device according to the present invention.

[0020] Figure 2 is a top-down illustration of a device according to the present invention.

[0021] Figure 3 is a diagrammatic representation of a competitive lateral flow test performed on a lateral flow test strip according to the present invention. Figure 4 is a histogram depicting the distribution of haemoglobin levels in blood sample taken from 180 donors.

[0022] Figure 5A depicts two side-by-side competitive assay lateral flow tests performed on a healthy and an unhealthy sample.

[0023] Figure 5B depicts two side-by-side sandwich assay lateral flow tests performed on a healthy and an unhealthy sample.

[0024] Detailed Description

[0025] Lateral flow device

[0026] In one aspect, the present invention relates to a lateral flow device for measuring the amount of a biomarker that is in a glycated form in a sample.

[0027] A lateral flow device refers to a device suitable for performing a lateral flow test on a liquid sample in order to detect the presence of a target analyte therein. Preferably the device is a handheld device, able to be operated in a human hand.

[0028] In general terms, a lateral flow test typically involves contacting a liquid sample with a test strip which comprises reactive molecules able to interact with a target analyte within the sample. The liquid sample can flow along the test strip, and any analyte therein can interact with the reactive molecules to ultimately produce a detectable signal upon the strip which can be used to determine the presence of the analyte within the sample.

[0029] The lateral flow device of the present invention comprises: a first test strip for measuring the level of a glycated biomarker in a competitive assay; a second test strip for measuring the level of the biomarker in a sandwich assay; and a sample inlet for receiving a sample and passing it to the first and second test strips.

[0030] In preferred instances, the lateral flow device is for measuring the amount of haemoglobin (Hb) that is in a glycosylated form (HbA1c) in a sample. The first test strip therefore measures the level of HbA1c in a competitive assay, while the second test strip measures the level of Hb in a sandwich assay. Alternative biomarkers that may be measured using the device include other glycated serum proteins ( / .e. a compound resulting from the glycation reaction between a sugar and a primary amine) such as glycated albumin. Glycated DNA may also be measured using the device.

[0031] The sample will typically be a biological sample and in particular a bodily fluid that can be readily obtained from a person, such as blood, saliva, sweat or urine, and which may be expected to contain biomarkers relevant to the person’s health. It will be appreciated that one or more processing steps may be performed on the sample prior to its use with the device of the present invention, for instance to allow release of intracellular biomarkers into solution.

[0032] Preferably, the sample is a whole blood sample. Where the glycated biomarker being measured by the device is HbA1c, the blood sample has preferably undergone a lysis step such that it comprises lysed red blood cells.

[0033] First test strip

[0034] The lateral flow device comprises a first test strip for measuring the level of a glycated biomarker in a competitive assay, i.e. the first test strip is configured to perform a competitive assay for the biomarker being measured by the device.

[0035] A test strip is any platform for performing a lateral flow assay. It will be appreciated that the term “strip” is not intended to impose any limitation on the dimensions of the test strip structure, though typical test strips are rectangular with a length i.e. the dimension along which the sample flows) that is longer than its width. The test strip will typically comprise a material which enables fluid to pass along it, such as materials based on a series of capillary beds such as porous paper, microstructured polymer, or sintered polymer. The first test strip may be one continuous material. However, in preferred embodiments, the test strip comprises more than one component such as a base membrane, a sample pad and a conjugate region. For instance, the first test strip may comprise a fibrous material such as nitrocellulose. Preferably, the first test strip comprises a nitrocellulose base membrane upon which a sample pad and a conjugate region, typically also in the form of a pad, are disposed. It will be appreciated that, where the first test strip is used in a device with another test strip, such as second test strip as described herein, a single sample pad may be disposed on both test strips.

[0036] A competitive assay performed on a sample refers to a configuration wherein the analyte of interest competes with an antigen in a detection portion of the test strip for binding to a labelled binding molecule (a “conjugate”). In the absence of analyte in the sample, the binding molecule is completely free to bind to the antigen in the detection portion, and thereby generate a signal. However, analyte present in the sample can bind to a portion of the population of the conjugate, preventing this portion from binding to the antigen in the detection portion. In this scenario, since fewer binding molecules bind to the antigens, an increasing amount of analyte results in a reduction in the signal.

[0037] In prior art POC devices which utilise sandwich assays, wherein a higher intensity signal is generated by higher amounts of HbA1c, there is a tendency for saturation to occur which hampers the ability to differentiate small variations - however, these small variations may be the difference between an accurate measurement within the prediabetic range or an inaccurate one in the diabetic range. By using a competitive assay in the first test strip, in which a healthier sample results in a more intense signal, this issue can be alleviated.

[0038] To perform a competitive assay, the first test strip will comprise a conjugate region. The conjugate region is the portion of the test strip which contains a glycated biomarker conjugate, permits a sample to pass along or through it, and permits the biomarker conjugate to move further along the test strip with the sample. While the conjugate region may be formed simply by a surface of a material which a sample may pass across, in lateral flow test strips the conjugate region will typically have a three-dimensional structure. As such, the conjugate region may be in the form of a pad comprising porous material. In the present invention, the conjugate region may comprise glass fibre, such as a glass fibre filter.

[0039] The glycated biomarker conjugate that is present in the conjugate region of the first test strip comprises a glycated biomarker binding molecule ( / .e. a molecule able to bind to a biomarker though a glycated biomarker binding moiety) which is linked to a reporter. Preferably, the glycated biomarker conjugate is an HbA1c conjugate, which comprises an HbA1c binding molecule. It will be appreciated that the conjugate that is used in the first test strip will binds to the biomarker only when it is in the form of a glycated biomarker.

[0040] The glycated biomarker conjugate is also able to bind to an antigen present in the one or more detection regions of the first test strip. Typically, the antigen present at the one or more detection portions is the glycated biomarker, and therefore is preferably HbA1c. However, antigen binding may occur at a secondary site - i.e. a site other than the biomarker binding moiety - of the glycated biomarker binding molecule. In these instances, a competitive assay may be carried out where, for instance, following binding of a biomarker to the binding moiety, the binding molecule undergoes a conformational change that renders the secondary site unable to bind the antigen. The glycated biomarker binding molecule preferably comprises an antibody or an antibody fragment which is capable of binding to a glycated biomarker as defined above. The glycated biomarker binding molecule may alternatively comprise an antibody mimic such as a molecularly imprinted polymer. The antibody will typically be a natural antibody (e.g. a monoclonal antibody), though a synthetic antibody may also be used (e.g. a recombinant antibody or a molecularly imprinted synthetic antibody). The antibody is preferably an anti- HbA1c antibody, and the biomarker binding moiety is preferably an HbA1c binding fragment of the antibody.

[0041] A reporter is present in the glycated biomarker conjugate and generates an output signal which can be used to measure the amount of glycated biomarker in the sample. The reporter is able to generate a signal which can be detected, in particular once the glycated biomarker binding molecule has bound to an antigen in a detection region. It will be understood that the reporter need not change in structure or conformation in order to generate the signal or modulate its intensity - the signal presence and intensity may result from e.g. the concentration of reporters in a detection portion.

[0042] Preferably, the reporter is a colorimetric reporter, meaning that the signal it produces can be detected optically or with the aid of e.g. a colorimeter. For instance, the reporter may produce a colour resulting from a particular absorption spectrum of the reporter. Suitable colorimetric reporters include: dyes such as fluorescent dyes; enzymes such as horseradish peroxidase and alkaline phosphatase; and nanoparticles such as metallic nanoparticles, carbon nanoparticles, silica nanoparticles and latex nanoparticles. Nanoparticles may be dye-doped nanoparticles, for instance of the colour green, blue, orange or red. Preferably, the reporter is a metallic nanoparticle such as a gold or silver nanoparticle, and more preferably is a gold nanoparticle.

[0043] Where the reporter is a metallic nanoparticle such as a gold nanoparticle, the nanoparticle may have a particle size in the range of from 10-100 nm, preferably from 20-80 nm, and more preferably from 30-50 nm. As used herein, particle size refers to the mean equivalent sphere hydrodynamic diameter of a nanoparticle as may be measured using dynamic light scattering, for instance according to ASTM E3247-20.

[0044] Accordingly, it is most preferred that the glycated biomarker conjugate comprises an anti- HbA1c antibody conjugated to a gold nanoparticle, preferably wherein the gold nanoparticle has a particle size of around 40 nm. Within the glycated biomarker conjugate, the glycated biomarker binding molecule is linked to the reporter. This linkage may directly connect the reporter and the binding moiety, but it is preferred that the linkage is indirect, such that the binding moiety and the reporter are separated by a portion of the binding molecule which does not directly take part in binding to the biomarker. For instance, where the glycated biomarker binding molecule is an anti-HbA1c antibody, the reporter may be linked to a constant domain of the antibody, for instance on the Fc region of the antibody.

[0045] The linkage may be monovalent or multivalent. It will be appreciated that multivalent linkages increase the number of glycated biomarker binding molecules per glycated biomarker conjugate and therefore the availability of binding sites per reporter.

[0046] Thus, more than one glycated biomarker binding molecules may be connected to a single reporter. For instance, a reporter may be linked to from 50 to 500, such as from 100 to 300 binding moieties, and more preferably from 150 to 250 binding moieties such as antibodies.

[0047] The reporter may be linked to the glycated biomarker binding molecule by direct adsorption, using a bifunctional linker or mediator linker (such as those based on EDC / NHS chemistry), or by adapter molecules such as streptavidin and biotin. The glycated biomarker binding molecule is generally linked to the reporter using a boronic acid linkage. In these instances, boronic acid serves as a bridge between the binding molecule and reporter.

[0048] The conjugate region may comprise the glycated biomarker conjugate in an amount of at least 5 pL, preferably at least 7 pL, and more preferably at least 10 pL as a solution at an optical density of OD5. The conjugate region may comprise the glycated biomarker conjugate in an amount of up to 25 pL, preferably up to 20 pL, and more preferably up to 15 pL as a solution at an optical density of OD5. Thus, the conjugate region may comprise the glycated biomarker conjugate in an amount of in the range of from 5 to 25 pL, preferably from 7 to 20 pL, and more preferably from 10 to 15 pL as a solution at an optical density of OD5. Optical density may be measured using a UV / Vis spectrophotometer at a wavelength of 550nm with a cuvette path length of 1cm.

[0049] The conjugate region will typically be in the form of a pad. The conjugate region may have a length of from 6 to 12 mm, preferably from 7 to 11 mm, and more preferably from 8 to 10 mm. The conjugate regional may have a width of from 1.5 to 4.5 mm, preferably from 2.0 to 4.0 mm, and more preferably from 2.5 to 3.5 mm. The conjugate region may have a depth of from 0.1 to 0.7 mm, preferably from 0.2 to 0.6 mm, and more preferably from 0.3 to 0.5 mm. The conjugate region may have a volume of from 5 to 15 mm3, preferably from 7 to 12 mm3, and more preferably from 8 to 11 mm3.

[0050] The conjugate region of the first test strip may comprise the glycated biomarker conjugate in an amount of from 3.6xio9to 5.5xio10molecules per test strip.

[0051] In addition to a conjugate region, the first test strip also comprises one or more detection regions. A detection region is a portion of the first test strip which comprises an antigen to which the glycated biomarker binding molecule of the glycated biomarker conjugate is able to bind, and which accumulates such antigen-bound binding molecules in order to generate a detectable signal. The one or more detection regions may be located on the base membrane which preferably forms part of the first test strip.

[0052] As mentioned above, the antigen is a molecule to which the glycated biomarker binding molecule of the glycated biomarker conjugate can bind. The antigen in the detection region is preferably the same as the glycated biomarker being tested for in the sample. Thus, the antigen is preferably a glycated biomarker and more preferably is HbA1c.

[0053] In order to provide a defined detection region from which a signal can reliably be read, the antigen in a detection region is typically immobilised within the detection region. Such immobilisation may be achieved by passive adsorption, particularly where a nitrocellulose membrane is used. Alternatively, linkers such as glutaraldehyde, succinimidyl 4-(N- maleimidomethyl)cyclohexane-1-carboxylate (SMCC) or sulfo-SMCC may be used.

[0054] The antigen may be present in the one or more detection regions of the first test strip at an average concentration ( / .e. the average across each of the one or more regions) of up to 1 mg / ml, preferably up to 0.75 mg / ml, and more preferably up to 0.45 mg / ml. The antigen may be present at an average concentration of at least 0.1 mg / ml, preferably at least 0.2 mg / ml, and more preferably at least 0.25 mg / ml. Thus, the antigen may be present in the one or more detection regions at an average concentration in the range of from 0.1 to 1 mg / ml, preferably from 0.2 to 0.75 mg / ml, and more preferably from 0.25 to 0.45 mg / ml. As these values represent an average across all detection regions, it will be appreciated that one or more of the detection regions may individually contain antigen levels outside of these ranges.

[0055] Alternatively, and most preferably, the antigen may be present in the one or more detection regions of the first test strip at an average concentration ( / .e. the average across each of the one or more regions) of up to 3 mg / ml, preferably up to 2.5 mg / ml, and more preferably up to 2.25 mg / ml. The antigen may be present at an average concentration of at least 1 mg / ml, preferably up to 1.5 mg / ml, and more preferably up to 1.75 mg / ml. Thus, the antigen may be present in the one or more detection regions at an average concentration in the range of from 1 to 3 mg / ml, preferably from 1.5 to 2.5 mg / ml, and more preferably from 1.75 to 2.25 mg / ml. As these values represent an average across all detection regions, it will be appreciated that one or more of the detection regions may individually contain antigen levels outside of these ranges.

[0056] Typically, each of the one or more detection regions contains from 0.1 to 0.5 pL, preferably from 0.2 to 0.4 pL, and more preferably from 0.25 to 0.35 pL of antigen.

[0057] Each of the one or more detection regions may have a length of from 0.6 to 1.2 mm, preferably from 0.7 to 1.1 mm, and more preferably from 0.8 to 1 mm. Each of the one or more detection regions may have a width of from 1.5 to 4.5 mm, preferably from 2.0 to 4.0 mm, and more preferably from 2.5 to 3.5 mm.

[0058] The antigen may be present in the one or more detection regions of the first test strip at an average amount ( / .e. the average across each of the one or more regions) of at least 3 x10’5mg, preferably at least 6 xw5mg, and more preferably at least 7.5 x10’5mg of antigen per detection region. The antigen may be present in the one or more detection regions of the first test strip at an average amount of up to 30 X10’5mg, preferably up to 22.5 X10’5mg, and more preferably up to 13.5 xw5mg of antigen per detection region. Thus, the antigen may be present in the one or more detection regions of the first test strip at an average amount of from 3 xw5to 30 xw5mg, from 6 xw5to 22.5 xw5mg, and more preferably from 7.5 xw5to 13.5 xw5mg of antigen per detection region.

[0059] Alternatively, and most preferably, the antigen may be present in the one or more detection regions of the first test strip at an average amount ( / .e. the average across each of the one or more regions) of at least 1 xw4mg, preferably at least 2.5xW4mg, and more preferably at least 5x1 O’4mg of antigen per detection region. The antigen may be present in the one or more detection regions of the first test strip at an average amount of up to 9x1 O'4mg, preferably up to 8x1 O’4mg, and more preferably up to 7xi(y4mg of antigen per detection region. Thus, the antigen may be present in the one or more detection regions of the first test strip at an average amount of from 1 xw4to 9x10’4mg, preferably from 2.5x1 O'4to 8x10’4mg, and more preferably from 5X10’4to 7xi(y4mg of antigen per detection region. The one or more detection regions and the conjugate region are preferably located on the first test strip such that the sample, once introduced to the strip, must pass through the conjugate portion in order to reach the first of the one or more detection regions.

[0060] Preferably, the first test strip comprises multiple detection regions. For instance, the first test strip may comprise at least 3 detection regions, preferably at least 4 detection regions, and more preferably at least 5 detection regions. In some instances, the first test strip comprises 5 detection regions. Such a configuration, as compared to a single test line, can assist in reducing the impact of saturation and thereby improve accuracy of the measurement.

[0061] Where the test strip comprises multiple detection regions, it is preferred that at least one of the detection regions comprises a different amount or concentration of antigen to another of the detection regions. For instance, the detection region first reached by the sample (the first detection region) may comprise a higher concentration or amount of antigen than the detection region last reached by the sample (the last detection region). Having a higher concentration of antigen in the detection regions which will be first contacted by the sample as it passes along the test strip helps to capture the baseline amount of biomarker (that would be present in every sample) within these regions, allowing detection regions further along the test strip to be more sensitive to any remaining biomarker which is more indicative of diabetic status.

[0062] Accordingly, the first detection region may comprise the antigen in a concentration of at least 0.3 mg / ml, preferably at least 0.4 mg / ml, and more preferably at least 0.45 mg / ml. The first detection region may comprise the antigen in a concentration of up to 1 mg / ml, preferably up to 0.75 mg / ml, and more preferably up to 0.55 mg / ml. Thus, the first detection region may comprise the antigen in a concentration in the range of from 0.3 to 1 mg / ml, preferably from 0.4 to 0.75 mg / ml, and more preferably from 0.45 to 0.55 mg / ml.

[0063] The first detection region may contain at least 9 x10’5mg, preferably at least 12 x10’5mg, and more preferably at least 13.5 X10’5mg of antigen. The first detection region may contain up to 30 x10’5mg, preferably up to 22.5 xw5mg, and more preferably up to 16.5 xw5mg of antigen. Thus, the first detection region may contain from 9 xw5to 30 xw5mg, from 12 xw5to 22.5 x -5mg, and more preferably from 13.5 xw5to 16.5 xw5mg of antigen.

[0064] Similarly, the last detection region may comprise the antigen in a concentration of at least 0.1 mg / ml, preferably at least 0.15 mg / ml, and more preferably at least 0.2 mg / ml. The last detection region may comprise the antigen in a concentration of up to 0.45 mg / ml, preferably up to 0.4 mg / ml, and more preferably up to 0.3 mg / ml. Thus, the last detection region may comprise the antigen in a concentration in the range of from 0.1 to 0.45 mg / ml, preferably from 0.15 to 0.4 mg / ml, and more preferably from 0.2 to 0.3 mg / ml.

[0065] The last detection region may contain at least 3 x10’5mg, preferably at least 4.5 x10’5mg, and more preferably at least 6 mg of antigen. The last detection region may contain up to 13.5 xio-5mg, preferably up to 12 xw5mg, and more preferably up to 9 xw5mg of antigen. Thus, the last detection region may contain from 3 xw5to 13.5 xw5mg, from 4.5 xw5to 12 xw5mg, and more preferably from 6 xw5to 9 xw5mg of antigen.

[0066] Therefore, the concentration or amount of the antigen in each of the multiple detection regions (where present) may stay the same or decrease with respect to the concentration or amount of antigen in the detection region that was reached by the sample before it. For instance, in the preferred embodiment where the first test strip comprises 5 detection regions, the first two regions contacted by the sample will preferably comprise the higher concentration of antigen in the ranges described above in relation to the first detection region, while the further three regions will preferably comprise a lower concentration of antigen in the ranges described above in relation to the last detection region.

[0067] Alternatively, and most preferably, it has been found that in the preferred embodiments where the first test strip comprises multiple, and more preferably five, detection regions, excellent results can be achieved where each of these detection regions comprises the same concentration or amount of the antigen. Specifically, each of the detection regions may comprise the antigen in a concentration of at least 1 mg / ml, preferably at least 1.5 mg / ml, and more preferably at least 1 .75 mg / ml. Each detection region may comprise the antigen in a concentration of up to 3 mg / ml, preferably up to 2.5 mg / ml, and more preferably up to 2.25 mg / ml. Thus, the first detection region may comprise the antigen in a concentration in the range of from 1 to 3 mg / ml, preferably from 1 .5 to 2.5 mg / ml, and more preferably from 1 .75 to 2.25 mg / ml.

[0068] Thus, each detection region may contain at least 3 xw4mg, preferably at least 4 x4mg, and more preferably at least 5 xw4mg of antigen. Each detection region may contain up to 9 xw4mg, preferably up to 8 xio-4mg, and more preferably up to 7 xw4mg of antigen. Thus, the first detection region may contain from 3 xw4to 9 xw4mg, from 4 xw4to 8 xw4mg, and more preferably from 5 xw4to 7 xw4mg of antigen.

[0069] It has been found that certain ratios of glycated biomarker in the sample (A), glycated biomarker binding molecule in the glycated biomarker conjugate (B), and antigen (C) can provide particular sensitivity of the first test strip within the lateral flow devices of the present invention. Preferably, in these ratios the glycated biomarker in the sample (A) is HbA1c, the binding molecule (B) is a HbA1c binding molecule, and the antigen (C) is HbA1c. While test strips having ratios giving high sensitivity are particularly suitable for use with the devices of the present invention, they may also be used in other devices and, as such, represent an aspect of the present invention.

[0070] Within the first test strip it is preferred that the ratio of the number of glycated biomarker binding molecules in the glycated biomarker conjugate (present in the conjugate region) to the number of antigen molecules in the one or more detection regions (B:C) is at least 1 :1000, and preferably at least 1 :1 107. Where the first test strip comprises multiple detection regions, it will be understood that the number of antigen molecules refers to the total amount of antigen across all of the multiple detection regions.

[0071] Accordingly, the ratio of the number of glycated biomarker binding molecules:antigen molecules present in the one or more detection regions (B:C) may be at least 1 :2000, preferably at least 1 :5000, and more preferably at least 1 :8,000. The ratio B:C may be up to 1 :25,000, preferably up to 1 : 15,000, and more preferably up to 1 : 12,000. Thus, the ratio of the number of binding molecules:antigen molecules present in the one or more detection regions may be in the range of from 1 :2000 to 1 :25,000, preferably from 1 :5000 to 1 : 15,000, and more preferably from 1 :8,000 to 1 :12,000.

[0072] Alternatively, and most preferably, the ratio of the number of glycated biomarker binding molecules:antigen molecules present in the one or more detection regions (B:C) may be at least 1 :2x107, preferably at least 1 :7.5xio7, and more preferably at least 1 :1 xi o8. The ratio B:C may be up to 1 :1 xio10, preferably up to 1 :7.5xio9, and more preferably up to 1 :5xio9. Thus, the ratio of the number of binding molecules:antigen molecules present in the one or more detection regions may be in the range of from 1 :2xio7to 1 :1 xio10, preferably from 1 :7.5xl07to 1 :7.5X 109, and more preferably from 1 :1 x8to 1 :5X109.

[0073] Similarly, in the first test strip the ratio of the number of glycated biomarker binding molecules to the number of glycated biomarker molecules (such as HbA1 c) in a typical sample (B:A) may be at least 1 :1 , preferably at least 1 :2, and more preferably at least 1 :5. The ratio B:A may be up to 1 :500, preferably up to 1 :100, and more preferably up to 1 :15. Thus, the number of glycated biomarker binding molecules to the number of glycated biomarker molecules in a typical sample may be in a ratio in the range of from 1 : 1 to 1 :500, preferably from 1 :2 to 1 : 100, and more preferably from 1 :5 to 1 :15. Alternatively, and most preferably, in the first test strip the ratio of the number of glycated biomarker binding molecules to the number of glycated biomarker molecules (such as HbA1 c) in a typical sample (B:A) may be at least 1 OO, preferably at least 1 :300, and more preferably at least 1 :500. The ratio B:A may be up to 1 :10,000, preferably up to 1 :7500, and more preferably up to 1 :5000. Thus, the number of glycated biomarker binding molecules to the number of glycated biomarker molecules in a typical sample may be in a ratio in the range of from 1 :100 to 1 :10,000, preferably from 1 :300 to 1 :7500, and more preferably from 1 :500 to 1 :5000.

[0074] In the first test strip, the ratio of the number of antigen molecules in the detection region to the number of glycated biomarker molecules in a typical sample (C:A) may be at least 1 :1 , preferably at least 10:1 , and more preferably at least 100:1. The ratio C:A may be up to 5,000:1 , preferably up to 2,000:1 , and more preferably up to 1000:1. Thus, the number of antigen molecules in the detection region to the number of glycated biomarker molecules in a typical sample may be in the range of from 1 :1 to 5,000:1 , preferably from 10:1 to 2,000:1 , and more preferably from 100:1 to 1000:1.

[0075] Alternatively, and most preferably, the ratio of the number of antigen molecules in the detection region to the number of glycated biomarker molecules in a typical sample (C:A) may be at least 10,000:1 , more preferably at least 50,000:1 , and more preferably at least 75,000:1. The ratio C:A may be up to 1 106:1 , preferably up to 800,000:1 , and more preferably up to 600,000:1. Thus, the number of antigen molecules in the detection region to the number of glycated biomarker molecules in a typical sample may be in the range of from 10,000:1 to 1 106:1 , preferably from 50,000: 1 to 800,000: 1 , and more preferably from 75,000: 1 to 600,000: 1 .

[0076] Therefore, the first test strip may comprise the glycated biomarker binding molecule of the glycated biomarker conjugate (A), antigen (B) and glycated biomarker in the sample (C) in a molecular ratio (A:B:C) of 1-500:1 :1000-25,000, preferably wherein the ratio is in the range of from 2-100:1 :5,000-15,000, and more preferably wherein the ratio is in the range of from 5- 15:1 :8,000-12,000.

[0077] Alternatively, and most preferably, the first test strip may comprise the glycated biomarker binding molecule of the glycated biomarker conjugate (A), antigen (B) and glycated biomarker in the sample (C) in a molecular ratio (A:B:C) of 100-10,000:1 :2x107-1 :1 x1010, preferably from 300-7500:1 :7.5x107-1 :7.5xl09, and more preferably from 500-5000:1 :1 x10® - 1 :5X109. For the purposes of the present invention, a typical sample may contain 5.6 nmol of glycated biomarker such as 5.6 nmol of HbA1c. A typical sample may contain 3.4 x 1015molecules of HbA1c.

[0078] The first test strip may also comprise a control region, which is preferably located after the one or more detection regions. The control region comprises a control molecule which binds to the glycated biomarker conjugate, typically the glycated biomarker binding molecule component, in order to provide an indication as to whether the lateral flow test is functioning correctly. Preferably, when the glycated biomarker binding molecule comprises an anti-HbA1c antibody, the control region comprises an anti-human IgG polyclonal antibody.

[0079] The control region may comprise the control molecule at a concentration in the range of from 0.1 to 0.45 mg / ml, preferably from 0.15 to 0.4 mg / ml, and more preferably from 0.2 to 0.3 mg / ml. The control region may contain from 3 x10’5to 13.5 x10’5mg, from 4.5 xw5to 12 xw5mg, and more preferably from 6 xw5to 9 xw5mg of the control molecule.

[0080] As mentioned above, the first test strip itself is also a focus of the present invention. Thus, in an aspect, the present invention is directed to a test strip for a lateral flow device, wherein the test strip is for measuring the level of a glycated biomarker in a sample in a competitive assay, comprising: a conjugate region comprising a glycated biomarker conjugate, the glycated biomarker conjugate comprising a glycated biomarker binding molecule linked to a reporter; and one or more detection regions comprising an antigen. In these aspects, the ratio of the number of binding molecules to the number of antigen molecules in the detection region is preferably at least 1 :1000, and more preferably at least 1 :1 xi o7. It will be appreciated that such a test strip preferably has the features of the first test strip as defined above.

[0081] Second test strip

[0082] The lateral flow device of the present invention also comprises a second test strip for measuring the level of non-glycated biomarker in a sandwich assay.

[0083] As with the first test strip, the second test strip may be one continuous material. However, in preferred embodiments, the second test strip comprises more than one component such as a base membrane, a sample pad and a conjugate region. For instance, the second test strip may comprise a fibrous material such as nitrocellulose. Preferably, the second test strip comprises a nitrocellulose base membrane upon which a sample pad and a conjugate region, typically also in the form of a pad, are disposed. As mentioned above, the sample pad that is disposed on the second test strip is preferably also disposed on the first test strip.

[0084] It will be appreciated that the biomarkers measured in the first and test strips are the same, except that the first test strip measures the glycated biomarker, whereas the second test strip measures the non-glycated biomarker. Typically, the second test strip will measure just the non-glycated biomarker ( / .e. not the glycated form of the biomarker) but in some instances the second test strip may measure the total amount of biomarker ( / .e. biomarker in its non-glycated and glycated form).

[0085] By comprising a second test strip, to measure the level of non-glycated biomarker, in combination with a first test strip, the lateral flow device can provide an accurate calibration of the glycated biomarker to the baseline level of non-glycated biomarker in the sample, since each of these can be quantified using their respective test strips.

[0086] As the preferred glycated biomarker is HbA1c, the non-glycated biomarker measured for in the second test strip is preferably haemoglobin (Hb).

[0087] As with the first test strip, the second test strip also preferably comprises a conjugate region, the conjugate region comprising a non-glycated biomarker conjugate. The non-glycated biomarker conjugate comprises a non-glycated biomarker binding molecule ( / .e. a molecule able to bind to a non-glycated biomarker through a binding moiety) linked to a reporter. Preferably, the non-glycated biomarker conjugate is an Hb conjugate, which comprises an Hb binding molecule.

[0088] The non-glycated biomarker binding molecule of the non-glycated biomarker conjugate preferably comprises an antibody or an antibody fragment which is capable of binding to a non-glycated biomarker as defined above. The non-glycated biomarker binding molecule may alternatively comprise an antibody mimic such as a molecularly imprinted polymer. The antibody will typically be a natural antibody (e.g. a monoclonal antibody), though a synthetic antibody may also be used (e.g. a recombinant antibody or a molecularly imprinted synthetic antibody). The antibody is preferably a Hb antibody, and the binding moiety is preferably an Hb binding fragment of the antibody. An exemplary antibody is BM302-S6B9, a B9 haemoglobin monoclonal antibody available from BBI solutions.

[0089] Any suitable molecule for generating a signal may be used as the reporter, for instance the molecules described above in connection with the first test trip. Preferably, the reporter is a metallic nanoparticle such as a gold or silver nanoparticle, and more preferably is a gold nanoparticle. The reporter may be the same for the first and second test strips.

[0090] Where the reporter is a metallic nanoparticle such as a gold nanoparticle, the nanoparticle may have a particle size in the range of from 10-100 nm, preferably from 20-80 nm, and more preferably from 30-50 nm. As used herein, particle size refers to the mean equivalent sphere hydrodynamic diameter of a nanoparticle as may be measured using dynamic light scattering, for instance according to ASTM E3247-20.

[0091] Accordingly, it is most preferred that the non-glycated biomarker conjugate is an anti-Hb antibody conjugated to a gold nanoparticle, preferably wherein the gold nanoparticle has a particle size of around 40 nm.

[0092] The reporter may be linked to the non-glycated biomarker binding molecule by the same means as described above in relation to the glycated biomarker conjugate that is used in the first test strip.

[0093] More than one non-glycated biomarker binding molecule may be connected to a single reporter. For instance, a reporter may be linked to from 50 to 500, such as from 100 to 300 non-glycated biomarker binding molecules, and more preferably from 150 to 250 non-glycated biomarker binding molecules such as antibodies.

[0094] The conjugate region in the second strip may comprise the non-glycated biomarker conjugate in an amount of at least 5 pL, preferably at least 7 pL, and more preferably at least 10 pL as a solution at an optical density of OD5. The conjugate region may comprise the non-glycated biomarker conjugate in an amount of up to 25 pL, preferably up to 20 pL, and more preferably up to 15 pL as a solution at an optical density of OD5. Thus, the conjugate region may comprise the non-glycated biomarker conjugate in an amount of from 5 to 25 pL, preferably from 7 to 20 pL, and more preferably from 10 to 15 pL as a solution at an optical density of OD5. Optical density may be measured using a UV / Vis spectrophotometer at a wavelength of 550nm with a cuvette path length of 1cm.

[0095] As with the first strip, the conjugate region in the second strip will typically be in the form of a pad. The conjugate region may have a length of from 6 to 12 mm, preferably from 7 to 11 mm, and more preferably from 8 to 10 mm. The conjugate regional may have a width of from 1 .5 to 4.5 mm, preferably from 2.0 to 4.0 mm, and more preferably from 2.5 to 3.5 mm. The conjugate region may have a depth of from 0.1 to 0.7 mm, preferably from 0.2 to 0.6 mm, and more preferably from 0.3 to 0.5 mm. The conjugate region may have a volume of from 5 to 15 mm3, preferably from 7 to 12 mm3, and more preferably from 8 to 11 mm3.

[0096] The conjugate region of the second test strip may comprise the non-glycated biomarker conjugate in an amount of from 3.6*109to 5.5*1010molecules per test strip.

[0097] The second test strip preferably also comprises one or more detection regions comprising a capture molecule to which the non-glycated biomarker can bind. Since the second test strip operates as a sandwich assay, the capture molecule binds to a different site on the non- glycated biomarker binding molecule of the non-glycated biomarker conjugate than that bound to in the conjugate region. Preferably, the capture molecule is able to bind Hb.

[0098] The capture molecule is preferably an antibody or antibody fragment, for instance an anti-Hb capture antibody.

[0099] As with the first test strip, the second test strip preferably comprises multiple detection regions. In the lateral flow device, the first and second test strips preferably comprise the same number of detection regions. This facilitates a comparison between the signals, for instance of their intensities, generated by the first and second test strips.

[0100] In order to provide a defined detection region from which a signal can reliably be read, the capture molecule in a detection region is typically immobilised within the detection region. Such immobilisation may be achieved by passive adsorption, particularly where a nitrocellulose membrane is used. Alternatively, linkers such as glutaraldehyde, succinimidyl 4- (N-maleimidomethyl)cyclohexane-l -carboxylate (SMCC) or sulfo-SMCC may be used.

[0101] The capture molecule may be present in the one or more detection regions of the first test strip at an average concentration ( / .e. the average across each of the one or more regions) of up to 1 mg / ml, preferably up to 0.5 mg / ml, and more preferably up to 0.3 mg / ml. The capture molecule may be present at an average concentration of at least 0.1 mg / ml, preferably at least 0.15 mg / ml, and more preferably at least 0.20 mg / ml. Thus, the capture molecule may be present in the one or more detection regions at an average concentration in the range of from 0.1 to 1 mg / ml, preferably from 0.15 to 0.5 mg / ml, and more preferably from 0.2 to 0.3 mg / ml. As these values represent an average across all detection regions, it will be appreciated that one or more of the detection regions may individually contain antigen levels outside of these ranges. However, in preferred embodiments, each of the detection regions of the first test strip comprise the capture molecule in this amount. Typically, each of the one or more detection regions contains from 0.1 to 0.5 pL, preferably from 0.2 to 0.4 pL, and more preferably from 0.25 to 0.35 pL of capture molecule.

[0102] Each of the one or more detection regions may have a length of from 0.6 to 1.2 mm, preferably from 0.7 to 1.1 mm, and more preferably from 0.8 to 1 mm. Each of the one or more detection regions may have a width of from 1.5 to 4.5 mm, preferably from 2.0 to 4.0 mm, and more preferably from 2.5 to 3.5 mm.

[0103] The capture molecule may be present in the one or more detection regions of the second test strip at an average amount ( / .e. the average across each of the one or more regions) of at least 3 x10'5mg, preferably at least 4.5 x10’5mg, and more preferably at least 6 mg of antigen per detection region. The capture molecule may be present in the one or more detection regions of the first test strip at an average amount of up to 30 x10’5mg, preferably up to 15 xw5mg, and more preferably up to 9 xw5mg of antigen per detection region. Thus, the capture molecule may be present in the one or more detection regions of the first test strip at an average amount of from 3 xw5to 30 xw5mg, from 4.5 xw5to 15 xw5mg, and more preferably from 6 xw5to 9 xw5mg of antigen per detection region. In preferred embodiments, each of the detection regions of the first test strip comprise the capture molecule in this amount.

[0104] The one or more detection regions and the conjugate region are preferably located on the second test strip such that the sample, once introduced to the strip, must pass through the conjugate portion in order to reach the first of the one or more detection regions.

[0105] Preferably, the second test strip comprises multiple detection regions. For instance, the second test strip may comprise at least 3 detection regions, preferably at least 4 detection regions, and more preferably at least 5 detection regions. In some instances, the second test strip comprises 5 detection regions. Such a configuration, as compared to a single test line, can assist in reducing the impact of saturation and thereby improve accuracy of the measurement. In preferred embodiments, the second test strip has the same number of detection regions as the first test strip.

[0106] Where the test strip comprises multiple detection regions, it is preferred that each of the detection regions contains the same amount of capture molecule. As with the first test strip, the second test strip may also comprise a control region, which is preferably located after the one or more detection regions. The control region comprises a control molecule which binds to the non-glycated biomarker conjugate in order to provide an indication as to whether the lateral flow test is functioning correctly. Preferably, the control region comprises an anti-human IgG polyclonal antibody.

[0107] The control region may comprise the control molecule at a concentration in the range of from 0.1 to 0.45 mg / ml, preferably from 0.15 to 0.4 mg / ml, and more preferably from 0.2 to 0.3 mg / ml. The control region may contain from 3 x10’5to 13.5 xw5mg, from 4.5 xw5to 12 xw5mg, and more preferably from 6 xw5to 9 xw5mg of the control molecule.

[0108] Sample inlet

[0109] The lateral flow device of the present invention also comprises a sample inlet for receiving a sample and passing it to the first and second test strips. As lateral flow test strips are typically contained within a housing, this sample inlet may be a port in the housing which is configured to permit fluid communication with the first and second test strips. If no such housing is present, and e.g. the two test strips are largely exposed, then an end of a test strip may still represent the sample inlet.

[0110] While the sample inlet may permit direct fluid communication of the sample from the external environment of the device to the first and second test strips, it will be appreciated that indirect fluid communication may also occur - i.e. intermediate structures may exist between the sample inlet and the first and second test strips.

[0111] The sample inlet may be sized so as to accommodate a fluid sample of an amount in the range of from 5-200 pL, preferably from 10-150 pL, and more preferably from 25-100 pL.

[0112] Preferably, the sample inlet comprises a sample port in fluid communication with a sample separation membrane that is in overlapping contact with the first and second test strips. This means that a first portion of the sample separation membrane overlaps with the first test strip, such that a portion of a sample entering the sample inlet passes through the first portion of the membrane into the first test strip. Likewise, a second portion of the sample separation membrane overlaps with the second test strip and allows a portion of a sample to pass into the second test strip through said second portion. Preferably, the sample separation is configured to divide the sample about equally between the first and second test strips. Any suitable sample separation membrane able to be used with a sample, in particular blood samples, may be used. For instance, the sample separation membrane may be a glass fibre filter, such as MF1 from Cytiva.

[0113] Within the lateral flow device, the first and second test strip are preferably arranged in parallel such that the first and second test strips may be contacted with the sample approximately simultaneously.

[0114] Further features

[0115] The lateral flow device of the present invention is able to accurately differentiate samples that differ in glycated biomarker level. For instance, the device may be able to differentiate samples that differ in HbA1 c levels by an amount of up to 5 mmol / mol, preferably up to 4 mmol / mol, and more preferably up to 3 mmol / mol.

[0116] The lateral flow device may comprise a housing which contains the first and second test strips, as shown in Figure 1 in an exploded view. The housing of the lateral flow device (101) may comprise a base (102) upon which the first test strip (103) and second test strip (104) are disposed. In this instance, each of the first (103) and second (104) test strips comprise five detection regions - the first detection region of the first test strip is denoted (103A), and the first detection region of the second test strip is denoted (104A). The first test strip (103) also comprises a control region (103B), and the second test strip (104) has a control region (104B). A sample separation membrane (105) is also present.

[0117] The housing can comprise a cover (106), which may be fastened to the base (102). For instance, the base (102) and cover (106) may be fastened with an interference fit, for instance using protrusions (107) in one which fit into notches (such as holes) in the other. Although depicted here substantially as a rectangular prism, it will be appreciated that the cover (106) and base (102) may be any suitable shape.

[0118] The cover (106) can comprise the sample inlet (108), which in this case is a port which is located such that, when the cover (106) and base (102) are fastened together, the inlet (108) is positioned above the sample separation membrane (105). In this way, the sample inlet (108) can direct the sample to the sample separation membrane (105), and thereafter to the first (103) and second (104) test strips. The cover (106) may also comprise a viewing port (109), which allows the detection regions of the first (103) and second (104) test strips to be viewed. The viewing port may be formed by a gap in the housing, or a transparent layer may be present within the viewing port that protects the test strips from exposure to the external environment, but still permits any signals that may be generated at the detection regions to be seen.

[0119] As shown in Figure 2, the lateral flow device (101) may comprise a first multicoloured barcode

[0120] (201) and a second multicoloured barcode (202), wherein the first multicoloured barcode (201) is preferably located adjacent to the first test strip (103) and the second multicoloured barcode

[0121] (202) is preferably located adjacent to the second test strip (104). The defined multicolour barcodes can be used to normalise an image taken (e.g. by a smartphone camera) of a signal generated at a detection region, if an image of it is taken at different lighting conditions. Ways of performing such image correction is described in EP4113429 A1.

[0122] The lateral flow device, as described herein, may be used for measuring the amount of a biomarker that is in glycated form within a sample. Preferably, the lateral flow device is used for measuring the amount of Hb that is in the form of HbA1c in a sample, more preferably wherein the sample is a blood sample.

[0123] Method for measuring the level of glycated biomarker

[0124] In another aspect, the present invention relates to a method for measuring the amount of biomarker that is in glycated form in a sample, comprising the steps of: adding a sample to a lateral flow device as defined herein; waiting for the sample to flow along the first test strip and the second test strip; and detecting an output from the first and second test strips.

[0125] Preferably, the method is for measuring the amount of haemoglobin (Hb) that is in the form of HbA1c in a sample, and the lateral flow device used is therefore configured to measure the amount of HbA1c, in the ways as have been described above.

[0126] The sample is preferably a blood sample, such as a whole blood sample. The volume of the blood sample may be in the range of from 1 to 20 pL, preferably from 5 to 15 pL, and more preferably from 7.5 to 12.5 pL.

[0127] A typical sample for the purposes of the present invention may contain 3.4 x 1015molecules molecules of glycated biomarker. However, a real-life sample will nearly always contain a different amount of glycated biomarker and it is this variation, as compared to the amount of non-glycated biomarker which will also vary from person to person, that the devices of the present invention are used to measure.

[0128] It will be appreciated that the sample may have undergone a sample preparation step prior to its addition to the lateral flow device. For instance, in particular where the glycated biomarker being measured by the device is HbA1c, the blood sample has preferably undergone a lysis step such that it comprises lysed red blood cells. The lysis step comprises adding the sample to a lysing solution, such as a lysing buffer. Preferably, the lysing solution is suitable for lysing red blood cells, such that the sample is prepared by mixing blood with a blood cell lysing buffer. This mixture of sample and lysing buffer would then be what is added to the lateral flow device.

[0129] Where a lysis step is present, the sample may be added to the lysing buffer in a ratio of sample:buffer of at least 1 :5, preferably of at least 1 :10, and more preferably at least 1 :25 by volume. The ratio of sample:buffer may be up to 1 :45, preferably up to 1 :40, and more preferably up to 1 :35 by volume. Thus, the sample may be added to the lysing buffer in a ratio in the range of from 1 :5 to 1 :45, preferably from 1 :10 to 1 :40, and more preferably from 1 :25 to 1 :35 by volume.

[0130] The sample is added to the sample inlet of the lateral flow device, which is in fluid communication with the first and second test strips. The sample may be introduced into the sample inlet in an amount of up to 200 pL, preferably up to 150 pL, and more preferably up to 125 pL. The sample may be introduced in an amount of at least 10 pL, preferably at least 50 pL, and more preferably at least 75 pL. Thus, the sample may be introduced into the sample inlet in an amount of from 10 to 200 pL, preferably from 50 to 150 pL, and more preferably from 75 to 125 pL. It will be appreciated that these amounts refer to the total amount of sample added during the method - this may take the form of a single addition, or multiple drops of sample, the combined total of which is within these ranges.

[0131] From the sample inlet of the lateral flow device, the sample is split such that a portion of the sample is delivered to each of the first and second test strips. This may be achieved using a sample separation membrane as described herein. For instance, the sample may be divided between the first and second test strips such that from 5 to 100 pL, preferably from 20 to 80 pL, and more preferably 40 to 60 pL of sample is delivered to each test strip.

[0132] Once the sample has been added to the lateral flow device, the next step is waiting for the sample to flow along the first test strip and the second test strip. For the first test strip, an exemplary description of the process is (with reference to Figure 3) as follows. As can be seen in Figure 3, the sample (301) which comprises a glycated biomarker such as HbA1c (302), is added to lateral flow device and, through the sample inlet, contacts the first test strip (103). Specifically, the sample (301) may first enter a sample pad (303), which is in communication with a conjugate region. In Figure 3, this is represented as the sample pad (303) partially overlapping with a conjugate pad (304). The conjugate pad (304) comprises a glycated biomarker conjugate (305), which in Figure 3 is exemplified as an anti-HbA1c antibody (306) conjugated to a 40nm gold nanoparticle reporter (307). The sample will flow from the sample pad (303) into the conjugate pad (304), wherein at least a portion of the HbA1c in the sample binds to the anti-HbA1c antibody (306) of the glycated biomarker conjugate (305). This forms a mixed sample, comprising HbA1c-bound binding molecule and an amount of glycated biomarker conjugate (305) which is not bound to HbA1c. This mixed sample can then flow into, and along, a base membrane (308) which the conjugate pad (304) is in contact with, for instance by partial overlap as in Figure 3.

[0133] The base membrane (308), which may be formed of a fibrous material such as nitrocellulose, comprises a detection region. In the embodiment shown in Figure 3, it comprises five detection regions (309), as well as a control region (310). Each of these detection regions (309) comprises an antigen (311), which here is also HbA1c. In a preferred embodiment, the first two of these detection regions (309) comprises a greater concentration of HbA1c than the other three.

[0134] The mixed sample first flows into the first detection region (309) - that closest to the conjugate pad (304) - then sequentially into the remaining detection regions (309). At each detection region, which comprises antigen (311), any unbound glycated biomarker binding molecule (305) can bind to the antigen (311). By virtue of the linked reporter (307), the accumulation of these antigen-bound glycated biomarker conjugates can generate a signal (in this case, a colorimetric signal) at the detection regions (309). Any HbA1c-bound binding molecule in the mixed sample is unable to bind to the antigen (311), and therefore does not accumulate in the detection region, and does not contribute to the signal generation. Instead, the HbA1c-bound binding molecule flows past the detection regions (309), for instance into an absorbent pad (312) which acts essentially as a reagent sink.

[0135] In the second test strip, which tests for the non-glycated biomarker (which would be Hb in the corresponding test strip to that exemplified in Figure 3), a similar process occurs. However, a conjugate region in the second test strip would comprise e.g. an anti-Hb antibody linked to a reporter, and the detection regions would comprise e.g. an Hb capture antibody. Therefore, Hb would bind to the anti-Hb antibody in the conjugate region to form an Hb-bound antibody. This Hb-bound antibody would then bind to the Hb capture antibody in the detection regions, typically by binding to another site on the Hb molecule, to generate a signal. The method then comprises a step of detecting an output from the first and second test strips. Typically, the output will be the signal from the one or more detection regions of the first and second test strips. Preferably, the output is a colorimetric output, more preferably wherein the colour intensity of the output is quantified to determine an amount of glycated biomarker, which is preferably HbA1c, present in the sample.

[0136] Examples

[0137] Example 1 - Variation in biomarker levels

[0138] The haemoglobin levels in blood samples from 180 donors were analysed, with the results being shown in Figure 4. The donors were a mixture of healthy, pre-diabetic and diabetic individuals.

[0139] As can be seen, Hb levels detected ranged from around 115-120 mg / ml to around 175-180 mg / ml. However, Hb levels in the range of 130 to 150 mg / ml were the most prevalent population, accounting for 111 out of 180 individuals. Thus, it can be seen that there are fairly significant deviations between the Hb levels of individuals. It likewise follows that other biomarkers, such as those described herein, can also exhibit such inter-patient variability.

[0140] However, HbA1c levels used for determining diabetic status, are typically provided as a ratio between HbA1c and Hb. Therefore, an HbA1c level calculated using an assumption that a sample has a “typical” Hb level (without actually measuring it) provides a high possibility of misclassification. This is illustrated in Table 1 (below), which contains exemplary HbA1c levels (as mmol / mol values) and corresponding classifications that would be generated from a set amount of HbA1c in a 10 pL sample, based on varying Hb levels (in a corresponding 10 pL sample) across the ranges found in Figure 4.

[0141]

[0142] Table 1

[0143] As can be seen from Table 1 , an assumption that e.g. a patient with a true amount of 3.65 x 10'6mmol HbA1c in a 10 pL sample has a 120 mg / ml level of Hb (corresponding to 7.4 x 10'8mol in a 10 pL sample) could lead to a diagnosis of full diabetes, whereas in actuality the patient is within the pre-diabetic range.

[0144] Example 2 - Competitive assay vs sandwich assay

[0145] In lateral flow devices of the present invention, utilising a competitive assay for the first test strip (which measures the glycated biomarker such as HbA1c) provides increased sensitivity which can allow for an improved ability to distinguish signal intensities between blood samples with similar HbA1c levels. This is particularly the case compared to sandwich assays which are typically used for these purposes.

[0146] Figures 5A and 5B depict images of lateral flow assay tests. Specifically, Figure 5A shows two side-by-side lateral flow tests which were run as competitive assays, using test strips in accordance with the first test strip as described herein. It can be seen that the 8% “unhealthy” HbA1c sample (48 mmol / mol) produced an intensity in the detection regions of the lefthand test strip, this intensity being noticeably fainter than for the 5% “healthy” HbA1c sample (31 mmol / mol - righthand test strip), even with purely visual inspection. The A value, indicating the degree of difference between the results, for the two results in Figure 5A is >15.

[0147] In contrast, Figure 5B shows two side-by-side lateral flow tests which were run as sandwich assays, using the same 8% “unhealthy” HbA1 c (48 mmol / mol) and 5% “healthy” HbA1c (31 mmol / mol) samples. The differences in intensity between the “healthy” and “unhealthy” samples are much less apparent. The A value for the two results in Figure 5B is approximately 3.

[0148] Example 3 - Components of the first test strip

[0149] Various components of the first test strip, such as the optical density and volume of the glycated biomarker conjugate, the blood / lysis buffer dilution and the antigen concentration in the test lines, were tested to determine their most efficacious ranges. The results are shown in Table 2 (below). Table 2

[0150] A A value in the range of 10 or higher was found to provide the most sensitive tests, although values lower than this could still provide effective tests.

Claims

Claims1 . A lateral flow device for measuring the amount of a biomarker that is in a glycated form in a sample, the device comprising: a first test strip for measuring the level of a glycated biomarker in a competitive assay; a second test strip for measuring the level of the non-glycated biomarker in a sandwich assay; and a sample inlet for receiving a sample and passing it to the first and second test strips.

2. The lateral flow device of Claim 1 , wherein the device is for measuring the amount of haemoglobin (Hb) that is in a glycated form (HbA1c) in a sample, the first test strip is for measuring the level of HbA1c in a competitive assay, and the second test strip is for measuring the level of Hb in a sandwich assay.

3. The lateral flow device of Claim 1 or 2, wherein the first test strip comprises: a conjugate region comprising a glycated biomarker conjugate, the glycated biomarker conjugate comprising a glycated biomarker binding molecule linked to a reporter; and one or more detection regions comprising an antigen, wherein the ratio of the number of glycated biomarker binding molecules to the number of antigen molecules in the one or more detection regions is preferably at least 1 : 1000, and more preferably at least 1 :1 107.

4. The lateral flow device of Claim 3, wherein the ratio of the number of glycated biomarker binding molecules:antigen present in the one or more detection regions is in the range of from 1 :2000 to 1 :25,000, preferably from 1 :5000 to 1 :15,000, and more preferably from 1 :8,000 to 1 :12,000.

5. The lateral flow device of Claim 3 or 4, wherein in the first test strip: the ratio of the number of glycated biomarker binding molecules to the number of glycated biomarker molecules in a typical sample is in a ratio in the range of from 1 :1 to 1 :500, preferably from 1 :2 to 1 OO, and more preferably from 1 :5 to 1 :15; and / or the ratio of the number of antigen molecules in the detection region to the number of glycated biomarker molecules in a typical sample is in the range offrom 1 :1 to 5,000:1 , preferably from 10:1 to 2,000:1 , and more preferably from 100:1 to 1000:1.

6. The lateral flow device of any of Claims 3 to 5, wherein the antigen is present in the one or more detection regions of the first test strip at an average concentration in the range of from 0.1 to 1 mg / ml, preferably from 0.2 to 0.75 mg / ml, and more preferably from 0.25 to 0.45 mg / ml.

7. The lateral flow device of any of Claims 3 to 6, wherein the conjugate region of the first test strip comprises the glycated biomarker conjugate in an amount of from 3.6x1 o9to 5.5x1 o10molecules per test strip.

8. The lateral flow device of Claim 3 or Claim 7, wherein the ratio of the number of glycated biomarker binding molecules:antigen molecules present in the one or more detection regions is in the range of from 1 :2xio7to 1 :1 xio10, preferably from 1 :7.5xio7to 1 :7.5xio9, and more preferably from 1 :1 xio8to 1 :5xio9.

9. The lateral flow device of Claim 3, Claim 7 or Claim 8, wherein in the first test strip: the ratio of the number of glycated biomarker binding molecules to the number of glycated biomarker molecules in a typical sample is in a ratio in the range of from 1 :100 to 1 :10,000, preferably from 1 :300 to 1 :7500, and more preferably from 1 :500 to 1 :5000; and / or the ratio of the number of antigen molecules in the detection region to the number of glycated biomarker molecules in a typical sample may be in the range of from 10,000:1 to 1 xio6:1 , preferably from 50,000:1 to 800,000:1 , and more preferably from 75,000:1 to 600,000:1.

10. The lateral flow device of any of Claims 3 to 9, wherein in the glycated biomarker conjugate: the glycated biomarker binding molecule is an antibody, preferably an anti- HbA1c antibody; and / or the reporter molecule is a colorimetric reporter, preferably a metallic nanoparticle, and more preferably a gold nanoparticle.11 . The lateral flow device of any of Claims 3 to 10, wherein the first test strip comprises multiple detection regions, preferably wherein at least one of the detection regions comprises a different concentration of antigen to another of the detection regions.

12. The lateral flow device of Claim 11 , wherein the concentration of antigen is higher in the first detection region than the last detection region reached by the sample; and preferably wherein: the first detection region comprises antigen in a concentration in the range of from 0.3 to 1 mg / ml, preferably from 0.4 to 0.75 mg / ml, and more preferably from 0.45 to 0.55 mg / ml; and / or the last detection region comprises antigen in a concentration in the range of from 0.1 to 0.45 mg / ml, preferably from 0.15 to 0.4 mg / ml, and more preferably from 0.2 to 0.3 mg / ml.

13. The lateral flow device of Claim 11 or Claim 12, wherein the concentration of antigen in each of the multiple detection regions in the first test strip stays the same or decreases as the sample moves along the test strip.

14. The lateral flow device of any of Claims 3 to 13, wherein: the glycated biomarker conjugate is a HbA1c conjugate which comprises a HbA1c binding molecule linked to a reporter; the antigen of the one or more detection regions is HbA1c; and the glycated biomarker in the sample is HbA1c.

15. The lateral flow device of any preceding claim, wherein the second test strip comprises: a conjugate region comprising a non-glycated biomarker conjugate, the nonglycated biomarker conjugate comprising a non-glycated biomarker binding molecule linked to a reporter; and one or more detection regions comprising a capture molecule to which the non- glycated biomarker conjugate can bind, preferably wherein: the non-glycated biomarker conjugate is a Hb conjugate which comprises a Hb binding molecule linked to a reporter, and the capture molecule is able to bind Hb.

16. The lateral flow device of any preceding claim, wherein the sample inlet comprises a sample port in fluid communication with a sample separation membrane that is in overlapping contact with the first and second test strips.

17. The lateral flow device of any preceding claim, wherein the first and / or second test strips comprises a fibrous material such as nitrocellulose.

18. The lateral flow device of any preceding claim, wherein the device comprises a first multicoloured barcode and a second multicoloured barcode, where the first multicoloured barcode is preferably located adjacent to the first test strip and the second multicoloured barcode is preferably located adjacent to the second test strip.

19. A test strip for a lateral flow device, wherein the test strip is for measuring the level of a glycated biomarker in a sample in a competitive assay and comprises: a conjugate region comprising a glycated biomarker conjugate, the glycated biomarker conjugate comprising a glycated biomarker binding molecule linked to a reporter; and one or more detection regions comprising an antigen, wherein the ratio of the number of glycated biomarker binding molecules in the conjugate region to the number of antigen molecules in the detection region is at least 1 : 1000, and more preferably at least 1 : 1 * 107, and wherein the test strip preferably has the features of the first test strip as defined in any of Claims 4 to 14.

20. Use of the lateral flow device of any of Claims 1 to 18 for measuring the amount of biomarker that is in glycated form in a sample, wherein the sample is preferably a blood sample, and preferably wherein the lateral flow device is for measuring the amount of haemoglobin (Hb) that is in the form of HbA1c.21 . A method for measuring the amount of biomarker that is in glycated form in a sample, comprising adding a sample to a lateral flow device as defined in any of Claims 1 to 18; waiting for the sample to flow along the first test strip and the second test strip; and detecting an output from the first and second test strips.

22. The method of Claim 21 , wherein the method is for measuring the amount of haemoglobin (Hb) that is in the form of HbA1c.

23. The method of Claims 21 or 22, wherein the output in step (c) is a colorimetric output, preferably wherein the colour intensity of the output is quantified to determine an amount of glycated biomarker, preferably HbA1c, present in the sample.

24. The method of any of Claims 21 to 23, wherein the sample is prepared by mixing blood with a blood cell lysing buffer.

25. The method of any of Claims 21 to 24, wherein the sample is introduced into the sample inlet in an amount of from 10 to 200 pL, preferably from 50 to 150 pL, and more preferably from 75 to 125 pL.

26. The method of any of Claims 21 to 25, wherein the sample is divided between the first and second test strips such that from 5 to 100 pL, preferably from 20 to 80 pL, and more preferably 40 to 60 pL of sample is delivered to each test strip.

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