Lateral flow printing, test, and detection method

Abstract

A lateral flow test strip for detecting the presence or absence of a first target analyte within a sample is described. The lateral flow test strip comprises: an input configured to receive the sample; a conjugate pad comprising a conjugate biomolecule configured to bind to a first target analyte in the sample; a membrane comprising one or more test zones, wherein a first test zone comprises a first soluble coloured compound substantially co-located with a first non-visible capture biomolecule, wherein the first non-visible capture biomolecule is attached to the membrane and is configured to bind to the first target analyte, thereby immobilising the first labelled conjugate assembly at the first test zone, and wherein the first soluble coloured compound is configured to be removed from the first test zone by a fluid flow of the sample and transported along the lateral flow test strip.

Description

LATERAL FLOW PRINTING, TEST, AND DETECTION METHODTECHNICAL FIELD

[0001] The present disclosure generally relates to the field of diagnostic and biomedical testing such as colorimetric or fluorescent immunoassay detection, lateral flow tests, immunoassay test instrument or apparatus suitable for use in medical diagnostics at a Point-of-Care (POC) setting and in a Physician’s Office Laboratories (POL).BACKGROUND

[0002] Lateral flow tests (also known as lateral flow immunochromatographic assays) are a simple device used to detect the presence (or absence) of a target analyte in a sample. Most commonly these tests are used for medical diagnostics either for home testing, point of care testing, or laboratory use. Often produced in a dipstick format, or packaged within a plastic cartridge, lateral flow tests are a form of immunoassay in which the test sample flows along a solid substrate or membrane, via capillary action. In a typical lateral flow test, the sample fluid is applied to the test strip and as it flows through the test strip it can bind to conjugate biomolecules that are also attached to detectable label particles, such as red coloured gold nanoparticles. This assembly bound to the sample analyte flows through the membrane of the lateral flow strip and encounters zones such as lines, spots which have been pre-treated with a capture reagent biomolecule that is immobilised in the zone. Depending upon the analytes present in the sample the labelled conjugate assembly can become bound at the test line, spot or zone and be detectable in this zone such as by the presence of a visible colour label particles or the presence of fluorescent label particles. Lateral flow tests can operate with alternative arrangements such as either competitive or sandwich assays but all result in an increase or decrease in the presence of the detectable label material or particles with the defined regions on the membrane.

[0003] This description of prior art covers a sandwich type assay construction, where the sample analyte is bound between the capture biomolecule and the conjugate biomolecule with an attached label particle. This type of assay results in an increasing population of label particles at the capture zone for a positive test. Other types of lateral flow assay construction include competitive assays which will result in a decreasing population of label particles on the capture zones for positive test result.

[0004] Both the presence of antigen or antibodies, examples of sample analytes, can be detected by lateral flow tests. The most well-known examples of lateral flow tests are home pregnancy tests. However, rapid tests or point of care tests are available for a wide range of applications including HIV tests, Troponin T test, Malaria tests, drugs of abuse tests, fertility tests, respiratory disease tests including SARS-CoV tests. Tests are available for both human and animal diagnostics. Tests are also available for non-clinical applications including testing food and water for contaminants. For example, when seeking to detect the presence of infection the concentration of antibodies specific to that pathogen can be measured. The pathogen protein antigen is used to capture the antibody, and subsequently detected by a secondary reagent that detects the presence of antibody binding. For measuring hormones such as insulin, the antibody against insulin is used to capture soluble insulin that is found in the sample.

[0005] In principle any colour or florescent label particle can be used for detection of the sample analyte (e.g. protein or antibody) once it bound in the capture zone of the lateral flow test. However, many tests use gold nanoparticles also referred to as colloidal gold particles as detection labels. The gold particles are red in colour due to localised surface plasmon resonance. Fluorescent or magnetic labelled particles can also be used - where these typically require the use of an electronic reader to detect the presence of these particles in zones and report a test result.

[0006] Most tests are intended to operate on a purely qualitative basis. However, it is possible to measure the intensity of the test line, zone or spot to determine the quantity of analyte in the sample. Implementing a magnetic immunoassay (MIA) in the lateral flow test form also allows for getting a quantified result. Generally, this involves the incorporation of known quantitated standards that can be used for comparative purposes.

[0007] Any discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification is not to be taken as an admission that any or all of these matters form part of the prior art base or were common general knowledge in the field relevant to the present disclosure as it existed before the priority date of each of the appended claims.

[0008] Throughout this specification the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.SUMMARY

[0009] Some embodiments relate to a lateral flow test strip for detecting the presence or absence of a first target analyte within a sample. The lateral flow test strip may comprise: an input configured to receive the sample; a conjugate pad in fluid communication with the input, the conjugate pad may comprise a conjugate biomolecule attached to a detection label, wherein the conjugate biomolecule may be configured to bind to a first target analyte in a sample applied to the input to form a first labelled conjugate assembly; a membrane in fluid communication with the conjugate pad, the membrane may comprise one or more test zones, wherein a first test zone of the one or more test zones may comprise a first soluble coloured compound substantially co-located with a first non- visible capture biomolecule, wherein the first non- visible capture biomolecule may be attached to the membrane and may be configured to bind to the first target analyte of a first labelled conjugate assembly in a sample transported along the lateral flow test strip, thereby immobilising the first labelled conjugate assembly at the first test zone, and wherein the first soluble coloured compound may be configured to be removed from the first test zone by a fluid flow of a sample applied to the input and transported along the lateral flow test strip.

[0010] Some embodiments relate to a lateral flow test strip for detecting the presence or absence of a first target analyte within a sample. The lateral flow test strip may comprise: an input configured to receive the sample; a membrane in fluid communication with the input, wherein the membrane may comprise one or more test zones, wherein a first test zone of the one or more test zones may comprise a first soluble coloured compound substantially co-located with a first non- visible capture biomolecule, wherein the first non-visible capture biomolecule may be attached to the membrane and may be configured to bind to a first target analyte in a sample applied to the input and transported along the lateral flow test strip, thereby immobilising the first target analyte at the first test zone, wherein the lateral flow test strip may be configured to direct a conjugate biomolecule attached to a detection label along the membrane, wherein the conjugate biomolecule may be configured to bind to the first target analyte to form a first labelled conjugate assembly, and wherein the first soluble coloured compound may be configured to be removed from the first test zone by a fluid flow of a sample applied to the input and transported along the lateral flow test strip.

[0011] The conjugate biomolecule may be configured to bind to the first target analyte after it is immobilised at the first test zone. The conjugate biomolecule may be configured to bind to the first target analyte before it is immobilised at the first test zone. The lateral flow test strip may comprisea conjugate biomolecule source in fluid communication with the input. The conjugate biomolecule source may be a conjugate pad containing the conjugate biomolecule.

[0012] The first soluble coloured compound may be water-soluble. The first test zone may exhibit at least one identifying characteristic selected from the group including: (i) a colour; (ii) a shape; (iii) a size; (iv) a relative position of the first test zone on the membrane. The first test zone may exhibit a circular shape. The at least one test zone may comprise a longitudinal shape extending along the length of the lateral flow test strip. The at least one test zone may comprise a longitudinal shape extending along the width of the lateral flow test strip. The one or more test zones may be arranged in an array on the membrane.

[0013] The conjugate biomolecule may be selected to be complimentary for binding to the first target analyte and may be selected from a group consisting of: antigens, antibodies, aptamers, and molecular recognition elements. The first target analyte may be an antigen selected from a group consisting of: protein, polysaccharide, lipid or nucleic acid. The first target analyte may be an antibody. The first target analyte may be a molecule or chemical moiety. The first non-visible capture biomolecule may be selected from a group consisting of: antigens, antibodies, aptamers, and molecular recognition elements.

[0014] The label may be a coloured particle, a gold particle, a florescent particle or a magnetic particle. Responsive to the first non-visible capture biomolecule binding to a respective first target analyte in the sample applied to the input of the lateral flow test strip and transported to the first test zone, the first test zone may be configured to transition from a first visible state to a second visible state to indicate the presence of the first target analyte in the sample.

[0015] The membrane may further comprise a control zone comprising an antibody configured to bind to a control analyte present in the sample. The control analyte may be present in the test fluid containing the sample and a wash buffer. The control analyte may be present in the buffer. The one or more test zones may comprise a second test zone, wherein the second test zone may comprise a second soluble coloured compound substantially co-located with a second non-visible capture biomolecule, wherein the second non-visible capture biomolecule may be configured to attach to the membrane and to bind to a respective second target analyte in the sample transported along the lateral flow test strip, thereby immobilising the second target analyte at the second test zone, wherein the second soluble coloured compound may be configured to be removed from the second test zone by a fluid flow of the sample applied to the input and transported along the lateralflow test strip; and wherein the first and second target analytes may be different from one another, and the first and second non-visible capture biomolecules may be different from one another.

[0016] A second labelled conjugate assembly may comprise the second target analyte bound to a second complimentary conjugate biomolecule attached to a second detection label, and the second complimentary conjugate biomolecule may bind to the second target analyte immobilised at the second test zone thereby forming the second labelled conjugate assembly at the second test zone. In other words, the second complimentary conjugate biomolecule may bind to the second target analyte after the second target analyte is immobilised at the second test zone.

[0017] A second labelled conjugate assembly may comprise the second target analyte bound to a second complimentary conjugate biomolecule attached to a second detection label, and the second non-visible capture biomolecule may bind to the second target analyte of the second labelled conjugate assembly thereby immobilising the second labelled conjugate assembly at the second test zone. In other words, the second complimentary conjugate biomolecule may bind to the second target analyte before the second target analyte is immobilised at the second test zone.

[0018] The second test zone may be positioned on the membrane at a location spaced apart from the first test zone. The first test zone may be positioned on the membrane in a location upstream of the second test zone. The first test zone and the second test zone may be positioned on the membrane in an interspersed arrangement.

[0019] Some embodiments relate to a method of manufacturing a lateral flow test strip for detecting the presence or absence of a first target analyte within a sample, the method may comprise: combining a first soluble coloured compound with a first non-visible capture biomolecule and a carrier fluid to form a first formulation, wherein the first non-visible capture biomolecule may be configured to bind to a respective first target analyte; applying the first formulation to one or more first locations of a membrane of the lateral flow test strip to form respective one or more first test zones, wherein, when the first target analyte comes into contact with the one or more first test zones, the first non-visible capture biomolecule may bind to the first target analyte, thereby immobilising the first target analyte at the one or more first test zones, and when the first soluble coloured compound comes into contact with a fluid flow of the sample comprising the first target analyte, the first soluble coloured compound may be removed from the one or more first test zones by the fluid flow.

[0020] A first labelled conjugate assembly may comprise the first target analyte bound to a first complimentary conjugate biomolecule attached to a first detection label, and wherein the method may further comprise configuring the test strip so that the first complimentary conjugate biomolecule attached to a first detection label comes into contact with the first target analyte immobilised at the one or more first test zones causing the first complimentary conjugate biomolecule to bind to the first target analyte, thereby forming the first labelled conjugate assembly at the one or more first test zones. In other words, the first complimentary conjugate biomolecule may bind to the first target analyte after the first target analyte is immobilised at the first test zone.

[0021] A first labelled conjugate assembly comprises the first target analyte bound to a first complimentary conjugate biomolecule attached to a first detection label, and wherein the method may further comprise configuring the test strip so that the first labelled conjugate assembly comes into contact with the one or more first test zones causing the first non- visible capture biomolecule to bind to the first target analyte, thereby immobilising the first labelled conjugate assembly at the one or more first test zones. In other words, the first complimentary conjugate biomolecule may bind to the first target analyte before the first target analyte is immobilised at the first test zone.

[0022] The method may further comprise: allowing the first formulation at the one or more first locations of the membrane to dry. Applying the first formulation to the one or more first locations of the membrane may comprise: printing the first formulation onto the membrane. Applying the first formulation to the one or more first locations of the membrane may comprise: applying the first formulation in droplets to the one or more first locations.

[0023] The method may further comprise applying multiple droplets to one or more of the one or more first locations on the membrane to thereby increase the concentration of the first non-visible capture biomolecule and to increase the colour concentration of the first soluble coloured compound in the one or more first test zones.

[0024] The method may further comprise: combining a second soluble coloured compound with a second non-visible capture biomolecule to form a second formulation, wherein the second non- visible capture biomolecule may be configured to bind to a respective second target analyte; and applying the second formulation to one or more second locations of a membrane of the lateral flow test strip to form respective one or more second test zones, and wherein, when the second target analyte comes into contact with the one or more second test zones, the second non-visible capture biomolecule may bind to the second target analyte, thereby immobilising the second target analyteat the one or more second test zones, and when the second soluble coloured compound comes into contact with a fluid flow of the sample comprising the second target analyte, the second soluble coloured compound may be removed from the one or more second test zones by the fluid flow.

[0025] A second labelled conjugate assembly may comprise the second target analyte bound to a second complimentary conjugate biomolecule attached to a second detection label, and wherein the method may further comprise configuring the test strip so that the second complimentary conjugate biomolecule comes into contact with the second target analyte immobilised at the one or more second test zones causing the second complimentary conjugate biomolecule to bind to the second target analyte, thereby forming the second labelled conjugate assembly at the one or more second test zones. In other words, the second complimentary conjugate biomolecule may bind to the second target analyte after the second target analyte is immobilised at the second test zone.

[0026] A second labelled conjugate assembly comprises the second target analyte bound to a second complimentary conjugate biomolecule attached to a second detection label, and wherein the method may further comprise configuring the test strip so that the second labelled conjugate assembly comes into contact with the one or more second test zones causing the second non- visible capture biomolecule to bind to the second target analyte, thereby immobilising the second labelled conjugate assembly at the one or more second test zones. In other words, the second complimentary conjugate biomolecule may bind to the second target analyte before the second target analyte is immobilised at the second test zone.

[0027] Some embodiments relate to a lateral flow test strip produced according to the abovedescribed method.

[0028] Some embodiments relate to a method for visual inspection of a manufactured lateral flow test strip prior to addition of a sample, the method may comprise: identifying a coloured region on the lateral flow test strip, wherein the coloured region may be indicative of a location of a first test zone comprising a first soluble coloured compound and a first non-visible capture molecule; inspecting the first test zone for at least one identifying characteristic; and based on the at least one identifying characteristic: (i) identifying if the associated capture biomolecule zone and its location and relationship to other zones on the strip is compatible with the sample to be tested; and / or (ii) determining if the lateral flow test strip satisfies one or more quality control parameters.

[0029] The inspecting may be performed by eye. The inspecting may be performed by a computer executing computer code configured to perform digital imaging or machine vision techniques.

[0030] The at least one identifying characteristic may be presence or absence of colour of the first test zone. The at least one identifying characteristic may comprise one of more of: (i) a colour of the first test zone; (ii) a shape of the first test zone; (iii) a size of the first test zone; (iv) a relative position of the first test zone on the membrane; (v) a size and position of the first test zone on the membrane with respect to the size and position of other further test zones of the membrane.

[0031] The method for visual inspection of a manufactured lateral flow test strip prior to addition of a sample may further comprise: determining a concentration of the first non-visible capture biomolecule based on a determination of a colour concentration of the first test zone. The lateral flow test strip may be the lateral flow test strip as previously described.

[0032] Some embodiments relate to a method for detecting the presence or absence of a first target analyte within a sample using the lateral flow test strip of as previously described, the method may comprise: applying the sample to the input of the lateral flow test strip, wherein the lateral flow test strip may be configured to convey the sample to at least the first test zone of the membrane of the lateral flow test strip; allowing the sample to remove the first soluble coloured compound from the first test zone; and after a first time interval, responsive to determining that the first test zone has transitioned from a first visible state to a second visible state, determining the presence of the first target analyte in the sample, and responsive to determining that the first test zone has not transitioned from the first visible state to the second visible state, determining the absence of the first target analyte in the sample.

[0033] Some embodiments relate to a cartridge, the cartridge may comprise: a lateral flow test strip assembly as previously described, wherein the lateral flow test strip assembly may be substantially enclosed within the cartridge; an inlet in fluid communication with the input and configured to convey the sample to the input; and a viewing port disposed in a wall of the cartridge and configured to allow for observation of the one or more test zones of the lateral flow test strip assembly.BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Some embodiments of the present disclosure are hereinafter described, by way of example only, with reference to the accompanying drawings, wherein:

[0035] FIG. 1 shows a schematic side view of a prior art lateral flow strip with sample;

[0036] FIG. 2 shows an image of lateral flow strip membrane of the lateral flow strip of Fig. 1 with detection zones represented by an array of spots where each level of shading represents the intended presence of a different capture biomolecule;

[0037] FIG. 3 is a schematic view of a lateral flow strip assembly, according to some embodiments;

[0038] FIG. 4A is a close-up top view of a membrane area of a lateral flow strip, according to some embodiments;

[0039] FIG. 4B depicts a set of three similarly printed lateral flow strips with coloured spot capture zones (prior to use), according to some embodiments;

[0040] FIG. 5A depicts an image illustrating stages of running a lateral flow assay using a lateral flow strip, according to some embodiments, wherein strip (i) shows where the sample fluid has been added and reached the coloured spots, strip (ii) sample fluid has been added and the fluid flow front has reached the top of the lateral flow membrane and (iii) shows where the sample fluid has been added and the fluid flow front has carried all of the soluble dye to the end of the lateral flow membrane;

[0041] FIG. 5B shows the strips of Fig. 4A after the flow has completed and the coloured dye has been transported to the waste pad; strip (i) shows one row as a set of spots that have bound label particles as positive test results, strip (ii) shows another row of as a set of spots that have bound label particle spots, and strip (iii) shows a different set of test spots as one row as a set of spots that have bound label particles as positive test results;

[0042] FIG. 6A and FIG. 6B are images of a printed array layout at the design stage displayed in design software. Different levels of shading are intended to indicate intended spot locations for different types of capture biomolecules are to be printed;

[0043] FIG. 7 is a process flow diagram of a method of manufacturing a lateral flow test strip for detecting the presence or absence of a first target analyte within a sample, such as the lateral flow test strip assembly of FIG. 3;

[0044] FIG. 8 is a process flow diagram of a method of visually inspecting an unused lateral flow test strip, such as the lateral flow test strip assembly of FIG. 3; and

[0045] FIG. 9 is shown a process flow diagram of a method of detecting the presence or absence of a target analyte within a sample using the lateral flow test strip assembly of FIG. 3.DETAILED DESCRIPTIONLateral flow tests

[0046] FIG. 1 shows a typical prior art lateral or capillary flow test strip assembly 100, running a sandwich type assay as commonly used in rapid diagnostic applications. As illustrated, the lateral flow test strip assembly 100 includes an absorptive sample application or input pad 101, a conjugate pad 102, a lateral flow membrane 103, and a waste absorbing pad 104. These components are bonded by an adhesive layer onto a carrier strip 105 that may be constructed from plastic sheet, for example. The lateral flow membrane 103 is typically a microporous nitrocellulose membrane with pore sizes ranging from 3 to 20 m. The conjugate pad 102 is preloaded with conjugate biomolecules 106, which are bound to detectable label particles 107. When the flow strip assembly 100 is operated by the addition of a sample 110 containing the target analyte 111 (commonly in the form of an antigen or antibody), shown in this example as a droplet, the sample 110 containing the target analyte 111 flows by capillary wetting through the input pad 101 into the conjugate pad 102 where it mixes with the conjugate biomolecules 106 attached to detectable labels 107, (collectively, labelled conjugates). These labelled conjugates are present on the conjugate pad 102 and are capable of being absorbed and carried by the sample 110. Where the target analyte 111 is present in the sample 110, it binds with conjugate biomolecules 106 or complimentary conjugate biomolecules 106 of the labelled conjugates forming assemblies now attached to the detectable labels 107, which can be referred to as labelled conjugate assemblies.

[0047] The labelled conjugate assemblies (that is, target analyte(s) 111 bound to the complimentary conjugate biomolecules 106 of the labelled conjugates) are capable of being conveyed or transported with or within the sample 110 as it flows along the lateral flow membrane 103 by capillary action. As a result, they can encounter zones on the lateral flow membrane 103 that have immobilised or capture regions comprising test zones 109 of capture biomolecules 108. The capture biomolecules 108 also bind to the target analyte 111, and in doing so, hold the labelled conjugate assemblies in place within the test zones 109. Accordingly, the attached labels 107 of the labelled conjugate assemblies are captured within the test zones 109 and become detectable test results, such as test lines or test spots specific to detection of the target analyte 111 of interest within the sample 110.

[0048] The detectable label 107 may be a specific molecule or particle, such as a coloured particle for example, a gold nanoparticle, or a latex bead dyed with colour such as blue or carbon black, or by attachment of a fluorescent label such as a europium chelate complex. This may allow reading of the results by colorimetric and fluorescent detection systems respectively.

[0049] Where a number of detectable labels 107 marking the presence of the target analyte 111 within the sample 110 are immobilised at the test zone 109, such as a line or a test line, the test zone 109 will present or display a coloured or fluorescent line, indicating positive samples. While not strictly necessary, many lateral flow test strip assembly 100 will incorporate a second line which contains an antibody that picks up a secondary analyte always present sample to confirm the test has operated correctly (positive control). This is commonly referred to as a control line or control feature.

[0050] Time to obtain the final test result is often a key driver of these products. Tests can take as little as a few minutes to develop. There is a trade-off between time taken to perform the test and sensitivity, with more sensitive tests taking longer to develop. A key advantage of this type of prior art lateral or capillary flow test strip assembly 100, in comparison to other immunoassays, is the simplicity of the test which requires little or no sample and reagent preparation.

[0051] The lateral flow test strip assembly 100, once manufactured may be enclosed within a carrier (not shown) or cartridge (not shown), such as two-part plastic cartridge that encloses the lateral flow test strip assembly 100, but may also provide ports (not shown) for receiving a sample 110 and allowing for observation of the capture zones on the lateral flow test strip assembly 100.

[0052] FIG. 2 shows an example lateral flow membrane 103 (typically nitrocellulose) within the lateral flow test strip assembly 100 of FIG. 1. As mentioned above, the lateral flow membrane 103 of the lateral flow test strip assembly 100 has one or more immobilised regions comprising tests zones 109 where immobilised capture biomolecules 108 are located. These test zones 109 may comprise an array 202 of test spots, such as shown in FIG 2. Each spot 204 of the array 202 contains a capture biomolecule 108 for a specific target analyte 111. The spots 204 of the array 202 may include or correspond with test zones 109 for a range of different target analytes 111 where these spot types can be printed in replicates. For the purposes of illustration, the spots 204 are shown on the example lateral flow membrane 103 with different colours or shading patterns depicting different immobilised capture biomolecules 108 for binding to different target analytes 111. However, in practice, once regions of the lateral flow membrane 103 are printed with capturebiomolecules 108 to form the spots 204 or test zones 109, and allowed to dry, the spots 204 or test zones 109 are not visible or readily detectable from the lateral flow membrane 103. For example, in the case of a nitrocellulose membrane, the lateral flow membrane 103 appears all white. Visual inspection of the spots 204 or immobilised regions or test zones 109 of the lateral flow membrane 103 is then no longer possible prior to running of the tests.

[0053] The development of lateral flow test strip assemblies 100, particularly ones comprising an array 202 of many spots 204 and involving visual or fluorescent based detection, may result in significant quality control and inspection challenges for manufacturers. This is because the printed immobilised regions or test zones 109, such as lines or spots, are not visually observable on the lateral flow membrane 103 of lateral flow test strip assembly 100 prior to use. The lack of visual cues for inspection creates difficulties in assessing the quality of the printed test zones 109 in lateral flow test assemblies 100. As a result, alternative methods for quality assurance and / or quality control may be necessary, such a running in test, or testing, a selection of samples 110 from batch of lateral flow test strip assemblies 100 and from the results, making inferred assessments of the quality of all of the lateral flow test strip assemblies 100 in the batch. However, running a lateral flow test strip assembly 100 test destroys it for further use and testing only a selection of strips from a batch does not provide full assurance for all of the lateral flow test strip assemblies 100 of the batch.

[0054] Described herein is a method for manufacturing one or more capture or immobilised regions comprising test zones on a lateral flow strip assembly. The method involves creating a formulation by combining a coloured compound with specific properties with the colourless capture biomolecules and then printing this formulation mixture onto the lateral flow membrane of a lateral flow test strip assembly. The coloured compound is selected to be suspended or soluble in the solution to be used for the printing process, most typically, but not limited to an aqueous solution. The colourless compound is selected such that it dries in place along with the capture biomolecules in the area of the test zone on the lateral flow membrane of the lateral flow test strip assembly. In some examples, the mixture is printed in the form of an array of test elements or spots. The coloured compound is selected so as not to bind in place on the lateral flow membrane, but to be suitable to be re-suspended or re-dissolved when subsequently wet or wetted by a sample comprising a target analyte, and become mobile such that it is carried away by fluid flow of the sample. The capture biomolecule of the mixture, printed along with the coloured compound, isconfigured to bind to the lateral flow membrane or be immobilised where it is printed on the lateral flow membrane and not be carried away by fluid flow of the sample.

[0055] This process facilitates the clear visualisation or independent detection of the immobilised regions or test zones where one or more non-visible capture biomolecules are printed and colocated with coloured compounds during the combined printing process. This approach supports inspection and quality control (QC) procedures while lateral flow test strip assembly is still within production, is being assembled into cartridges, and right up until the time the lateral flow test strip assembly it is actually being used by a user applying a sample to an input or input pad of the lateral flow test strip assembly. The soluble or suspended compounds or dyes in the printing formulation that are co-located with the capture biomolecules 108 in the test zones are selected to be removed or displaced during the assay flow process, ensuring that they do not inhibit or disturb the final reading of the test results by the user or a test instrument once the sample has been added and is conveyed along the lateral flow membrane allowing the lateral flow test strip assembly to increase or decrease the level of the detectable label particles in the test zones dependent on the presence of the target analyte.

[0056] The method for manufacturing one or more immobilised regions comprising test zones on a lateral flow test strip assembly therefore produces an improved lateral flow membrane of a lateral flow test strip assembly, and accordingly, produces an improved lateral flow test strip assembly. The lateral flow test strip assembly according to described embodiments comprises a lateral flow membrane having one or more immobilised regions comprising test zones, each test zone comprising a non-visible capture biomolecule co-located with a soluble coloured compound. The non-visible capture biomolecule is configured to attach to the lateral flow membrane and to bind to a respective target analyte in a sample applied to the lateral flow test strip assembly, and in doing so, hold a labelled conjugate assembly (comprising the target analyte, complimentary conjugate biomolecule and attached label) in place within the immobilised region or test zone. However, the soluble coloured compound is configured to be detached, removed or displaced in response to the application of a fluid, such as that of the sample. Accordingly, as the sample is conveyed along the lateral flow membrane of the lateral flow test strip assembly, and comes into contact with the test zone, the labelled conjugate assembly is captured or immobilised by the non-visible capture biomolecule, whereas the soluble coloured compound is removed from the test zone by the fluid flow of the sample being conveyed along the lateral flow test strip assembly.

[0057] The present disclosure describes a method for performing quality control of such a lateral flow test strip assembly with one or more immobilised regions or test zones, according to some embodiments. This method enables the user to assess the presence or correctness of the non- visible capture biomolecule that has been applied or printed on the lateral flow membrane as part of the same manufacturing or printing process. Typically, the capture biomolecule is invisible on the lateral flow membrane once deposited, posing challenges in assessing the production quality without resorting to methods that test only some strips in batch of strips. For example, a faulty print may result in an incomplete or inadequate test zone such as a missing spot. If the coloured compound was not present, the non-visible nature of the capture biomolecules in the test zone would mean that this faulty print may go undetected by random sampling quality control processes.

[0058] Some embodiments of the present disclosure involve formulating soluble coloured compounds in a fluid carrier as a mixture or formulation along with the non-visible capture biomolecule that is to be printed. The manufacturing or printing process places this formulation mixture onto test zones on a lateral flow test strip assembly where a carrier fluid of the formulation can evaporate, leaving the non-visible capture biomolecules immobilised in test zone(s) and the coloured compounds also substantially co-located in the same zones. This approach allows for continuous quality control during the manufacturing process. By combining the soluble coloured compound with the non-visible capture biomolecule, a visual or otherwise observable indicator is created at the test zone. This provides a relatively straightforward means of evaluating the presence and distribution of the capture biomolecule 108 on the lateral flow membrane. Consequently, this facilitates real-time quality control by enabling immediate inspection and / or identification of the capture biomolecule on the lateral flow membrane, thereby ensuring the accuracy and reliability of the production process for later flow test strip assembly.

[0059] The space, position and / or size of the coloured test zone on a lateral flow test strip assembly according to the present disclosure can be validated by visually checking the test zone by eye or by subjecting the lateral flow strip assembly to a digital imaging and / or image analysis procedure, as may be executed by a computer executing computer code, for example, or any suitable instrument. In some embodiments, the coloured mobile water-soluble or water suspended coloured compound is combined with the capture biomolecule and applied or printed onto the lateral flow membrane as a coloured test zone. When the test zone has dried, the coloured compound is visible in the test zone.

[0060] Embodiments of the present disclosure include a method for detecting the presence of a non- visible capture biomolecule on a lateral flow test strip assembly. In use, a test operator adds a test fluid containing a sample, such as a fluid sample, and some cases test fluid in the form of a wash buffer, to the lateral flow test strip assembly. The test fluid enters the conjugate pad, where the target analyte in the sample binds with the conjugate biomolecules that are also attached to label particles forming a conjugate-target analyte-label particle assemblies or labelled conjugate assemblies. The test fluid then moves along the lateral flow membrane the via capillary action carrying the target analyte-label particle assemblies along with the flow, whereby it encounters the coloured test zone(s). In this flow process, the fluid flow re-suspends or dissolves the coloured compound and conveys or transports it away from the test zone and into a waste pad of the lateral flow test strip assembly. As the test fluid progresses, leaving the test zone(s) uncoloured, it also enables the carried conjugate-target analyte-label particle assemblies to encounter capture biomolecules in the tests zone(s) and become immobilised. As a result, the colour of the coloured compound has been removed cannot mask or be mistaken for the colour or detection of the visible or fluorescent label (if the label subsequently binds via the immobilised capture biomolecules in the area of the test region). In a positive test, the target analyte of interest binds to the printed capture biomolecule at the test zone(s) on the lateral flow test strip assembly. The capture biomolecule, bound to the lateral flow membrane, for example, a nitrocellulose membrane, remains relatively stationary as the sample (and buffer where present) flow through the lateral flow test strip assembly. The immobilised capture biomolecules can, for example, be antigens or antibodies. The detectable label particles or molecules may be a colloidal gold particles, fluorescent particles, or similar labels.

[0061] Referring again now to the prior art lateral flow membrane 103 of FIG. 2. As previously discussed, in such prior art lateral flow test strip assemblies, when spots are physically printed and dried, the capture biomolecules 108 are designed to be immobilised on the lateral flow membrane 103 and these spots are colourless and are not visible.Lateral flow test strip assembly

[0062] Referring to FIG. 3, there is shown a lateral flow test strip assembly 300, according to some embodiments. The lateral flow test strip assembly 300 is an unused lateral flow test strip assembly 300. In other words, a sample 310 comprising a target analyte 311 has not yet been applied or provided to the lateral flow test strip assembly 300 for testing.

[0063] The lateral flow test strip assembly 300 is substantially elongate and is configured to convey fluid from a first end 313 toward a second end 314. The lateral flow strip test assembly 300 may comprise an input 301, such as an input pad at or toward the first end 313. The input 301 is configured to receive a sample 310 comprising the target analyte 311 to be tested using the lateral flow test strip assembly 300.

[0064] The target analyte 311 may be an antigen selected from a group consisting of: protein, polysaccharide, lipid, or nucleic acid. The target analyte may be an antibody. The target analyte may be a molecule or chemical moiety.

[0065] The input 301 is in fluid communication with a conjugate pad 302 and is configured to convey or transport a fluid flow of the sample 310 to the conjugate pad 302. The conjugate pad 302 comprises one or more conjugate biomolecules 306 attached to respective detection labels 307. The conjugate biomolecule 306 is configured to bind to a target analyte 311, such as a complimentary target analyte 311, in the sample 310 applied to the input 301 to form a labelled conjugate assembly.

[0066] The detection label may be a coloured particle, a gold particle, a florescent particle or a magnetic particle.

[0067] The conjugate biomolecule 306 may be selected to be complimentary for binding to the target analyte. The conjugate biomolecule 306 may be selected from a group consisting of: antigens, antibodies, aptamers, and molecular recognition elements.

[0068] The conjugate pad 302 is in fluid communication with a membrane or lateral flow membrane 303. The lateral flow membrane 303 comprises a capture or immobilisation region 312 comprising one or more test zones 309. A first test zone of the one or more test zones 309 comprises a first soluble coloured compound 316 substantially co-located with a first non-visible capture biomolecule 308. The first soluble coloured compound 316 may be water-soluble. The first non- visible capture biomolecule 308 may be selected from a group consisting of: antigens, antibodies, aptamers, and molecular recognition elements.

[0069] In some examples, the first test zone exhibits at least one identifying characteristic selected from the group including: (i) a colour; (ii) a shape; (iii) a size; (iv) a relative position of the first test zone on the membrane. For example, the first test zone exhibits a circular shape. In some embodiments, at least one test zone 309 comprises a longitudinal shape extending along the lengthor width of the lateral flow test strip, such as a line, band, or strip. The test zone(s) 309 may be arranged in an array on the membrane 303.

[0070] The first non- visible capture biomolecule 308 is attached to the lateral flow membrane 303 and is configured to bind to a respective target analyte 311 of a labelled conjugate assembly in a sample 310 conveyed along the lateral flow test strip assembly 300, thereby immobilising the labelled conjugate assembly at the first test zone. The first soluble coloured compound 316 is configured to be removed from the first test zone by the fluid flow of the sample 310 applied to the input 301 and conveyed along the lateral flow test strip 300.

[0071] The lateral flow test strip assembly 300 may further comprise an end or waste pad 304 provided at or toward the second end 314. As the first soluble coloured compound 316 is washed away from the first test zone with a flow front of the fluid flow of the sample 310 and conveyed or transported to the waste pad 304.

[0072] When the first non-visible capture biomolecule 308 binds to a respective target analyte of a labelled conjugate assembly in the applied sample 310, the test zone 309 is configured to transition from a first visible state to a second visible state to indicate the presence of the target analyte in the sample. On the other hand, if the first non-visible capture biomolecule 308 does not bind to a respective target analyte of a labelled conjugate assembly in the applied sample 310, the test zone 309 does not transition to the second visible state. Instead, the test zone 309 may remain in the first state, indicating the absence of the target analyte 311 in the sample 310.

[0073] In some embodiments, the membrane 303 also comprises a control region 317 comprising one or more control zones 318. Each control zone 318 comprises an antibody configured to bind to a control analyte 319 that may be present in the sample 310. The control analyte 319 may be present in the test fluid comprising the sample 310 and the wash buffer. The control analyte 319 may be present in the buffer. When the antibody binds to a control analyte 319 in the applied sample 310, the control zone 318 is configured to transition from a third visible state to a fourth visible state to indicate the efficacy of the lateral flow test strip assembly 300. On the other hand, if antibody does not bind to the control analyte 319 in the applied sample 310, the control zone 318 does not transition to the fourth visible state. Instead, the control zone 318 may remain in the third state, indicating an ineffective test. For example, the test may be ineffective because the lateral flow test strip assembly 300 is faulty or because the user has not used it properly, for example, by not applying sufficient sample 310 to the lateral flow test strip assembly 300.

[0074] In some embodiments, the lateral flow test strip assembly 300 comprises more than one test zone 309. For example, the lateral flow test strip assembly 300 may comprise a second test zone 309. The second test zone may be positioned on the membrane at a location spaced apart from the first test zone. The first test zone may be positioned on the membrane in a location upstream of the second test zone. The first test zone and the second test zone may be positioned on the membrane in an interspersed arrangement.

[0075] In some embodiments, the second test zone 309 comprises a second soluble coloured compound 316 substantially co-located with a second non- visible capture biomolecule 308. For example, the second soluble coloured compound 316 and the second non- visible capture biomolecule 308 may be mixed together before being applied to the membrane 303. The first and second non-visible capture biomolecules are different from one another. Similar to the first non- visible capture biomolecule 308, the second non-visible capture biomolecule 308 is configured to attach to the membrane 303 and is configured to bind to a respective second target analyte 311 of a second labelled conjugate assembly in the sample 310 transported along the lateral flow test strip 300, thereby immobilising the second labelled conjugate assembly at the second test zone 309. The second soluble coloured compound is configured to be removed from the second test zone by a fluid flow of the sample applied to the input and transported along the lateral flow test strip. The first and second target analytes are different from one another.

[0076] In some embodiments, the input 301 is in fluid communication with the membrane 303 and is configured to convey or transport the fluid flow of the sample 310 to the membrane 303. The first non-visible capture biomolecule 308 is attached to the lateral flow membrane 303 and is configured to bind to the respective target analyte 311 in the sample 310 applied to the input 301 and transported along the lateral flow test strip assembly 300, thereby immobilising the respective target analyte 311 at the first test zone. The input 301 may also be in fluid communication with a conjugate biomolecule source, such as the conjugate pad 302, for example.

[0077] In this embodiment, the lateral flow test strip assembly 300 is configured to direct the one or more conjugate biomolecules 306 attached to the respective detection labels 307 along the membrane 303. The one or more conjugate biomolecules 306 are configured to bind to the respective target analyte 311, such as the complimentary target analyte 311, in the sample 310 applied to the input 301 to form the labelled conjugate assembly. The one or more conjugate biomolecules 306 may be configured to bind to the respective target analyte 311 after it is immobilised at the one or more test zones 309. The one or more conjugate biomolecules 306 maybe configured to bind to the respective target analyte 311 before it is immobilised at the one or more test zones 309.

[0078] The lateral flow test strip assembly 300, once manufactured may be enclosed within a carrier (not shown) or cartridge (not shown), such as two-part plastic cartridge that encloses the lateral flow test strip assembly 300. One or more input ports or inlets may be provided in the carrier (not shown) or cartridge (not shown) for receiving the sample 110. One or more viewing ports (not shown) may be provided in the carrier (not shown) or cartridge (not shown) for allowing for observation of the capture region 312 and / or the control region 317 on the membrane 303. In some embodiments, the lateral flow test strip assembly 300 is provided in a dipstick format.

[0079] Referring to FIG. 4A, there is illustrated a test zone 402 of a lateral flow membrane 400 of a lateral flow strip or lateral flow strip assembly, such as lateral flow strip assembly 300 (see FIG. 3), according to some embodiments. In this example, a formulation or solution comprising a soluble coloured compound, such as a water-soluble dye, and a non-visible capture biomolecule, is formulated and printed or applied onto the membrane 400 of the lateral flow test strip assembly. After the printing, the solution or formulation is allowed to dry on a surface of the membrane 400, where it can be visualised, in this embodiment as shaded spots.

[0080] Referring to FIG. 4B, there is shown a set of three separate lateral flow membranes 402B 1, 402B2, 402B3 of respective lateral flow test strip assemblies 400B 1, 400B2, 400B3. Here, it is demonstrated that a variety of different test zone 318 arrays or spot arrays can be produced, depending on the required application. In an array of spots, as exemplified, the location relationship between the spots and the presence of all of the spots in the array defines or can be used to identify each spot. This can be more important than the absolute position of each individual spot or the overall grid on the membrane 400A, 400B, 400C. The use of two or more different coloured compounds (represented in figures as different shading patterns) can be used to identify different spots with different capture biomolecules. Specific fixed reference spots can provide important advantages over use of a single-coloured compound in subsequent quality control and inspection steps, for example.

[0081] Referring to FIG. 5A, there is shown an image 500A of three separate lateral flow membranes of a lateral flow test strip assembly 502 such as lateral flow test strip assembly 300, according to some embodiments, which illustrates the stages of running a lateral flow test assay using the lateral flow strip assembly 500 by a user. In this example, a detection label (attached toa conjugate biomolecule provided on a conjugate pad of the lateral flow test strip 500) used is gold nanoparticle label particles, which are often referred to as colloidal gold label particles. These label particles are initially located in the conjugate pad and bound to a mobile conjugate biomolecule. The sample applied to the lateral flow test strip 502, which may be an aqueous sample fluid, carries the conjugated label particles within the flow. The fluid flow front can advance ahead of the conjugate bound colloidal gold particles being carried within the flow. The advancing fluid front can be observed washing away the soluble coloured compound (such as a water mobile, soluble or re-suspended coloured compound) used in the spot application or printing. In the later stages of the use of the assay, only the colloid gold-labelled capture analyte spots remain visible. These captured locations display the results for the array in each case. It is important to note that the soluble coloured compound (e.g., water soluble or water suspended coloured compounds or dyes) have been transported away and for this reason do not interfere with the reading of the target analyte bound to the capture molecule on the lateral flow array strip 500.

[0082] Referring again to FIG. 5A, the image 500A shows the printed array where sample fluid has been added to each strip in a succession, showing the early-stage flow front just arriving at the spot locations and progressing through the spot locations with soluble dye spots present. Strip (i), shows where the sample fluid has been added and the flow front has just reached the coloured spot locations and the coloured dye in the first rows of spots as dissolved into the sample fluid and is being carried forward by the flow. This image also shows where the colloidal gold label particles are also being carried along the lateral flow membrane with the flow, but lag behind the leading edge of flow. These particles are visibly red within the membrane as they flow. Strip (ii) shows where sample fluid has been added and the fluid flow front has reached the top of the lateral flow membrane, and the soluble colour dyes have been transported forward within the membrane. These dyes have moved away from the original printed spot capture biomolecule locations on the membrane and are approaching the waste pad. These dyes may appear as a blurred or bleeding line that is approaching the waste pad. At this stage the conjugate bound, sample analyte with colloidal gold label particles show up as a red region on the membrane as they are carried within the fluid flow and are starting to flow over the test zones, but insufficient label particles have accumulated at any spot location to show as positive test results. Strip (iii) shows where sample fluid has been added and the fluid flow front has carried all of the soluble dye to the end of the lateral flow membrane. The blurred or bleeding line of soluble colour dyes has travelled from the original printed spot capture biomolecule locations on the membrane and has reached the waste pad. Theflow has had more time for the conjugate bound sample analyte with bound label particles to start to accumulate and one row of spots with positive result are just becoming visible.

[0083] Referring to FIG. 5B, there is shown an image 500B of an embodiment of the printed array, where the same strips 502 as in FIG. 5A have had more time for the flow to complete and the test result to stabilise. The flow has completed allowing all of the coloured dye to be transported to a waste pad of the lateral flow strip 502 and most of the remaining colloidal gold marker particles that are not bound to test zones to also clear the membrane area and be absorbed into the waste pad. The colloidal gold particles that have been absorbed into the waste pad may appear as a stain on the waste pad. The strip result has stabilised where only the colloidal gold that has been selectively bound to some spot locations is visible for the three test cases. Strip (i) shows one row as a set of spots that have bound label particles as positive test results. Strip (ii) shows a faint signal for a different row of spots that have bound label particles as positive test results for a different analyte. Strip (iii) shows a different set of test spots as one row as a set of spots that have bound label particles as positive test results.Lateral flow matrix design

[0084] A lateral flow test assay assembly 300 can be designed for the simultaneous detection of multiple target analytes 311 from a sample 310. In some embodiments, the design includes a nitrocellulose membrane with a printed configuration being an array of spots. Referring to FIG. 6A, an array configuration of multiple spots has the advantage that it can support increased multiplexing of different target analyte 311 detection capabilities without requiring an increase in sample volume or dilution of the sample 310. The array may comprise at least one test zone 309 or spot. In some embodiments, the array comprises a diagonal arrangement of test spots 309. In some embodiments, the array comprises a regular grid arrangement of the test spots 309. In some embodiments, each spot in the array may comprise a specific capture biomolecule 308, such as an antigen, antibody, aptamer, or another molecular recognition element, each capable of binding to a distinct target analyte 311. The array is typically structured with multiple sets of test zones 309 or test spots. Each set of spots may be configured to target a specific target analyte. The multiple sets of test spots may be positioned or located alongside at least one set of control zones 318 or control spots. In some embodiments, each test spot targets a particular target analyte 311 of interest, enabling simultaneous testing of various analytes in a single test assay. In some embodiments, 30 to 50 analytes may be tested simultaneously, wherein the test assay comprises a corresponding number of test spots. In other embodiments, a set of test spots can be used for aspecific target analyte 311. This approach using replicates allows averaging or other algorithms to be applied to the replicates reducing the overall variance in the test result or provide an improved reliability of the test result, such as reduce false positives or reduced false negatives. In some embodiments, control zones 318 spots are interspersed within the array to act as position references or quality controls for the assay. A significant advantage of this design is its multiplexing ability with the detection of multiple targets within a sample 310. This feature is especially valuable in medical diagnostics, where limited sample volumes necessitate comprehensive testing. The spot array format substantially increases the number of detection sides on a single strip while maintaining a compact form factor and utilising the available sample fluid flow volume.

[0085] In some embodiments, the lateral flow test assay assembly 300 utilises lines for the test zones 309 and / or control zones 318. In other embodiments, spots or an array of spots can be used. The advantage of using an array or grid of spots is that it allows for additional multiplexing of multiple different target analytes 311 within a single lateral flow test strip assembly 300. Additionally, the use of replicates for each target analyte 311 can enhance test reliability, reducing variance or the coefficient of variation (CV). Furthermore, digital analysis of multiplex lateral flow test arrays can be conducted using lateral flow reader instruments. These instruments capture a digital image of the test strip and utilises image analysis software to evaluate the array of spots for multiple multiplexed test results. This approach can also allow more complex algorithms to be implemented within the software of the reader instrument where the test result can be made up of multiple replicates for multiple target analyte 311 types and multiple control and position reference spot results.

[0086] In some embodiments, a single spot location is used to capture a biomolecule 308, (and associated coloured compound). In some embodiments, multiple spots for each capture biomolecule 308, (and associated coloured compound) are printed in the array to form replicates. Replicates can be used to improve test reliability and reduce test variance. Capture biomolecules 308 can serve as either the defining capture substances in the sample 310 or as control analytes for the test. The lateral flow test strip assembly 300 may comprise an array of detection spots (not shown) deployed on the membrane 303. For example, the array of detection spots may be deployed within a region of the test strip in which the test and control zone 318 spots are located. The array of detection spots (not shown) may form a quadrilateral shape. There may be four detection spots. For example, a detection spot may be positioned toward or at each comer of the quadrilateral. A detection spot may be positioned on or toward each corner of membrane303. The detection spots (not shown) may be used to determine a relative location of the test zones 309 to ensure correct reading of the test zones 309 by image processing or machine vision techniques.

[0087] The simplicity and reliability of the lateral flow test array format can be extended by using array and spot patterns, which may provide higher functionality and more test outputs than may be achieved with lines alone. There is a practical limit to the number of lines that can be run in series across a flow strip without degrading the flow and capture efficiency of successive lines. Arrays of spots effectively overcome this limitation. In some embodiments, the spots are between 0.05 mm to 5 mm in diameter. In some embodiments, the array spots are 0.5 mm in diameter printed on a lateral flow membrane 103 that is assembled as a test strip with a width of 6 mm. The lateral flow membranes 103 may be composed of nitrocellulose membrane, however, alternative substrates are commonly used and known to the skilled person. In some embodiments, the membranes or strips are between 2 mm to 12 mm in width.

[0088] Referring to FIG. 6B, in some embodiments the lateral flow test array 300 may comprise an offset grid arrangement of the test spots. The offset grid arrangement may comprise a first row of the test zones 309, wherein the first row comprises a plurality of first separations that respectively space apart adjacent ones of the test zones 309 in the first row. The offset grid arrangement may further comprise a second row of the test zones 309, wherein the second row comprises a plurality of second separations that respectively space apart adjacent ones of the test zones 309 in the second row. The first row and the second row may be offset in a longitudinal direction of the lateral flow test strip array 300 substantially corresponding to a flow direction from the input 301 and conjugate pad 302 to the waste pad 304. Each of the second separations may be laterally aligned with a test zone 309 in the first row so that a test zone 309 in the second row is exposed to the flow direction. The array comprises at least one cluster of the test zones 309. Each one of the first separations may vary in size. Each one of the second separations may vary in size.

[0089] In some embodiments, each spot is printed with a unique capture biomolecule 303 type for binding of a target analyte 311. Since not all spots are in series (whereby the spots are offset from each other) and as several spots are printed across the strip width, each spot only occupies a small area within the overall flow width. This format facilitates efficient multiplexing of a large number of target analytes 311 and allows for identification and quantification of individual target analytes 311. This use of arrays in this manner addresses the demands for higher sensitivity, increased multiplexing capabilities, and reduced sample volume in lateral flow tests.

[0090] There is no specific limitation to the geometric design of the array spot layout in the lateral flow test arrays. The layout may be a regular grid, or it can adopt other configurations including grouped patterns, clusters forming identifiable shapes, or even random or pseudo-random arrays. In some embodiments, the spot locations must be identifiable to the reading and image analysis system to accurately interpret the individual target analyte test outcomes and report overall test results. Utilising diagonal row of spots, also referred to as an offset grid layout, offers advantages in lateral flow test arrays 300. This design may better utilise the flow width for spot placements and may enable higher density printing of spots or layouts that improve sample flow over the spots. Such an arrangement may be particularly beneficial for diagnostics requiring multiple analytes to be tested simultaneously within a single assay, for example, 30 to 50 analytes. The advantages of this approach can include enhanced sensitivity, reduced sample volume requirements, and / or the capability to detect a broader range of analytes in a single test. The offset grid design may minimise cross -reactivity between adjacent detection zones, thereby improving the accuracy of the test and / or reducing the likelihood of false positives. Additionally, this layout may allow for increased exposure of the test zones 309 to target analyte 311 within the sample 310 fluid to the flow lines.

[0091] In further embodiments, the target analyte 311 array may be printed on a functionalised glass or plastic surface within a plastic test cartridge. This setup utilises a combination of capillary flow and pressure assisted flow to drive the test sample, conjugate and buffer reagents across the array. This process results in the formation of a similar array of test zones 309, as seen in diagnostics tests. This advancement allows for more efficient and precise testing, leveraging the unique properties of the functionalised surface and fluid flow in the cartridge to enhance test performance.

[0092] In some embodiments, the total print volume or number of droplets deposited within a specific test spot area can be used to adjust the concentration of the capture biomolecules 103 that will be bound within the spot area. This concentration can be expressed as the number of capture biomolecules 308 per unit area within the test zone 309. This process adjusts the overall concentration of the capture biomolecules 303 immobilised in the spot area and can be used to adjust the response of the spot area to sample analyte and label particles.

[0093] In printing different concentrations, the colour density of coloured print fluid formulation will also change, with a higher concentration having a darker colour density for the spot area once it has dried in location. The colour density of the spot can be used to confirm or calibrate the response of the spot prior to the test being run. Using this method, spot replicates can be printedwhere specific spots in the set of replicates can be printed at different capture biomolecule 308 (and associated colour compound concentrations). This use of spots at different concentrations can be used to extend the dynamic range of the test system when it is intended to measure the concentration of the target analyte 311. Since each concentration of capture biomolecules 308 on a spot will have different optimal range for measurement of target analyte 111 concentration, printing a number of spots with different concentrations can provide an improved measurement of target analyte 311 concentration over a wide range of concentrations.

[0094] In some embodiments, a reader instrument may be used to measure the different concentrations. The instrument can first use digital imaging and image analysis in software to read the position, colour and / or the colour density of each spot in the dry unused lateral flow strip array to determine or confirm the printed concentration. The test is then run by applying the sample 310 and allowing time for the fluid to flow and transport away the coloured compounds and selectively bind the detectable labels at the spot capture locations. The instrument software algorithm can use the colour information for spot locations and concentrations from the first image to provide an improved test outcome calculation of the target analyte 311 and target analyte 311 concentration detected on the test strip from the second image that shows the concentration of the label particles captured at each spot, after the colour compounds have been washed away.Lateral flow matrix printing

[0095] Referring now to FIG. 7, there is shown a process flow diagram of a method 700 of manufacturing a lateral flow test strip assembly 300 for detecting the presence or absence of a first target analyte within a sample 310. For example, method 700 may be used to produce lateral flow test strip 300 described above with reference to FIG. 3.

[0096] At 702, a first soluble coloured compound 316 is combined or mixed with a first non- visible capture biomolecule 308 to form a first formulation. The first non-visible capture biomolecule 308 is configured to bind to a respective first target analyte 311.

[0097] At 704, the first formulation is applied to one or more first locations of a lateral flow membrane 303 of the lateral flow test strip assembly 300 to form respective one or more first test zones 309. In some embodiments, applying the first formulation to the one or more first locations of the lateral flow membrane 303 may comprises printing the first formulation onto the lateral flow membrane 303. For example, applying the first formulation to the one or more first locations of the lateral flow membrane 303 may comprise applying the first formulation in droplets to the oneor more first locations. In some examples, each droplet may form a spot. Each spot may correspond with a respective test zone 309.

[0098] When a first labelled conjugate assembly (the first labelled conjugate assembly comprising the first target analyte 311 bound to a first conjugate biomolecule 306 attached to a first detection label 307) comes into contact with the one or more first test zones 309, the first non- visible capture biomolecule binds 308 to the first target analyte 311, thereby immobilising the first labelled conjugate assembly at the one or more first test zones 309. When the first soluble coloured compound 316 comes into contact with a fluid flow of a sample 310 comprising the first labelled conjugate assembly, the first soluble coloured compound 316 is removed from the one or more first test zones 309 by the fluid flow.

[0099] In some embodiments, the method 700 further comprises allowing the first formulation at the one or more first locations of the lateral flow membrane 303 to dry.

[0100] In some embodiments, multiple droplets are applied to one or more of the one or more first locations on the lateral flow membrane 303 to thereby increase the concentration of the first non- visible capture biomolecule 308 and to increase the colour concentration of the first soluble coloured compound 316 in the one or more first test zones 309.

[0101] A second formulation may also be applied to the lateral flow membrane 303 to produce the lateral flow test strip assembly 300. For example, a second soluble coloured compound 316 may be combined with a second non-visible capture biomolecule 308 to form a second formulation. The second non-visible capture biomolecule 308 is configured to bind to a respective second target analyte 311. The first and second non-visible capture biomolecule 308 may be different from one another. The first and second target analytes 311 may be different from one another. The second formulation may be applied to one or more second locations of a lateral flow membrane 303 of the lateral flow test strip assembly 300 to form respective one or more second test zones 309. The second locations may be different to the first locations.

[0102] When a second labelled conjugate assembly (the second labelled conjugate assembly comprising the second target analyte 311 bound to a second conjugate biomolecule 306 attached to a second detection label 307) comes into contact with the one or more second test zones 309, the second non-visible capture biomolecule binds 308 to the second target analyte 311, thereby immobilising the second labelled conjugate assembly at the one or more second test zones 309. When the second soluble coloured compound 316 comes into contact with a fluid flow of thesample 310 comprising the second labelled conjugate assembly, the second soluble coloured compound is removed from the one or more second test zones 309 by the fluid flow.

[0103] In some embodiments, the present disclosure describes a method for printing the mixture of the dissolved or suspended coloured compound and the non-visible capture biomolecules 308 within, for example water-based, carrier a fluid to form spots in a multitarget analyte array on lateral flow test strip assembly 300. The printing method can use any known method for depositing controlled small volumes of the fluid mixture to form printed zones on the lateral flow membrane 303 of the lateral flow test strip assembly 300. Known printing methods include but are not limited to droplets, pressure assisted droplets controlled by a solenoid valve, or fluid pressure or air pressure assisted spraying, pumped jetting or droplets piezo ink-jet printing or contact printing or striping. In one embodiment, a solenoid valve is used to control the release of a pressurised fluid mixture, where the time of electrical signal opening the solenoid valve is used to control the volume of the expressed fluid volume that is released as a spot onto the lateral flow membrane 303. For example, a commercial value can be used for this embodiment, such as a VHS series 2-way dispense valve manufactured by the LEE Company, Westbrook, United States. This is 6 mm dispensing valve that combines inkjet printing technology with inert materials to achieve precision droplets in the nanolitre to millilitre range. The valve has an ultra-fast response time (as fast as 250 ps) suitable for accurate fluid regulation and dispensing repeatable droplets.

[0104] Volumetric dispensing of the non-visible capture biomolecule 308 and colour compound formulation using a solenoid valve is typically through a narrow metallic or ceramic nozzle using non-contact droplet printing method of the with the droplets contacting the lateral flow membrane 303 as they are formed. The size of each test zone 309 spot that is formed is controlled by adjusting the dispensed volume, which can range from as low as InL up to 500nL. Spots may be formed by a single droplet or by printing multiple droplets at the same location to deposit more volume at the spot location.

[0105] In some embodiments, a piezo type inkjet printing nozzle such as the MD-K-130 microdrop dispenser head, manufactured by Microdrop Technologies GmbH, Norderstedt, Germany can be used to print the test zones on the lateral flow membrane 303. Microdrop Dispenser Heads are based on piezo-driven inkjet printing technology. The integrated piezo actuator induces a shockwave into the fluid contained in the head, which causes a droplet to be emitted from the nozzle. Piezo inkjet print nozzle droplet volumes are as small as 20pL to 200pL and larger spot volumes can be created by deposition multiple spots.In an embodiment, the spot area created on the lateral flow membrane 303 from a single droplet may be only 0.05 mm in diameter or smaller. Larger spot areas sizes can then be created by moving the print head in a localised pattern while dispensing multiple droplets to create a final spot that may have a larger area, for example, a circular spot with a 0.2 mm diameter.Quality control

[0106] Referring now to FIG. 8, there is shown a process flow diagram of a method 800 of visually inspecting an unused lateral flow test strip assembly 300. In other words, a sample 310 comprising a target analyte 311 has not yet been applied or provided to the lateral flow test strip 300 for testing. For example, the lateral flow test strip assembly 300 may be the lateral flow test strip assembly 300 of FIG. 6.

[0107] At 802, a coloured region on the lateral flow test strip assembly 300 is determined or identified. The coloured region is indicative of a location of a first test zone 309 comprising a first soluble coloured compound 316 and a first non- visible capture molecule 308.

[0108] At 804, the first test zone 309 is inspected for at least one identifying characteristic. For example, the visual inspection can be performed by a human, that is “by eye’ . Alternatively, or in addition image processing or machine vision techniques may be used to inspect the first test zone 309 for the at least one identifying characteristic.

[0109] At 806, based on the at least one identifying characteristic, (i) determining or identifying whether the associated capture biomolecule zone and its location and relationship to other zones on the strip is compatible with a sample to be tested; and / or (ii) determining if the lateral flow test strip assembly 300 satisfies one or more quality control parameters. For example, the at least one identifying characteristic is presence or absence of colour of the first test zone 309. The identifying characteristics may comprise one of more of: (i) a colour of the first test zone 309; (ii) a shape of the first test zone 309; (iii) a size of the first test zone 309; (iv) a relative position of the first test zone on the lateral flow membrane 303; (v) a size and position of the first test zone 309 on the lateral flow membrane 303 with respect to the size and position of other further test zones 309 of the lateral flow membrane 303.

[0110] In some embodiments, a concentration of the first non-visible capture biomolecule 610 is determined based on a determination of a colour concentration of the first test zone 303.

[0111] The printing of non-visible capture biomolecules 308 on lateral flow test assay assembly 300, whether visual or fluorescent-based, presents challenges in process control and inspection. Printed protein capture biomolecules 308 lack colour at the time of printing onto the lateral flow membrane 303. The printed non-visible capture biomolecules 308 on the lateral flow membrane 303 only acquire colour during use of the test strip when the conjugated detection particles, such as colloidal gold, are bound to the non-visible capture biomolecules 308 via the target analyte 311 to indicate a positive test. Consequently, these printed biomolecules 308 are not immediately observable post-printing. A similar scenario occurs with fluorescence labels, where the capture biomolecules 308 remain colourless and do not exhibit fluorescence after printing. This aspect of lateral flow test strip assembly 300 manufacturing necessitates specific control in an effort methods to ensure effective and reliable operation of lateral flow assays.

[0112] The present disclosure describes a method of combining distinct coloured compounds with the solution used to print each non-visible capture biomolecule 308, resulting in each non-visible capture biomolecule 308 printed area also displaying a unique spot colour when dried, prior to use. This approach of integrating coloured dyes with the analyte solution at the time of spot printing offers significant advantages in the printing process, inspection, and quality control compared to existing methods. In the prior techniques, a coloured compound or dye may be used initially to test the printer, which is then removed before printing the colourless analyte. However, this approach does not inspect the actual printing of the test analyte itself. Typically, a selected number of strips from each batch are tested with control materials that represent sample material, allowing for the detection of the intended visual-colorimetric or fluorescence test labels. These tested strips are then analysed using statistical techniques to assess the reliability of the entire batch. However, this method is destructive, consumes the tested strips, and does not guarantee that every test region on every strip is printed correctly and in the correct location. By contrast, the new method provides a non-destructive, more reliable, and comprehensive way to ensure the accuracy of printing on each strip.

[0113] In some embodiments, each capture biomolecule 308 can be identified by a distinct colour, facilitating the tracking of individual capture biomolecules 310 during the manufacturing process. For example, capture biomolecule A 308 may be marked with a blue colour compound, while capture biomolecule B may be identified by a green colour compound. In some embodiments, the lateral flow test strip assembly 300 comprises a first test zone 309 having a first colour, and a second test zone 309 having a second colour. The first test zone 309 may be part of a set of firsttest zones 309, and the second test zone 309 may be part of a set of second test zones 309. The strip may comprise at least two test zones 309, wherein each test zone 309 has a different colour to assist with identification the capture analyte at the respective test zone 309. The colour-coding ensures that the correct analyte is applied to each test strip and position, preventing errors such as the application of the wrong analyte, omissions, or insufficient quality that could impact the readability and correct operation of the test strip by a user or reader instrument interpreting the test.

[0114] Some coloured compounds can be configured to remain immobilised or bind in location when printed and dried as spots on the lateral flow membrane 303. In some embodiments, the spot colour will be permanent and will not be washed away by the sample 310. These permanentcoloured spots are topically printed as coloured compounds only with no capture biomolecules present. These permanently coloured spots are used to provide fixed, always present fiducial markers to reference the array locations. The provision of permanent fiducial markers means that even when the coloured compounds have been washed away, the array layout can still be interpreted from the permanent printed spots.

[0115] The incorporation of coloured water-soluble compounds or dyes aids in quality control measures within the manufacturing processes of lateral flow tests. These coloured, water soluble compounds or dyes can be combined with colourless analytes prior to printing of each spot on the lateral flow strip. Alternatively, they can be added during the printing stage, either mixed with the analyte or applied as an overlay. In another embodiment, the coloured, water soluble compound or dye is added after the print spots have dried. The cartridge assembly or test packaging process includes multiple stages for visual inspection and quality control, suitable for digital image acquisition and software -based image analysis.

[0116] In some embodiments, digital images of the dried formulated analyte are captured, and analysed using image analysis software. Machine vision methods inspect various properties of each printed spot and the overall array layout on the strip, such as the spot position along the strip and proximity to the strip edge, against established acceptance and rejection criteria.

[0117] In quality control processes, digital imaging and image analysis assess parameters like colour analysis, object counting, position determination, shape recognition, and / or size measurements. Additional quality control tests include characterising and measuring spot diameter, spot dose, spot form, circularity and uniformity, spot location on strip, presence ofsatellite droplets or splatter, detection of partially printed or misformed spots, for example, forming a ring or crescent and not a full circular spot. For example, spot location is determined by each spot being correctly located within the array relative to other spots, and the array of spots is within the correct region limits on the strip. Spot dose is determined by the correct colour density, ensuring the printed spot has the correct fluid volume and is not partially filled. All these measurements and parameters are assessed against established pass criteria as part of the quality control limits.

[0118] Imaging of coloured water-soluble dyes combined with the capture analyte can significantly aid in quality control processes in lateral flow test manufacturing. Quality control imaging is capable of detecting a wide range of errors and issues in the lateral flow printing process. These include missing analyte dots, incorrect size and volume, incorrect position, artifacts like splattering, poor quality of the analyte, and inadequacy of reprints. When the control failures occur, the affected test can either be discarded or remedied through repeated deposition of the target analyte onto the lateral flow test strip assembly 300. If an error is detected on the lateral flow test strip assembly 300 is irreparable or indicates a significant problem, the test is discarded. Conversely, if the issues are correctable, the capture biomolecule can be re-deposited to rectify the quality control issue, thereby ensuring accurate results.

[0119] In some embodiments, quality control algorithms are used to establish pass criteria based on individual spots, averages of spots and variance in spot characteristics. Quality control algorithms facilitate determination of whether individual spots or the overall quality of a sample 310 meet predefined standards. The criteria consider factors such as the characteristics for individual spots, the average of multiple spots, the variation in spot attributes. These algorithms aid users in automating the quality control decision-making process. In one embodiment, machine vision technology is used to automatically inspect the printed array. This inspection process checks for issues such as missing analyte dots, incorrect size and / or volume, misplaced spots, artifacts like splattering, poor quality of the capture biomolecule 308, and inadequacies in reprints, ensuring comprehensive quality assurance in the manufacturing of lateral flow test strip 300.

[0120] In some embodiments, additional coloured spots can be used as markers to define the dimensions of the strip, aiding in the cutting process to ensure that the array is accurately positioned on each strip. The cutting mechanism can be either manual, performed by the user, or an automated process known to those skilled in the field. In certain embodiments, these spots act as markers fora laser cutter, indicating where to cut the strip. This allows the laser to precisely cut the strips while they are still positioned in the printer.

[0121] The incorporation of coloured water-soluble compounds or dyes can significantly enhance quality control processes integrated into the execution of a lateral flow test. These coloured spots, present on the strips for final use, play a crucial role in the quality control during the lateral flow test procedure. The lateral flow strips may be designed as dip strips or assembled into cartridges. In the final stage of packaging or shipping, the assembled strip or cartridge array can be visually inspected by a user or automatically examined through machine vision. For example, array strips can be analysed using a reader instrument, such as the Axxin AX-2X-S. This instrument has multispectral capabilities and can confirm that the spots are correctly placed.

[0122] The use of coloured compounds or dyes within each spot enhances quality control and testing capabilities within reading instruments for lateral flow tests. These instruments are designed to capture an image of the detection region, with image analysis software analysing the spots. In one embodiment, the instruments allow for the insertion of the cartridge, positioning the detection region of the strip or cartridge window within the field of view of the internal image sensors, while keeping the sample addition port external to the instrument. In this configuration, the operator starts the test on the instrument with a new, unused cartridge in place. The instrument captures images of the detection area and validates the correct placement of the spots before the test is started. This validation ensures that the cartridge is unused, as indicated by the presence of the coloured spots, which would otherwise be washed away.

[0123] The instrument’s software and user interface can guide the user to add the sample 605 or sample buffer solution to the sample port and, if necessary, additional wash buffer. The instrument can capture multiple images over time to monitor the sample addition and the flow of the sample fluid through the lateral flow membrane 303. As the non-binding soluble dyes dissolve and are washed away by the sample, the instrument can in software analysis of captured digital images and incorporate analysis of these changes to confirm the test has run correctly and incorporate this information into the overall test result. This analysis can verify that adequate sample is added and that there is sufficient fluid flow through the lateral flow membrane 303, ensuring the test operation is valid and within the limits for displaying a correct test result. Additionally, if the instrument uses monochromatic detection, such as green absorption for detecting colloidal gold, the presence of red or other coloured non-binding soluble dyes can still be detected by visual absorption within the sample limited spectral range.Using the lateral flow test strip to detect the presence or absence of a target analyte within a sample

[0124] Referring now to FIG. 9, there is shown a process flow diagram of a method 900 of detecting the presence or absence of a target analyte 311 within a sample using the lateral flow test strip assembly 300 of FIG. 3.

[0125] At 902, a sample 310 is applied to the lateral flow test strip assembly 300. The lateral flow test strip assembly 300 is configured to convey or transport the sample 310 to at least the first test zone 320 of the lateral flow membrane 303 of the lateral flow test strip assembly 300.

[0126] At 904, the sample 310 is allowed to remove, or causes removal of, the first soluble coloured compound 316 from the first test zone 309, and may for example, convey or transport the first soluble coloured compound 316 to the waste pad 304.

[0127] At 906, after a first time interval, responsive to determining that the first test zone 309 has transitioned from a first visible state to a second visible state, determining the presence of the target analyte 311 in the sample 310, and responsive to determining that the first test zone 309 has not transitioned from the first visible state to the second visible state, determining the absence of the target analyte 311 in the sample 310.Coloured compounds

[0128] The addition of coloured compounds or dyes to the capture biomolecule 308 solution printed on test zones 309 can aid in quality control in lateral flow tests. In some embodiments, the colour compound or dye is water soluble or suitable for suspension and transport in a water-based solution. These coloured compounds or dyes are selected for their ability to dry alongside the printed capture analyte while not binding to the substrate membrane material and retaining water solubility or capacity to be re-suspended into the aqueous flow after drying. A number of substrate membrane materials are known to the skilled person in the art. For example, a typical lateral flow membrane 303 is nitrocellulose. Other types of membranes or surfaces that support the required fluid flow of the sample and conjugate are known to the skilled person. The capture analytes are configured to remain in place on the lateral flow membrane 303, ensuring they are not washed away by the sample 310 or by the test fluid (comprising the sample and wash buffers that carry the sample and conjugated detection analyte and detection molecule) through the lateral flow membrane 303 over the test zone 309.

[0129] The coloured compound is configured to be washed away with the flow front of the sample fluid, migrate through the lateral flow membrane 303, and be transported past the test zones 309 and into the waste pad 304. As a result, the coloured compounds are removed during the use of the test, leaving the test strip free of these dyes in the detection regions. This process ensures that upon completion of the test, only the captured colorimetric or fluorescence labels are observable. The initial printed dyes, having been removed by the flow front, are no longer present in the vicinity of the detection zones and therefore cannot be mistaken for a captured visible label (to falsely indicate a positive test) or interfere with the detection of the intended label particles in the test zones 320.

[0130] The coloured water-soluble compounds or compounds suitable to be suspended within a water-based carrier fluid refer to a class of substances that readily dissolve or suspend in water and impart distinct hues or pigments to the solution. These compounds are characterised by their ability to disperse uniformly in the aqueous carrier fluid, but after printing and evaporation of the carrier fluid, create visually observable colours or enhancements. These properties in water allows for incorporation into a wide range of products and processes, facilitating tasks such as colouring, staining, marking and visual detection. For example, food dyes are food grade water-soluble dyes that are used to colour various food and beverages. Examples include Red 40 (Allura Red), Blue 1 (Brilliant Blue) and Yellow 5 (Tartrazine). In certain embodiments of the present disclosure, soluble coloured compounds do not interact with either the capture analyte, target analyte or the test membrane or matrix. Non-limiting examples of water-soluble compounds suitable for incorporation into the printing solution for the lateral flow membrane are erioglaucine, tartrazine, and fast green, for instance. A variety of other water-soluble or water suspended coloured compounds can be employed for this purpose.

[0131] Coloured compounds can be configured to soluble or suspended in the water-based printing mixture but bind or become immobilised once they are absorbed into the printed spot area of the lateral flow membrane 303. One way to achieve this is to setup the coloured compound as particles or are chemical substances that possess distinct colouration but do not readily dissolve or mix with water. These compounds exhibit a visual colour or pigmentation when observed but remain insoluble in aqueous solutions. They are typically characterised by their inability to form a homogenous solution when added to water. Instead, they tend to separate and settle as solids or particulates in water. Insoluble coloured compounds do not wash out when the test is run and define particular characteristics of the final test readout or of the array format itself. Insoluble coloured compounds can be used to define fixed array dimensions, assist with gridding andreading. Insoluble coloured compounds can be used to define fixed analyte responses such as quantification standards or other forms of comparator. The insoluble coloured compounds may bind to the test analytes or to test matrix directly. Insoluble coloured compounds can comprise of dyes or other coloured particles. Non-limiting examples of water-insoluble compounds are colloidal gold, silver, latex, carbon, europium, fluorophores, and quantum dots. A skilled person in the field would recognise that a variety of water insoluble coloured compounds can be employed for this purpose.

[0132] In some embodiments of the present disclosure, different coloured dyes can be used to distinguish various protein types within the printed array spots for visual inspection and quality control procedures. For example, array spot layout for a malaria test may be configured differently to a respiratory viral test so that it can be identified or validated within the instrument for test type. In further embodiments, the proteins may be printed in replicates, further enhancing the quality control procedure. The combination of multiple colours and replicates may not only simply the identification of proteins but also allow for the detection of anomalies or inconsistencies in the printed array strips.

[0133] In some embodiments of the present disclosure machine visions systems can be used for automated inspection of the lateral flow test strip assembly 300. The machine vision system is designed to analyse the lateral flow strips systematically, capturing high-solution images, and processing data with accuracy. It can identify, classify, and quantify printed array spots, making it an ideal tool for quality control processes and rapid diagnostic applications.

Claims

CLAIMS1. A lateral flow test strip for detecting the presence or absence of a first target analyte within a sample, the lateral flow test strip comprising: an input configured to receive the sample; a conjugate pad in fluid communication with the input, the conjugate pad comprising a conjugate biomolecule attached to a detection label, wherein the conjugate biomolecule is configured to bind to a first target analyte in a sample applied to the input to form a first labelled conjugate assembly; a membrane in fluid communication with the conjugate pad, the membrane comprising one or more test zones, wherein a first test zone of the one or more test zones comprises a first soluble coloured compound substantially co-located with a first non-visible capture biomolecule, wherein the first non-visible capture biomolecule is attached to the membrane and is configured to bind to the first target analyte of a first labelled conjugate assembly in a sample transported along the lateral flow test strip, thereby immobilising the first labelled conjugate assembly at the first test zone, and wherein the first soluble coloured compound is configured to be removed from the first test zone by a fluid flow of a sample applied to the input and transported along the lateral flow test strip.

2. A lateral flow test strip for detecting the presence or absence of a first target analyte within a sample, the lateral flow test strip comprising: an input configured to receive the sample; a membrane in fluid communication with the input, the membrane comprising one or more test zones, wherein a first test zone of the one or more test zones comprises a first soluble coloured compound substantially co-located with a first non-visible capture biomolecule, wherein the first non-visible capture biomolecule is attached to the membrane and is configured to bind to a first target analyte in a sample applied to the input and transported along the lateral flow test strip, thereby immobilising the first target analyte at the first test zone, wherein the lateral flow test strip is configured to direct a conjugate biomolecule attached to a detection label along the membrane, wherein the conjugate biomolecule is configured to bind to the first target analyte to form a first labelled conjugate assembly, andwherein the first soluble coloured compound is configured to be removed from the first test zone by a fluid flow of a sample applied to the input and transported along the lateral flow test strip.

3. The lateral flow test strip of claim 2, wherein the conjugate biomolecule is configured to bind to the first target analyte after it is immobilised at the first test zone.

4. The lateral flow test strip of claim 2, wherein the conjugate biomolecule is configured to bind to the first target analyte before it is immobilised at the first test zone.

5. The lateral flow test strip of any one of claims 2 to 4, wherein the lateral flow test strip comprises a conjugate biomolecule source in fluid communication with the input.

6. The lateral flow test strip of any one of the preceding claims, wherein the first soluble coloured compound is water-soluble.

7. The lateral flow test strip of any one of the preceding claims, wherein the first test zone exhibits at least one identifying characteristic selected from the group including:(i) a colour;(ii) a shape;(iii) a size;(iv) a relative position of the first test zone on the membrane.

8. The lateral flow test strip of any one of the preceding claims, wherein the first test zone exhibits a circular shape.

9. The lateral flow test strip of any one of the preceding claims, wherein the at least one test zone comprises a longitudinal shape extending along the width of the lateral flow test strip.

10. The lateral flow test strip of any one of the preceding claims, wherein the one or more test zones are arranged in an array on the membrane.

11. The lateral flow test strip of any one of the preceding claims, wherein the conjugate biomolecule is selected to be complimentary for binding to the first target analyte, and is selected from a group consisting of: antigens, antibodies, aptamers, and molecular recognition elements.

12. The lateral flow test strip of any one of the preceding claims, wherein the first target analyte is an antigen selected from a group consisting of: protein, polysaccharide, lipid or nucleic acid.

13. The lateral flow test strip of any one of the preceding claims, wherein the first target analyte is an antibody.

14. The lateral flow test strip of any one of the preceding claims, wherein the first target analyte is a molecule or chemical moiety.

15. The lateral flow test strip of any one of the preceding claims, wherein the first non-visible capture biomolecule is selected from a group consisting of: antigens, antibodies, aptamers, and molecular recognition elements.

16. The lateral flow test strip of any one of the preceding claims, wherein the detection label is a coloured particle, a gold particle, a florescent particle or a magnetic particle.

17. The lateral flow test strip of any one of the preceding claims, wherein responsive to the first non-visible capture biomolecule binding to a respective first target analyte in the sample applied to the input of the lateral flow test strip and transported to the first test zone, the first test zone is configured to transition from a first visible state to a second visible state to indicate the presence of the first target analyte in the sample.

18. The lateral flow test strip of any one of the preceding claims, wherein the membrane further comprises a control zone comprising an antibody configured to bind to a control analyte present in the sample.

19. The lateral flow test strip of any one of the preceding claims, wherein the one or more test zones comprises a second test zone, wherein the second test zone comprises a second soluble coloured compound substantially co-located with a second non-visible capture biomolecule, wherein the second non- visible capture biomolecule is configured to attach to the membrane and to bind to a respective second target analyte in the sample transported along the lateral flow test strip, thereby immobilising the second target analyte at the second test zone, wherein the second soluble coloured compound is configured to be removed from the second test zone by a fluid flow of the sample applied to the input and transported along the lateral flow test strip; and wherein the first and second target analytes are different from one another, and the first and second non-visible capture biomolecules are different from one another.

20. The lateral flow test strip of claim 19 when dependent on claim 3, wherein a second labelled conjugate assembly comprises the second target analyte bound to a second complimentary conjugate biomolecule attached to a second detection label, and the second complimentary conjugate biomolecule binds to the second target analyte immobilised at the second test zone thereby forming the second labelled conjugate assembly at the second test zone.

21. The lateral flow test strip of claim 19 when dependent on claim 4, wherein a second labelled conjugate assembly comprises the second target analyte bound to a second complimentary conjugate biomolecule attached to a second detection label, and the second non- visible capture biomolecule binds to the second target analyte of the second labelled conjugate assembly thereby immobilising the second labelled conjugate assembly at the second test zone.

22. The lateral flow test strip of any one of claims 19 to 21, wherein the second test zone is positioned on the membrane at a location spaced apart from the first test zone.

23. The lateral flow test strip of any one of claims 19 to 22, wherein the first test zone is positioned on the membrane in a location upstream of the second test zone.

24. The lateral flow test strip of any one of claims 19 to 23, wherein the first test zone and the second test zone are positioned on the membrane in an interspersed arrangement.

25. A method of manufacturing a lateral flow test strip for detecting the presence or absence of a first target analyte within a sample, the method comprising: combining a first soluble coloured compound with a first non- visible capture biomolecule and a carrier fluid to form a first formulation, wherein the first non- visible capture biomolecule is configured to bind to a respective first target analyte; applying the first formulation to one or more first locations of a membrane of the lateral flow test strip to form respective one or more first test zones, wherein, when the first target analyte comes into contact with the one or more first test zones, the first non- visible capture biomolecule binds to the first target analyte, thereby immobilising the first target analyte at the one or more first test zones, and when the first soluble coloured compound comes into contact with a fluid flow of the sample comprising the first target analyte, the first soluble coloured compound is removed from the one or more first test zones by the fluid flow.

26. The method of claim 25, wherein a first labelled conjugate assembly comprises the first target analyte bound to a first complimentary conjugate biomolecule attached to a first detection label, and wherein the method further comprises configuring the test strip so that the first complimentary conjugate biomolecule attached to a first detection label comes into contact with the first target analyte immobilised at the one or more first test zones causing the first complimentary conjugate biomolecule to bind to the first target analyte, thereby forming the first labelled conjugate assembly at the one or more first test zones.

27. The method of claim 25, wherein a first labelled conjugate assembly comprises the first target analyte bound to a first complimentary conjugate biomolecule attached to a first detection label, and wherein the method further comprises configuring the test strip so that the first labelled conjugate assembly comes into contact with the one or more first test zones causing the first non- visible capture biomolecule to bind to the first target analyte, thereby immobilising the first labelled conjugate assembly at the one or more first test zones.

28. The method of any one of claims 25 to 27, further comprising: allowing the first formulation at the one or more first locations of the membrane to dry.

29. The method of any one of claims 25 to 28, wherein applying the first formulation to the one or more first locations of the membrane comprises: printing the first formulation onto the membrane.

30. The method of any one of claims 25 to 29, wherein applying the first formulation to the one or more first locations of the membrane comprises: applying the first formulation in droplets to the one or more first locations.

31. The method of any one of claims 25 to 30, further comprising applying multiple droplets to one or more of the one or more first locations on the membrane to thereby increase the concentration of the first non- visible capture biomolecule and to increase the colour concentration of the first soluble coloured compound in the one or more first test zones.

32. The method of any one of claims 25 to 31, further comprising: combining a second soluble coloured compound with a second non-visible capture biomolecule to form a second formulation, wherein the second non-visible capture biomolecule is configured to bind to a respective second target analyte; and applying the second formulation to one or more second locations of a membrane of the lateral flow test strip to form respective one or more second test zones, and wherein, when the second target analyte comes into contact with the one or more second test zones, the second non-visible capture biomolecule binds to the second target analyte, thereby immobilising the second target analyte at the one or more second test zones, and when the second soluble coloured compound comes into contact with a fluid flow of the sample comprising the second target analyte, the second soluble coloured compound is removed from the one or more second test zones by the fluid flow.

33. The method of claim 32 when dependent on claim 26, wherein a second labelled conjugate assembly comprises the second target analyte bound to a second complimentary conjugate biomolecule attached to a second detection label, and wherein the method further comprises configuring the test strip so that the second complimentary conjugate biomolecule comes into contact with the second target analyte immobilised at the one or more second test zones causing the second complimentary conjugate biomolecule to bind to the second target analyte, thereby forming the second labelled conjugate assembly at the one or more second test zones.

34. The method of claim 32 when dependent on claim 27, wherein a second labelled conjugate assembly comprises the second target analyte bound to a second complimentary conjugate biomolecule attached to a second detection label, and wherein the method further comprises configuring the test strip so that the second labelled conjugate assembly comes into contact with the one or more second test zones causing the second non- visible capture biomolecule to bind to the second target analyte, thereby immobilising the second labelled conjugate assembly at the one or more second test zones.

35. A lateral flow test strip produced according to the method of any one of claims 25 to 34.

36. A method for visual inspection of a manufactured lateral flow test strip prior to addition of a sample, the method comprising: identifying a coloured region on the lateral flow test strip, wherein the coloured region is indicative of a location of a first test zone comprising a first soluble coloured compound and a first non- visible capture molecule; inspecting the first test zone for at least one identifying characteristic; and based on the at least one identifying characteristic:(i) identifying if the associated capture biomolecule zone and its location and relationship to other zones on the strip is compatible with the sample to be tested; and / or(ii) determining if the lateral flow test strip satisfies one or more quality control parameters.

37. The method of claim 36, wherein the inspecting is performed by eye.

38. The method of claim 36, wherein the inspecting is performed by a computer executing computer code configured to perform digital imaging or machine vision techniques.

39. The method of any one of claims 36 to 38, wherein the at least one identifying characteristic is presence or absence of colour of the first test zone.

40. The method of any one of claims 36 to 39, wherein the at least one identifying characteristic comprises one of more of:(i) a colour of the first test zone;(ii) a shape of the first test zone;(iii) a size of the first test zone;(iv) a relative position of the first test zone on the membrane;(v) a size and position of the first test zone on the membrane with respect to the size and position of other further test zones of the membrane.

41. The method of any one of claims 36 to 40, further comprising: determining a concentration of the first non- visible capture biomolecule based on a determination of a colour concentration of the first test zone.

42. The method of any one of claims 36 to 41, wherein the lateral flow test strip is the lateral flow test strip of any one of claims 1 to 24 or claim 35.

43. A method for detecting the presence or absence of a first target analyte within a sample using the lateral flow test strip of any one of claims 1 to 24, claim 35, the method comprising: applying the sample to the input of the lateral flow test strip, wherein the lateral flow test strip is configured to convey the sample to at least the first test zone of the membrane of the lateral flow test strip; allowing the sample to remove the first soluble coloured compound from the first test zone; and after a first time interval, responsive to determining that the first test zone has transitioned from a first visible state to a second visible state, determining the presence of the first target analyte in the sample, and responsive to determining that the first test zone has not transitioned from the first visible state to the second visible state, determining the absence of the first target analyte in the sample.

44. A cartridge comprising: a lateral flow test strip assembly according to any one of claims 1 to 24 or claim 35, wherein the lateral flow test strip assembly is substantially enclosed within the cartridge; an inlet in fluid communication with the input and configured to convey the sample to the input; and a viewing port disposed in a wall of the cartridge and configured to allow for observation of the one or more test zones of the lateral flow test strip assembly.

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