Analyte detection

Abstract

Provided herein is a lateral flow test device for detecting an analyte in a test sample comprising a sample-receiving region, a conjugate pad, and at least one detection region. The conjugate pad comprises a lectin conjugated to a detectable label (lectin-label conjugate), wherein the lectin-label conjugate is capable of mobilisation on contact with the sample, and binding to the analyte. The first detection region comprises an immobilised capture molecule which is also capable of binding to the analyte when bound to the lectin-label conjugate. The detectable label of the lectin-label conjugate is capable of producing a detectable signal in the first detection region. The test sample may be mixed with at least a first surfactant and a second surfactant to facilitate extraction of the analyte, wherein the first surfactant comprises a PEGylated sorbitan or fatty acid ester thereof, and wherein the second surfactant comprises a PEGylated alcohol.

Description

[0001] ANALYTE DETECTION

[0002] Field of the Invention

[0003] The present invention relates to a lateral flow test device for detecting an analyte in a sample, a method of detecting an analyte in a sample, and a kit comprising the lateral flow test device.

[0004] Background of the Invention

[0005] Lateral flow test

[0006] A lateral flow test enables rapid detection and quantification of an analyte in a liquid sample. Lateral flow tests are widely used in medical diagnostics for point of care testing (for example, home testing) or laboratory use. A typical lateral flow test device is represented by Figure 1 and includes the following components: 1) sample pad (106); 2) conjugate pad (108); 3) nitrocellulose membrane (112); and 4) absorbent pad (116). The components may be housed in a cassette or provided on a backing card (102) for ease of handling.

[0007] With reference to Figure 1 , a common type of lateral flow test is based on a ‘sandwich’ immunoassay. In this format, the conjugate pad (108) typically comprises a primary antibody that is specific for the target analyte conjugated to a detectable label (primary antibody-label conjugate) deposited therein. The primary antibody-label conjugate is capable of mobilisation when the sample (104) flows through the conjugate pad. The nitrocellulose membrane (112) typically includes a test region (110) (for example, a test line) comprising an immobilised antibody specific for the target analyte. The nitrocellulose membrane may also include a control region (114) (for example, a control line) comprising an immobilised secondary antibody which binds to the primary antibodylabel conjugate.

[0008] In use, a test sample comprising a target analyte is added to the sample pad (106) of the lateral flow test device (100), and the sample flows through to the conjugate pad (108), thereby mobilising the primary antibody-label conjugate. The target analyte and the primary antibody-label conjugate come into contact and form a complex (analyte-primary antibody-label complex). The complex migrates towards the antibodies immobilised on the nitrocellulose membrane (112) by capillary action. These immobilised antibodies capture the analyte-primary antibody-label complex, thereby producing a visible line indicative of a positive response. If the test sample does not comprise any target analyte (or the target analyte is present in an amount which is below the limit of detection of the assay), a visible line will not appear on the test region. Any primary antibody-label conjugate which has not bound to the target analyte may bind to the secondary antibodies immobilised in the control region (114). A detectable signal in the control region indicates that the primary antibody-label conjugate has flowed correctly, regardless of the result of the test. In other words, the signal intensity of the control line is independent of the signal intensity of the test line, and serves only to indicate that the lateral flow test is viable.

[0009] Another type of lateral flow test is based on a “competitive” immunoassay. In competitive assays, the conjugate pad comprises a labelled conjugate which specifically binds to the target analyte in the test sample. For example, the labelled conjugate may comprise a conjugate of a primary antibody and detectable label. The test line comprises an immobilised capture molecule which may bind to the labelled conjugate. For example, the capture molecule may comprise a secondary antibody which binds to the labelled conjugate, or the target analyte. When a test sample which does not contain a target analyte is applied to the sample pad of the test device, the labelled conjugate flows through the membrane and binds to the capture molecule at the test line, thereby producing a detectable signal at the test line. When target analyte is present in the test sample, the labelled conjugate binds to the target analyte, and this, in turn, inhibits the binding of the labelled conjugate to the immobilised capture molecule of the test line. (That is to say, the immobilised capture molecule binds only to the labelled conjugate when it is not bound to a target analyte.) Thus, the presence of target analyte in the sample is indicated by the absence of a detectable signal at the test line.

[0010] Fuel contamination

[0011] Fuel may become contaminated with microorganisms, including bacteria and fungi, usually following distillation. Microorganisms are typically found in a free water phase existing at the bottom of fuel tanks, and at the fuel / water interface. The microorganisms utilise alkanes and additives in fuel as a food source. Microorganisms may also exist in water that is suspended in the fuel phase which is present above the free water phase (for example, as “pockets” or “microdroplets” of water). The presence of microorganisms in fuel may result in equipment damage and consequent malfunction. In particular, microbial biomass may cause blockages in system components (for example, in filters), and acid by-products of microbial metabolism may cause exfoliation and pitting corrosion in tanks. Microbial biofilms may also cause electrochemical corrosion of system components. A major contaminant of fuel in the aviation industry is the fungus Hormoconis resinae (H.res) (also known as the “Jet Fuel Fungus”). However, many other harmful bacterial and fungal contaminants may exist in fuel and cause comparable damage to fuel systems. Therefore, it is important to detect microbial contamination in fuel in order to be able to minimise or eradicate contamination with appropriate biocide treatment before there is damage to equipment. Moreover, regulatory bodies such as the International Air Transport Association (IATA) recommend acceptable levels of fuel contamination.

[0012] Standard laboratory culture techniques are used to evaluate and quantify microbial contamination. However, this requires transporting samples to a laboratory or other testing facility which may result in cross-contamination and / or changes to microbial populations during transit. Additionally, performing time-consuming culturing of microorganisms may result in undesirable delays. As such, expensive biocide treatment is often applied after suspected contamination and before laboratory confirmation. Depending on the test results, the biocide treatment may be deemed unnecessary or inadequate.

[0013] US 10, 145, 833 B2 describes a faster method of evaluating microbial contamination based on a competitive-type immunoassay. Specifically disclosed is a method of detecting Hormonoconis resinae in a fuel sample comprising contacting the sample with a primary polyclonal antibody specific for Hormonoconis resinae, and detecting the presence of bound antibody using a labelled secondary antibody in an ELISA system or using a lateral flow test device.

[0014] Fuelstat® Plus is a test kit comprising a lateral flow test device for microbial detection in fuel based on a competitive-type immunoassay described US 10, 145, 833 B2. In this test device, the conjugate pad comprises a mobilisable labelled polyclonal antibody which is specific for certain hydrocarbon-degrading microorganisms including Hormoconis resinae (the analyte). The test line contains an immobilised antigen to which the labelled polyclonal antibody may bind. The control line contains an immobilised secondary antibody which is capable of binding to the labelled polyclonal antibody. When a test sample which does not contain analyte is applied to the device, the labelled polyclonal antibody becomes mobilised from the conjugate pad and migrates along the device to the test line where it is bound by the antigen at the test line. This results in a detectable signal at the test line. When a test sample which contains analyte is applied to the device, the labelled polyclonal antibody becomes mobilised from the conjugate pad and binds to the analyte from the sample. Labelled polyclonal antibody which is bound to analyte cannot bind to the antigen on the test line due to occupancy of the antigen binding site on the antibody. This results in a reduced or absent signal at the test line. Any excess labelled polyclonal antibody which is not captured by the antigen at the test line becomes captured by the secondary antibody at the control line, thus producing a detectable signal at the control line, confirming proper flow of reagents through the device.

[0015] However, whilst the kit comprising the lateral flow test device enables the detection of the presence of certain hydrocarbon- degrading microorganisms including Hormoconis resinae, preferentially in the free water phase, the kit may have certain disadvantages. For example, the disadvantages include the following: 1) Other microorganisms may remain undetected in view of the specificity of the antibodies used; 2) microorganisms present in the fuel phase (for example, within “pockets” or “microdroplets” of water suspended in the fuel) may remain undetected; and 3) the test device enables only a semi-quantitative assessment of microbial contamination. (Specifically, the test device includes different devices configured to have different thresholds for detection where each threshold corresponds to a given level of contamination (such as “low”, “moderate” and “high”).

[0016] Objectives

[0017] Accordingly, there remains a need for an improved method for detecting the presence of microorganisms in a sample, particularly a fuel sample. In particular, it would be desirable to provide a method and / or device which enables a reliable and consistent detection of the presence of microorganisms in both the free water phase and the fuel phase of fuel samples within a short time-frame (for example, less than 15 minutes). The method and / or device should also enable a broad recognition and detection of harmful microorganisms to enable an accurate assessment of microbial contamination in a fuel sample.

[0018] The present invention seeks to achieve one or more of the above objectives.

[0019] Summary of the Invention

[0020] Aspects of the invention are predicated on the unexpected finding that efficient extraction of microorganisms and microbial products from fuel samples may be achieved when the samples are contacted with a combination of a first surfactant comprising a PEGylated sorbitan or fatty acid ester thereof, and a second surfactant comprising a PEGylated alcohol. In particular, the present inventors have found that the detection of the presence of microorganisms is unreliable or inefficient when extraction is performed with only one of the above surfactants or alternative surfactants. Specifically, different levels of microbial contamination may be detected from the same fuel sample using the same test device when either one of the above surfactants (or no surfactants) is used for extraction. However, when the aforementioned combination of surfactants is used, the presence of microorganisms may be detected more efficiently and consistently with a given test device.

[0021] Further aspects of the invention are predicated on the finding that lectins such as mannan-binding lectin may be incorporated within a lateral flow test device for the detection of the presence of contaminating and harmful microorganisms in fuel samples. The lectins not only provide a broader recognition of microorganisms as compared to antibodies that are conventionally used in lateral flow test devices, but they also enable the detection of microbial products including microbial polysaccharides, thus enabling a more accurate evaluation of microbial status in a sample.

[0022] Accordingly, in a first aspect of the invention, there is provided a lateral flow test device for detecting an analyte in a test sample, the device comprising a solid support structure, the solid support structure comprising a sample-receiving region, a conjugate pad, and at least one detection region, wherein the at least one detection region comprises a first detection region, wherein the solid support is configured to permit liquid to flow sequentially from the sample-receiving region to the first detection region via the conjugate pad, wherein: i) the conjugate pad comprises a lectin conjugated to a detectable label (lectin-label conjugate), wherein the lectin-label conjugate is capable of mobilisation on contact with the sample, wherein the lectin moiety of the lectin-label conjugate is capable of binding to the analyte to form a first complex comprising the analyte and lectin-label conjugate; ii) the first detection region comprises an immobilised capture molecule which is capable of binding to the analyte in the first complex to immobilise the first complex and to form a second complex comprising the capture molecule, analyte and lectin-label conjugate; and iii) the detectable label of the lectin-label conjugate in the second complex is capable of producing a detectable signal in the first detection region.

[0023] Advantageously, the lectin conjugated to the detectable label comprises mannan-binding lectin.

[0024] Advantageously, the immobilised capture molecule comprises a lectin, preferably mannan-binding lectin.

[0025] Conveniently, the detectable label of the lectin-label conjugate comprises a nanoparticle, preferably a gold nanoparticle.

[0026] Preferably, the conjugate pad further comprises a primary antibody conjugated to a detectable label (primary antibody-label conjugate), wherein the primary antibody-label conjugate is capable of mobilisation on contact with the sample, and wherein the primary antibody does not bind to the analyte, and wherein, the lateral flow test device comprises a second detection region, wherein the second detection region comprises an immobilised secondary antibody which is capable of binding to the primary antibody to immobilise the primary antibody-label conjugate and form a third complex, and wherein the detectable label of the primary antibody-label conjugate in the third complex is capable of producing a detectable signal in the second detection region. Conveniently, the detectable label of the primary antibody-label conjugate comprises a nanoparticle, preferably a gold nanoparticle.

[0027] In a second aspect of the invention, there is provided a method for detecting an analyte in a test sample, the method comprising the steps of: i) mixing the test sample with at least a first surfactant and a second surfactant, wherein the first surfactant comprises a PEGylated sorbitan or fatty acid ester thereof, and wherein the second surfactant comprises a PEGylated alcohol, to obtain a treated sample; ii) allowing the treated sample to contact a lectin conjugated to a detectable label (lectin-label conjugate), wherein the lectin is capable of binding to the analyte, wherein the lectin-label conjugate binds to the analyte in order to form a first complex comprising the analyte and lectin-label conjugate; iii) allowing the first complex obtained in step ii) to contact a capture molecule, wherein the capture molecule is capable of binding to the analyte in the first complex, wherein the capture molecule binds to the analyte in the first complex in order to form a second complex comprising the capture molecule, analyte and lectin-label conjugate; and iv) detecting a signal from the detectable label of the lectin-label conjugate in the second complex formed in step iii), wherein a detected signal indicates the presence of analyte in the test sample.

[0028] Conveniently, the test sample comprises a fuel sample, preferably a hydrocarbon fuel sample or a biodiesel fuel sample, or a lubrication fluid sample.

[0029] Preferably, the method comprises separating the fuel sample into a fuel phase and a free water phase, such that the test sample that is mixed with the first and second surfactants comprises the separated fuel phase or the separated free water phase.

[0030] Conveniently, in step i), the test sample is mixed with an aqueous composition comprising the first and second surfactants. The aqueous composition may further comprise one or more components selected from a buffer, a salt, a dye and a preservative. Preferably, the aqueous composition comprises a dye and a calcium salt. Advantageously, in step i), after the test sample is mixed with an aqueous composition comprising the first and second surfactants, the test sample is separated into a free water phase enriched with analyte and at least one other phase, and wherein the treated sample comprises the separated free water phase enriched with analyte.

[0031] Preferably, each of the first surfactant and second surfactant is present in the aqueous composition in an amount of from 0.01 to 0.05 wt.%, or from 0.02 to 0.04 wt.%, or from 0.030 to 0.035 wt.% by total weight of the composition.

[0032] Conveniently, the test sample comprises is non-aqueous, and in step i), the ratio of aqueous composition to test sample is from 1 :100 to 1 :200, or from 1 :130 to 1 :160, or 1 :150 by volume.

[0033] Alternatively, the test sample is aqueous, and in step i), the ratio of aqueous composition to test sample is from 1 :3 to 1 : 1 , or from 1 :2 to 1 : 1 , or 1 : 1 , by volume.

[0034] Advantageously, the lectin conjugated to the detectable label comprises mannan-binding lectin.

[0035] Further advantageously, the capture molecule comprises a lectin, preferably mannan- binding lectin.

[0036] Preferably, the lectin-label conjugate is deposited on a solid support, and the lectin-label conjugate is mobilised when brought into contact with the sample comprising the analyte in step ii).

[0037] Conveniently, the capture molecule is immobilised on a solid support, and immobilises the first complex on binding to the analyte in the first complex.

[0038] Advantageously, the first surfactant comprises a fatty acid ester of PEGylated sorbitan.

[0039] Preferably, the PEGylated sorbitan comprises one to four fatty acid esters, or one to two fatty acid esters, or one fatty acid ester. More preferably, the fatty acid ester is a C6-20 fatty acid ester, C10-16 fatty acid ester, C12-14 fatty acid ester, or a C12 fatty acid ester. Most preferably, the first surfactant comprises polyethylene glycol sorbitan monolaurate. Conveniently, the PEGylated sorbitan or fatty acid ester thereof comprises 2 to 50, 5 to 40, 10 to 30, 15 to 25, or 20 PEG linkages.

[0040] Advantageously, the PEGylated alcohol comprises a PEGylated C1-C20 alcohol, preferably, a PEGylated Ce-Cie alcohol.

[0041] Preferably, the PEGylated alcohol further comprises a polypropylene glycol (PPG) linkage.

[0042] Conveniently, the PEGylated alcohol comprises a first PEGylated alcohol and a second PEGylated alcohol, wherein the first PEGylated alcohol comprises a PEGylated C6-C12 alcohol and the second PEGylated alcohol comprises a PEGylated C10-C16 alcohol.

[0043] Preferably, the first PEGylated alcohol and the second PEGylated alcohol each comprises a polypropylene glycol (PPG) linkage.

[0044] Advantageously, the first surfactant and second surfactant are mixed with the test sample in a 1 :1 weight ratio.

[0045] Conveniently, the method further comprises allowing the treated sample to contact a primary antibody conjugated to a detectable label (primary antibody-label conjugate), wherein the antibody moiety of the primary antibody-label conjugate does not bind to the analyte, and wherein the method further comprises contacting the primary antibody-label conjugate with a secondary antibody which binds to the primary antibody to form a third complex comprising the secondary antibody and primary antibody label-conjguate bound thereto, and detecting a signal from the detectable label in the third complex.

[0046] Advantageously, the primary antibody-label conjugate is provided on a solid support, and the primary-label conjugate is mobilised when brought into contact with the sample obtained from step i).

[0047] Advantageously, the secondary antibody is immobilised on a solid support, and immobilises the third complex on binding to the primary antibody in the third complex. Preferably, the detectable label of the lectin-label conjugate and / or of the primary antibody-label conjugate comprises a nanoparticle. More preferably, the nanoparticle comprises a gold nanoparticle.

[0048] Advantageously, the analyte comprises a microorganism and / or or a microbial product such as a microbial polysaccharide. Preferably, the microorganism comprises a bacterium and / or fungus.

[0049] Optionally, the bacterium comprises one or more of: Acinetobacter venetianus RAG-1 (ATCC 31012), Alcaligenes species, Bacillus licheniformis, Nocardiodes luteus, Pseudomonas aerigunosa (ATCC 27853), Pseudomonas putida (ATCC 12633), Pseudomonas fluorescens, Kocuria kristinae (ATCC 27570), Brevundimonas vesicularis (ATCC 11426), Rhodococcus erythropolis.

[0050] Further optionally, the fungus comprises one or more yeast selected from Candida guilliermondii (ATCC 6260), Rhodotorula mucilaginosa (ATCC 2510), Yarrowia lipolytica (ATCC 9773) and Candida kersosiniea.

[0051] Still further optionally, the fungus comprises one or more filamentous fungi selected from: Hormoconis. resinae (ATCC 20495), Aspergillus niger (ATCC 9642) and Penicillium coryphilum.

[0052] Conveniently, the microorganism comprises Hormoconis. resinae (ATCC 20495).

[0053] In a third aspect of the invention, there is provided a method as defined above, wherein the method comprises using the lateral flow test device as defined above.

[0054] In a fourth aspect of the invention, there is provided a kit comprising: i) a lateral flow test device as defined herein; and ii) a first surfactant comprising a PEGylated sorbitan or fatty acid ester thereof and a second surfactant comprising a PEGylated alcohol.

[0055] Advantageously, the first surfactant comprising the PEGylated sorbitan or fatty acid ester thereof is as defined above. Advantageously, the second surfactant comprising the PEGylated alcohol is as defined above.

[0056] Preferably, the first surfactant and the second surfactant are provided in an aqueous composition which may be as defined above.

[0057] Brief Description of the Figures

[0058] Figure 1 is a schematic representation of a conventional LFT device.

[0059] Figure 2 is a schematic representation of a LFT device according to one embodiment of the invention.

[0060] Figure 3 is a schematic representation of one aspect of a method of detecting an analyte according to an embodiment of the invention.

[0061] Figure 4 is a bar chart illustrating analyte detection from diesel sample 1 using an extraction buffer comprising Tween™ 20 (T) or Ecosurf™ SA-9 (S).

[0062] Figure 5 is a bar chart illustrating analyte detection from diesel sample 2 using an extraction buffer comprising Tween™ 20 (T) or Ecosurf™ SA-9 (S).

[0063] Figure 6 is a bar chart illustrating analyte detection from diesel sample 3 using an extraction buffer comprising Tween™ 20 (T) or Ecosurf™ SA-9 (S).

[0064] Figure 7 is a bar chart illustrating analyte detection from diesel sample 1 using an extraction buffer comprising Tween™ 20 and Ecosurf™ SA-9 (TAS) or comprising Tween™ 20 (T).

[0065] Figure 8 is a bar chart illustrating analyte detection from diesel sample 4 using an extraction buffer comprising varying amounts of Tween™ 20 and Ecosurf™ SA-9 (TAS) or comprising Tween™ 20 (T). Figure 9A is a bar chart illustrating analyte detection from different jet fuel samples using an extraction buffer comprising Tween™ 20 (T20), Ecosurf™ SA-9 (SA9) or Tween™ 20 and Ecosurf™ SA-9 (TAS), as assessed by a visual scoring method.

[0066] Figure 9B is a bar chart illustrating analyte detection from different jet fuel samples using an extraction buffer comprising Tween™ 20 (T20), Ecosurf™ SA-9 (SA9) or Tween™ 20 and Ecosurf™ SA-9 (TAS), as assessed by optical density measurements.

[0067] Figure 10 is a schematic representation of a computing system for quantifying a concentration of analyte molecules in a test sample based on signal intensity in the detection region.

[0068] Figure 11 is a flow chart illustrating steps involved in the treatment of a fuel sample containing an analyte according to an embodiment of the invention prior to detecting an analyte using a lateral flow test.

[0069] Definitions

[0070] The term “immobilised”, as used herein, means that a molecule is retained or fixed on the relevant part of the device using any suitable means known to the person skilled in the art. The immobilised molecule is not released or mobilised upon contact with the test sample or by the flow of the test sample. In particular, an “immobilised” capture molecule is one which will normally be retained in the detection region in use, and will hence bind to relevant complementary molecules contained within a liquid contacting the test region.

[0071] The term “mobilisable” or “capable of mobilisation” as used herein, means that the relevant molecule is releasably retained or fixed on the relevant part of the device prior to use of the device, and is released from the part of the device upon contact with the test sample. A molecule which is capable of mobilisation can be carried away from the original point of retention / fixation by the lateral flow of the test sample.

[0072] The term “microbial product” as defined herein means a product derived from a microorganism. The product may be produced and / or secreted by the microorganism, (for example, as a metabolite), or the product may be a cellular component such as a component of a cell wall.

[0073] The term “aqueous composition” as used herein refers to a water-based composition comprising more than 50 wt.% water. The term “non-aqueous composition” as used herein refers to a composition comprising water in amount of 50 wt.% or less.

[0074] The term “phase” as used herein refers to a distinctive form of matter that is obtained from a liquid composition, for example, a fuel sample, by a method such as gravity separation. In the context of the present invention, a phase can be mechanically separated or isolated from another phase of the liquid composition.

[0075] The term “free water phase” of a liquid composition, for example, a fuel sample, as used herein, refers to a water-based phase comprising at least 50 wt.% water that is obtained by gravity separation of the liquid composition. The free water phase excludes water which may be fixed within an emulsion and thus which is not available for reaction. In a fuel sample which has been subjected to gravity separation, any free water phase settles below a fuel phase. The fuel phase of a fuel sample may contain “pockets” or “microdroplets” of water which are not part of the free water phase.

[0076] The term “aqueous phase” of a liquid composition, for example, a fuel sample, as used herein, refers to a water-based phase comprising at least 50 wt.% water and includes the free water phase defined above and any water which may be fixed within an emulsion and thus which is not available for reaction in the liquid composition. By way of example, “pockets” or “microdroplets” of water which may be found in the fuel phase as described above, are considered to be part of the aqueous phase.

[0077] The term “non-aqueous phase” of a liquid composition as used herein refers to the matter which is not contained in the aqueous phase as defined above.

[0078] The term “fuel / water interface” as used herein refers to a distinct phase that may include microorganisms and products thereof, and that is found in between the free water phase and the fuel phase in a fuel sample. The fuel / water interface may comprise a microbial biofilm. Detailed Description

[0079] The invention relates, in general terms, to a lateral flow test device for detecting an analyte in a test sample. The device comprises a solid support structure, the solid support structure comprising a sample-receiving region, a conjugate pad, and at least a first detection region. The solid support structure is configured to permit liquid to flow sequentially from the sample-receiving region to the first detection region via the conjugate pad. The conjugate pad comprises a conjugate of a lectin and a detectable label (lectin-label conjugate), wherein the lectin-label conjugate is capable of mobilisation on contact with the sample, and wherein the lectin moiety of the lectin-label conjugate is capable of binding to the analyte to form a first complex comprising the analyte and lectin-label conjugate. The first detection region comprises an immobilised capture molecule which is capable of binding to the analyte in the first complex to immobilise the first complex and to form a second complex comprising the capture molecule, analyte and lectin-label conjugate. The label of the lectin-label conjugate is capable of producing a detectable signal in the first detection region.

[0080] A schematic representation of a lateral flow test device according to an embodiment of the invention is illustrated in Figure 2.

[0081] Solid support structure

[0082] With reference to Figure 2, the device (200) comprises a solid support structure, upon or within which the other components and reagents of the lateral flow test are placed. For example, the solid support structure may comprise a backing card (202) upon which are arranged the other components and reagents for the lateral flow test. The backing card (202) may then be contained within a housing. In some instances, the solid support structure may itself comprise a housing, within which are arranged the other components and reagents for the lateral flow test. The solid support structure may be made of any suitable material known to the person skilled in the art, such as plastic. Preferably, the solid support structure is arranged to allow at least a portion of the sample pad and each detection region to be visible. The solid support structure is configured to allow liquid (204) (for example, a test sample, and any other liquid reagents or components added to the device) to flow sequentially from the sample-receiving region (206) to the conjugate pad (208), and then to the first detection region (210). The device (200) is configured to allow the liquid to contact and mix with the lectin-label conjugate of the conjugate pad (208), and subsequently, to contact the immobilised capture molecule of the first detection region (210).

[0083] Sample-receiving region

[0084] The sample-receiving region is configured to receive a test sample which is preferably in liquid form. The sample-receiving region is also configured to receive any other liquids, such as liquid reagents which may be added to the device of the invention.

[0085] In some embodiments, the sample-receiving region comprises a sample pad. The sample-receiving region may be made of any suitable material known to the person skilled in the art. For example, the sample-receiving region may be composed of glass fibre, cellulose, or cotton. In preferred embodiments, the sample-receiving region is composed of a blend of cotton and rayon. The composition of the sample-receiving region may be varied according to the test sample medium.

[0086] In some embodiments, the sample-receiving region does not undergo any pre-treatment. In alternative embodiments, the sample-receiving region may be pre-treated with buffer components to enhance assay performance. The pre-treatment may serve to mitigate sample variability (pH, viscosity, protein concentration, salt concentration etc.), and to improve flow and reproducibility. Sample pad treatment can be performed by any suitable means known to the person skilled in the art, such as by spraying or immersion. The buffer components may be dried onto the sample-receiving region, such that addition of a liquid, such as a liquid test sample, causes the buffer components to be released from the sample-receiving region and to mix with the test sample. Any suitable buffer components known to the person skilled in the art, such as salts, sucrose and glycerol, may be used.

[0087] The sample-receiving region may be a part of a single structure comprising the conjugate pad, and optionally also the first detection region, or more preferably, the samplereceiving region may be a separate structure from each of the conjugate pad and the first detection region. In some embodiments, the sample-receiving region may be a region of the conjugate pad, such that there is no distinction between the samplereceiving region and the conjugate pad (i.e. the test sample is added to the conjugate pad).

[0088] Conjugate pad

[0089] The conjugate pad comprises a conjugate of a lectin and a detectable label (lectin-label conjugate) which is capable of mobilisation on contact with the sample. The lectin-label conjugate is disposed on or within the conjugate pad so that lectin-label conjugate is released by, and mixes with, a test sample which contacts the conjugate pad. For example, the lectin-label conjugate may be deposited and dried onto the conjugate pad, so that it is released or mobilised by, and mixes with, the test sample upon contact therewith. The conjugate may be deposited on the conjugate pad using an air jet dispenser. Alternatively, the conjugate may be deposited on the conjugate pad by immersing the pad into a solution comprising the conjugate followed by drying. The solution comprising the conjugate which is applied to the conjugate pad by the means described above may comprise a sugar to ensure long-term stability of the conjugate in the dried form and re-solubilisation on contact with the sample. The sugar is preferably selected such that it is not bound by the lectin moiety of the lectin-label conjugate and therefore, does not interfere with the assay. In some embodiments, the solution comprises 10 to 20 wt.% trehalose and / or sucrose, preferably 20 wt.% trehalose.

[0090] The conjugate pad may be composed of any suitable material known to the person skilled in the art. There are many materials commercially available for use as a conjugate pad from suppliers such as Millipore™ and Ahlstrom™. The material selected will determine the volume of conjugate that can be adsorbed, as well as the speed of release. Therefore, the selection can be made according to the type of sample to be tested. A preferred conjugate pad is composed of polyester.

[0091] It may be necessary to pre-treat the conjugate pad before dispensing or depositing the conjugate. The pre-treatment components may include a buffer for pH adjustment (for example, Tris buffer). Proteins, polymers and surfactants may also added to aid in the release of the lectin-label conjugate and flow of the assay. When running the assay, these components may move through the device faster than the conjugate and thus may help block protein-binding sites on the nitrocellulose membrane prior to conjugate interaction. This reduces non-specific interactions. In a preferred embodiment, the pretreatment buffer comprises Tris buffer, Tween 20, and BSA (for example, 100mM Tris, 0.2-0.3 wt.% Tween™ 20, 0.3 to 0. 7 wt.% BSA, pH8). In some embodiments, the pretreatment buffer may comprise a source of calcium ions (for example, calcium chloride) to facilitate binding of the lectin to the analyte. This is particularly relevant to calcium-ion dependent lectins (C-type lectins) such as MBL. Alternatively, as described below, a source of calcium ions may be present in the test sample before applying to the lateral flow test device. The pre-treatment may be carried out using routine methods as described above in relation to the sample receiving-region.

[0092] In some embodiments, the conjugate pad is a region of the same structure comprising the first detection region. Alternatively, it may be a separate structure from the first detection region. When the conjugate pad is a separate structure from the first detection region, it may be attached to the structure comprising the first detection region by any suitable means known to the person skilled in the art, for example, by means of a backing card.

[0093] Lectin-label conjugate

[0094] Lectins are proteins with a wide distribution in nature. Lectins recognize and reversibly bind to carbohydrates and glycoconjugates, often in the presence of metal ions. In most cases, lectins are multivalent, and are able to bind to carbohydrates or glycoconjugates in solution or in association with cell membranes. Their multivalent structure accounts for their well-known ability to agglutinate cells. The ability to agglutinate cells distinguishes lectins from other macromolecules which are able to bind carbohydrates.

[0095] In some embodiments, the lectin of the lectin-label conjugate comprises mannan-binding lectin (MBL). Mannan-binding lectin may also be referred to as mannose-binding lectin. MBL is a protein of the innate immune system belonging to the C-type family of lectins. MBL recognizes and binds various pathogens (including bacteria, viruses, fungi, and parasites) in a calcium ion-dependent manner, and is able to deploy a variety of antimicrobial activities. The human MBL gene (MBL2) product is a 24 kDa polypeptide characterised by a 248- amino acid sequence comprising four distinct domains: 1) a cysteine-rich N-terminal domain; 2) a collagenous domain; 3) a short a-helical coiled-coil domain (neck region), and a carbohydrate-recognition domain which forms the prominent globular head of the molecule. Three polypeptide chains form a triple helix through the collagenous region, stabilized by hydrophobic interactions and interchain disulphide bonds within the N- terminal cysteine-rich domains. The trimeric form is the basic structural subunit of all circulating forms of MBL. Larger molecules may be obtained by the oligomerization of these homotrimeric subunits. This oligomerization allows high-affinity interaction between MBL and carbohydrates on microorganisms. The highly-ordered oligomeric structure, the spacing between carbohydrate-recognition domains, and the orientation of the carbohydrate-recognition domains also define the targets to which MBL binds. MBL binds to a range of sugars including N-acetyl-D-glucosamine, mannose, N-acetyl- galactosamine, and galactose. MBL may bind to polysaccharides comprising these sugars and may also bind to sugars which are complexed with other molecules such as DNA, lipid (glycolipid) or protein (glycoprotein). These sugars are widely distributed across different microbial groups, thus enabling a broad recognition of microorganisms and microbial products in test samples which is not typically achieved with antibodies.

[0096] Other lectins are also envisaged in the present invention. These include but are not limited to: P-type lectins, S-type lectins, l-type lectins and other C-type lectins. The lectins may also comprise a genetically modified lectins or synthetic lectins.

[0097] The lectin is conjugated to a detectable label (referred to hereinafter as the “first” detectable label). The first detectable label may be any suitable molecule known to the person skilled in the art which produces a detectable signal, the intensity of which can be detected or measured. For example, the first detectable label may be a dye particle, a carbon particle, a fluorescent label, a latex particle, a gold particle, a magnetic particle, or the like. In some embodiments, the first detectable label produces an optical signal, such as a colour change.

[0098] In some embodiments, the first detectable label comprises a nanoparticle, preferably a gold nanoparticle. The lectin is conjugated to the first detectable label using any suitable means known to the person skilled in the art. In particular, the first detectable label continues to be detectable while the lectin is conjugated thereto. In preferred embodiments, passive adsorption is used to conjugate a label such as a gold nanoparticle to the lectin. The mechanism of passive adsorption is based on Van der Waals forces, hydrophobic interactions, and ionic interactions between lectin molecules and the detectable label (for example, the surface of nanoparticles) to enable spontaneous coupling of the lectin to the label. The resulting interactions between the lectin molecule and the label molecule may be influenced by the coupling environment. Thus, for example, the pH may be adjusted to optimise the efficiency of the adsorption. An excess of lectin with respect to the label is generally typically used to ensure dense surface binding and high stability post-conjugation.

[0099] In other embodiments, the lectin is conjugated to the detectable label through covalent attachment. Covalent attachment may be accomplished using routine methods. In some embodiments, the lectin is covalently conjugated to the detectable label via a linker. The linker may be any suitable linker known to the person skilled in the art. For example, the linker may comprise biotin and / or BSA. In some embodiments, the linker is biotin-BSA.

[0100] Once the conjugation is complete, the lectin-label conjugate may be re-suspended in an appropriate solution for deposition onto the conjugate pad, as described above.

[0101] Detection region(s)

[0102] With further reference to Figure 2, the lateral flow test device comprises at least a first detection region (210) in which a signal may be detected to indicate the presence of the analyte in the test sample. In some embodiments, the lateral flow test device comprises a first detection region (210) and a second detection region (214). In these embodiments, the first and second detection regions may be different areas of a single structure or may each be separate structures or areas of separate structures. When the first and second detection regions are separate structures or areas of separate structures, the structures may be fixed to one another using any suitable means known to the person skilled in the art (for example, by means a backing card) to allow liquid to flow from the structure comprising the first detection region to the structure comprising the second detection region or vice versa. In some embodiments, the first and second detection regions are arranged such that, during use of the lateral flow test, liquid flowing through / along the lateral flow test device reaches the first detection region before the second detection region, or liquid flowing through / along the lateral flow test device reaches the second detection region before the first detection region. Preferably, liquid flowing through / along the lateral flow test device reaches the first detection region before the second detection region.

[0103] As illustrated in Figure 2, preferably, the first and second detection regions (210, 214) are separate regions of a single structure (212), which may be a permeable or semi- permeable membrane which permits liquid flow on or through it. Preferably, the first detection region (210) is an area of a nitrocellulose membrane (212). When first and second detection regions (210, 214) are present, the first and second detection regions are preferably separate areas of a single nitrocellulose membrane (212).

[0104] The first detection region comprises an immobilised capture molecule. The capture molecule is capable of binding to the analyte. In use, the lectin-label conjugate of the conjugate pad becomes mobilised on contact with the test sample and binds to the analyte in the sample to form a first complex. The first complex is subsequently able to migrate to the first detection region where the capture molecule binds to the analyte in the first complex, and immobilises the first complex. The first detectable label from the lectin-label conjugate in the first complex produces a detectable signal in the first detection region, confirming the presence of the analyte in the test sample. This is illustrated in Figure 3. If there is no analyte present in the sample, then the lectin-label conjugate which has mobilised from the conjugate pad will not bind to the capture molecule (because the capture molecule binds to the analyte and not any other component of the lectin-label conjugate) and thus, no signal will be detected in the first detection region.

[0105] The first detection region may be any suitable shape, such as a square, rectangle, circle, triangle, oval or line. Preferably, the first detection region is a line, formed by immobilising the relevant capture molecule in a line. Preferably, the first detection region stretches across the entire width of the flow path of the liquid along / through the membrane (i.e. perpendicular to the direction of liquid flow). This provides the advantage that the lectin- label conjugate which is bound to analyte cannot circumvent the first detection region before migrating further along the device, thereby ensuring an accurate detection of the analyte. In embodiments where a second detection region is present, the second detection region may have comparable features to the first detection region as described above. However, as described below, the second detection region comprises an immobilised molecule which is different to the capture molecule of the first detection region.

[0106] In preferred embodiments, the capture molecule comprises a lectin. More preferably, the capture molecule comprises MBL. In these embodiments, it may be desirable to deposit the lectin-label conjugate on / within the conjugate pad in an amount sufficient to ensure that all the analyte in the test sample is captured by the lectin-label conjugate, thus preventing free analyte migrating to the first detection region and occupying binding sites on the capture molecule.

[0107] In embodiments where a second detection region is present, the conjugate pad may additionally comprise a conjugate of a control molecule which does not bind to the analyte and a detectable label. For example, the conjugate pad may additionally comprise a conjugate of a primary antibody conjugated to a detectable label (primary antibody-label conjugate). In a similar manner to the lectin-label conjugate, the primary antibody-label conjugate is capable of mobilisation on contact with the sample. The primary antibody-label conjugate is disposed on or within the conjugate pad so that primary antibody-label conjugate is released by, and mixes with, a test sample which contacts the conjugate pad. For example, the primary antibody-label conjugate may be deposited and dried onto the conjugate pad, so that it is released or mobilised by, and mixes with, the test sample upon contact therewith. However, in contrast to the lectin- label conjugate, the primary antibody-label conjugate does not bind to the analyte. Therefore, as the primary antibody-label conjugate (without any bound analyte) migrates to the first detection region on contact with the test sample, it will not be captured by the capture molecule in this region. In these embodiments, the second detection region may comprise a molecule which binds to the control molecule-label conjugate. For example, the second detection region may comprise an immobilised secondary antibody. The immobilised secondary antibody will bind to the primary antibody of the mobilised primary antibody-label conjugate (or indeed any other control molecule-label conjugate), thereby immobilising the primary antibody-label conjugate (or other control molecule-label conjugate) in the second detection region. The secondary antibody is preferably specific to the species from which the primary antibody is derived. Thus, if the primary antibody is a mouse antibody, the secondary antibody is preferably an anti-mouse antibody. In preferred embodiments, the primary antibody is IgY which is readily available from chicken egg yolk, and the secondary antibody is an anti-chicken antibody. The primary antibody may be conjugated to the second detectable label as described above for the lectin-label conjugate. The detectable label of the primary antibody-label conjugate (referred to hereinafter as the “second” detectable label) may be as described above, and is preferably the same as the first detectable label such that the same method may be used to detect the signal in the first detection region and second detection region. The primary antibody-label conjugate may be deposited onto the conjugate pad simultaneously with or sequentially to the lectin-label conjugate using the methods described above.

[0108] A signal will be detectable in the second detection region regardless of whether or not there is analyte in the sample. The primary antibody-label conjugate in the conjugate pad and secondary antibody in the detection region may collectively serve as a control for the assay which demonstrates that the assay is functional and that the results are valid.

[0109] Other control molecules are envisaged in accordance with the lateral flow test device. For example, the second detection region may comprise an immobilised anti-lectin antibody which is capable of binding to the lectin-label conjugate. In another embodiment, the second detection region may comprise mannan. However, in these embodiments, a significant excess of lectin-label conjugate molecules would be required on / within the conjugate pad to ensure free lectin-conjugate molecules (i.e. those not bound to analyte) were available to bind to the anti-lectin antibody or mannan in the second detection region to produce a detectable signal. This may be particularly difficult to achieve when a sample comprises a high level of analyte. Therefore, an independent control system using a primary and secondary antibody, as described above, is preferred.

[0110] Immobilisation of molecules to the detection region(s)

[0111] The capture molecule (for example, MBL) and secondary antibody may be immobilised onto the first detection region (for example, as a test line) and second detection region (for example, as a control line), respectively, by routine methods known to a person skilled in the art. For example, striping of the test and control lines onto a nitrocellulose membrane may be accomplished with the use of a dispensing instrument. Such instruments are commercially available (for example, Kinematic™, Biodot™, and Imagene™) which may use contact or non-contact dispensing. Handling of the membrane prior to and after striping is important for the performance. For example, nitrocellulose membranes that are too dry may result in spotty, non-uniform lines, whilst nitrocellulose membranes that are too damp may result in a widened test line that results in a decreased signal intensity. Therefore, the nitrocellulose membrane is preferably equilibrated to a controlled humidity environment of suitably humidity level. After striping, the capture molecule or secondary antibody is fixed to the membrane by standard drying and curing protocols. Once the nitrocellulose has been striped and dried, it is important that the membranes are stored in a moisture-free environment. Dried nitrocellulose will readily absorb any moisture in the environment, and this can result in proteins detaching from the striped lines. Accordingly, the lateral flow test device is preferably stored in a sealed pouch with a desiccant prior to use.

[0112] Signal detection

[0113] The signal produced by the detectable label of each of the lectin-label conjugate and primary antibody-label conjugate may be detected by any suitable means for the type of signal to be detected. In some embodiments, the detectable label produces an optical signal and the signal is detected, and the intensity thereof measured, using an optical signal sensor and / or reader. For example, an optical signal may be detected, and the intensity thereof measured, using a smartphone (e.g. using the camera of a smartphone). Alternatively, the optical signal may be detected, and the intensity thereof measured, using a reader including lateral low test device holder configured to receive the lateral flow test device for measurement. The holder may include one or more walls configured to enclose the lateral flow test device (e.g. to block external light). The reader may include a light source to illuminate the lateral flow test device during measurement. This may allow the amount of light incident on the lateral flow test device to be controlled during measurement. In other embodiments, the signal may be detected and intensity thereof assessed by the naked eye. Further discussion of the means of signal detection is provided below with reference to Figure 10.

[0114] The detection of the signal and optional assessment of the intensity thereof in each detection region may be performed on the signal present in each detection region at a minimum time after the test sample has been applied to the sample-receiving region. It will be understood that the detection of the signal intensities can be performed using the detection regions themselves (i.e. direct detection from the detection region) or using a reproduction, such as a photograph, of each detection region. In particular, sufficient time must elapse before the detection of the signal is performed in order to permit the test sample to migrate to the conjugate pad, and then to the detection region(s). The detection of the signal in each detection region may be performed after any suitable minimum time from the point of applying the sample to the sample-receiving region, for example, after at least 1 minute, at least 2 minutes, at least 3 minutes, at least 4 minutes, at least 5 minutes, at least 10 minutes, at least 15 minutes, at least 20 minutes, at least 25 minutes or at least 30 minutes. Preferably, the detection of the signal and optional assessment of the intensity thereof is performed at least 15 minutes, for example 15 to 20 minutes, after the test sample has been applied to the sample-receiving region.

[0115] The detection of the signal and optional assessment of the intensity thereof in each detection region may be based on the signal present in the detection region before a maximum time has elapsed from the application of the test sample to the samplereceiving region. For example, the detection of the signal and intensity thereof may be based on the signal present at the detection region(s) no more than 1 hour, no more than 50 minutes, no more than 45 minutes, no more than 40 minutes, no more than 35 minutes or no more than 30 minutes after the test sample has been applied to the samplereceiving region. In some embodiments, the detection of the signal and intensity thereof may be based on the signal present in each detection region no more than one hour after the test sample has been applied to the sample-receiving region.

[0116] The lateral flow test device of the present invention can be run qualitatively. A qualitative test only examines the sample for the presence or absence of the analyte, and may be assessed by the naked eye.

[0117] Alternatively, the lateral flow test device may be used to provide a semi-quantitative or a quantitative result. In particular, the lateral flow test device may be manufactured to have a known amount of each component, namely, the mobilisable lectin-label conjugate and capture molecule, in order to provide an appropriate detection threshold for the target analyte in question. The person skilled in the art will know how to adjust the amounts of the lectin-label conjugate and capture molecule in order to provide a certain threshold for a positive response (i.e. detection of target analyte in the test sample). Thus, a positive response would indicate that the threshold has been met or exceeded.

[0118] In some embodiments, the quantitation of analyte molecules in the test sample is carried out by measuring the signal intensity in the first detection region and optionally, in the second detection region. This may be achieved by obtaining one or more photographs of the detection region. The location of each detection region within each of the one or more photographs may be obtained through user input or using an image recognition method, such as edge detection and / or machine learning. The signal intensity of each detection region may be determined based on the intensity of one or more pixels representing the first detection region. This may be based on one pixel, or may be an accumulation of intensity values of multiple pixels (e.g. an average intensity). The pixel intensity values may then be converted into a signal intensity which is proportional to the concentration of analyte in the sample. Typically, signal intensities are compared to a calibration standard and converted to an analyte concentration value. Relevant calibration data may be encoded into the appropriate software. In some embodiments, the concentration of analyte is determined based on the ratio of signal intensity at the first detection region to the signal intensity at the second detection region (control region). This is to ensure that any differences in the performance between devices used for detection of the analyte (for example, smart phone, camera etc.) do not result in variations in the quantification of the analyte. In some embodiments, the determined concentration of analyte is categorised as negligible contamination, moderate contamination or heavy contamination based on existing calibration data.

[0119] The above-discussed possible minimum time for the detection of the signal intensities and possible maximum time that has elapsed from the application of the test sample to the sample-receiving region apply equally to the quantitation of the concentration of analyte in the test sample.

[0120] Measurement system

[0121] Figure 10 shows a schematic representation of a computing system 300 for quantifying the concentration of analyte molecule in a test sample based on the signal intensities of at least the first detection region, and optionally, the second detection region according to an embodiment of the invention.

[0122] The computing system 300 comprises a processor 310, memory 320, non-volatile memory 330 and an input / output (I / O) interface 340. Optionally, the computing system 300 may comprise an optical sensor 350 (e.g. a digital camera). A bus 310a connects the processor 310 to the memory 330 and other components of the computing system 300. The computing system 300 may be any form of computing system, such as a personal computer, a server, a smartphone, a tablet, etc.

[0123] The computing system 300 is controlled by the processor 310. The processor 310 is configured to quantify the concentration of an analyte in a test sample based on executable code stored in the non-volatile memory 330 and loaded for execution into memory 320 (e.g. Random Access Memory, RAM).

[0124] The quantified concentration is determined based on one or more measurements of signal intensity at the first detection region and optionally, the second detection region. These measurements may be obtained from one or more images (e.g. one or more photographs) of the lateral flow test device showing each of the detection regions. This image may be obtained by an optical sensor, such as a digital camera. The optical sensor may be integrated within the computing system (e.g. optical sensor 350) or may be within an external system. That is, the computing system 300 may be configured to obtain one or images of the lateral flow test device through an integrated optical sensor, or may be configured to obtain one or more images of the lateral flow test device from an external system (e.g. over a network, such as the internet).

[0125] Where the user makes use of a mobile device (e.g. smartphone or tablet) to obtain an image of the lateral flow test device, the mobile device may be configured, through software, to perform the steps described herein to quantify the concentration of analyte molecule in the test sample. Alternatively, the image may be transferred to another computing system (the computing system 300), e.g. in the cloud, for quantitation.

[0126] The quantified concentration determined by the processor 300 may be output via the I / O interface 340 (e.g. a screen or to an external system via a network, such as the internet), may be utilised by the processor 310 for further calculations, or may be stored into nonvolatile memory 330 for later use.

[0127] Absorbent Region

[0128] With further reference to Figure 2, in some embodiments, the lateral flow test device (200) comprises an absorbent region (216), which may be an absorbent pad. The absorbent region (216) absorbs excess sample containing reagents that have not been bound or immobilised in the assay, whilst maintaining capillary flow through the detection regions to clear the background and optimise signal intensity. In a preferred embodiment, an absorbent pad (216) is composed of cotton or cellulose.

[0129] Further Components

[0130] The lateral flow test device of the present invention may include further components for the optimisation of the lateral flow test device, in line with conventional practice in the art. These additional components may be any suitable components known in the art for use with lateral flow tests or ELISA assays. For example, the lateral flow test device may comprise a buffer for modifying the flow rate of the test sample along the solid support structure, or a blocking agent for reducing non-specific binding of lectin-label conjugate and reducing background signals. The blocking agent may be any suitable blocking agent known in the art, such as a blocking agent comprising BSA or PBS. In some embodiments, the lateral flow test device may include a buffer comprising a source of calcium ions (for example, calcium chloride) to facilitate binding of the lectin-label conjugate to the analyte, and binding of the first complex comprising the analyte and lectin-label complex to the capture molecule. This may be particularly desirable when the lectin of the lectin-label conjugate and the capture molecule comprise a calcium iondependent lectin such as MBL.

[0131] Sample containing analyte

[0132] The test sample that is applied to the lateral flow device may be any liquid sample of interest. In preferred embodiments, the sample containing the analyte to be detected is a fuel sample, and in particular, a hydrocarbon fuel sample. Examples of hydrocarbon fuel include, but are not limited to, gasoline, jet fuel and diesel. In other embodiments, the sample may be a biodiesel sample or a lubrication fluid sample. In yet further preferred embodiments, the sample may an aqueous or water-based sample such as a free water phase of a fuel sample which, for example, has been obtained by subjecting the fuel sample to gravity separation and isolating the free water phase. The test sample, and processing thereof is discussed in more detail below.

[0133] The analyte to be detected is not particularly limited. In preferred embodiments, the analyte comprises a microorganism or a microbial product which is derived from the microorganism. The microbial product may comprise a polysaccharide. For example, the polysaccharide may include, but is not limited to, a component of biofilm, lipopolysaccharide from a cell wall, or a secreted glycoprotein.

[0134] Microorganisms require the presence of water to grow and proliferate in fuel systems. Fuel systems typically contain small amounts of water that may arise from condensation within the systems. Due to the higher density of water compared to the fuel itself, water typically exist as a “free water phase” at the bottom of vessels and tanks in fuel systems, below the “fuel phase”. Microorganisms tend to be found in the water phase and at the fuel / water interface, feeding on alkane and additives in the fuel. Fuel samples which do not have a free water phase are known as “fuel only” samples. However, even in “fuel only” samples, microbial contamination can be significant. This is because variable levels of emulsified water may be found in the fuel phase (that is to say, “pockets” or “microdroplets” of water are suspended in the fuel phase), thus supporting microbial growth.

[0135] When obtaining a fuel sample to be tested using the lateral flow test device and / or in accordance with the method of the invention, the sample is preferably taken from the lowest point of any tank or fuel delivery system where any free water phase (even if not visible to the naked eye) is expected to exist and particulates may gather. This is likely to give the most representative result of microbial contamination. However, the fuel sample to be tested may also be taken from any other part of a tank or fuel delivery system. For example, the fuel sample may be taken from a fuel phase which lies above a fuel / water interface, or the fuel sample may be taken from a fuel / water interface. Accordingly, in order to detect an analyte (particularly, a microorganism and / or a microbial product) in a fuel sample using the lateral flow test device of the invention, preferably, it may first be determined whether the fuel sample contains a free water phase. For example, a fuel sample may be agitated and left to settle for 10 to 15 minutes to see whether a free water phase appears at the bottom of the sample. This method is known as gravity separation. If a free water phase does appear, then in some embodiments, the water phase is separated from the fuel phase, and tested independently of, and in addition, to the fuel phase. This may enable a more representative determination of microbial contamination. The water phase may be separated from the fuel phase using standard methods such as decanting the (upper) fuel phase, or using a pipette, syringe or the like to extract the water phase from the vessel containing the sample. If the fuel sample does not contain a free water phase, then the sample may be tested and / or treated directly in accordance with the methods below. Thus, any of a separated free water phase, a separated fuel phase, a fuel sample which has not undergone any separation process including a “fuel only” sample (that is to say, a sample in which there is no apparent water phase after settling) may be considered a test sample for detecting an analyte or a test sample in accordance with the invention. The possible methods of preparing a fuel sample prior to detection of the analyte are illustrated in Figure 11.

[0136] In preferred embodiments, the test sample is subjected to a treatment to enhance the extraction of the analyte before application to the lateral flow test device to obtain a treated sample. Specifically, the separated free water phase, separated fuel phase, a fuel sample which has not undergone any separation step, including a “fuel only” sample, may be mixed with a first surfactant comprising a PEGylated sorbitan or fatty acid ester thereof, and a second surfactant comprising a PEGylated alcohol. If the test sample is a separated fuel phase or a fuel sample which has not undergone any separation such as a “fuel only” sample, the first surfactant and the second surfactant are preferably provided in an aqueous composition. This ensures efficient extraction of the analyte from the fuel phase into a water phase. However, if the test sample is a separated free water phase, the first surfactant and the second surfactant may be provided in an aqueous composition or mixed directly with the test sample (that is to say, without providing the surfactants in an aqueous composition). The present inventors have unexpectedly found that when a sample comprising an analyte (particularly when the sample is a separated free water phase or a separated fuel phase derived from fuel, a fuel sample which has not undergone any separation such as a “fuel only” sample,) is mixed with the combination of the first and second surfactants prior to detecting the analyte, the accuracy and efficiency of detection is improved. In particular, the accuracy and efficiency of the detection of the analyte is improved when using the lateral flow test device as described herein to detect the analyte. More specifically, the present inventors have found that when using either of the first and second surfactants alone, variability in analyte detection is observed across different samples. Thus, for example, it has been discovered that whilst the first surfactant may outperform the second surfactant in a given fuel sample, the second surfactant may outperform the first surfactant in another fuel sample. (The term “outperform” refers to the amount of analyte extracted, and consequently, detected). In contrast, the combination of first and second surfactants consistently outperforms each of the first and second surfactants across different fuel samples. That is to say, the combination of the first and second surfactants consistently allows effective and improved detection of analytes across different fuel samples, particularly when the analyte is detected using the lateral flow test device as defined herein.

[0137] Accordingly, in a further aspect, the invention relates, in general terms, to a method for detecting an analyte in a test sample. The method comprises the following steps: i) mixing the test sample with at least a first surfactant and a second surfactant, wherein the first surfactant comprises a PEGylated sorbitan or fatty acid ester thereof, and wherein the second surfactant comprises a PEGylated alcohol, to obtain a treated sample; ii) allowing the treated sample to contact a lectin conjugated to a detectable label (lectin-label conjugate), wherein the lectin is capable of binding to the analyte, wherein the lectin-label conjugate binds to the analyte in order to form a first complex comprising the analyte and lectin-label conjugate; iii) allowing the first complex obtained in step ii) to contact a capture molecule, wherein the capture molecule is capable of binding to the analyte in the first complex, wherein the capture molecule binds to the analyte in the first complex in order to form a second complex comprising the capture molecule, analyte and lectin-label conjugate; and iv) detecting a signal from the detectable label of the lectin-label conjugate in the second complex formed in step iii), wherein a detected signal indicates the presence of analyte in the test sample.

[0138] It will be understood that in preferred embodiments, the method of the invention is performed using the lateral flow test device of the invention, as described above. However, other detection systems such as ELISAs are also envisaged.

[0139] First and second surfactants

[0140] The first surfactant comprises a PEGylated sorbitan or a fatty acid ester thereof. Sorbitan may be produced by routine methods involving the dehydration of sorbitol. The dehydration reaction usually produces sorbitan as a mixture of five- and six-membered cyclic ethers (1 ,4-anhydrosorbitol, 1 ,5-anhydrosorbitol and 1 ,4, 3, 6- dianhydrosorbitol) with the five-membered 1 ,4-anhydrosorbitol form being the dominant product. In preferred embodiments, the sorbitan moiety of the PEGylated sorbitan comprises 1 ,4-anhydrosorbitol, illustrated below as Formula I.

[0141] Formula I - 1 ,4-anhydrosorbitol

[0142] The term “PEGylated” refers to the covalent attachment of one or more polyethylene glycol (PEG) polymer chains, illustrated as Formula II below, to a given molecule (for example, sorbitan or alcohols in the present invention). PEGylation may also be carried out using routine methods.

[0143] Formula II - Polyethylene glycol (where n represents the number of ethylene glycol monomers)

[0144] In some embodiments, the first surfactant comprises a fatty acid ester of PEGylated sorbitan. The PEGylated sorbitan may comprise one to four fatty acid esters, one to three fatty acid esters, or one to two fatty acid esters. Preferably, the PEGylated sorbitan comprises one fatty acid ester.

[0145] In the above embodiments, the fatty acid ester may be a C6-20 fatty acid ester (e.g. C6-20 alkyl fatty acid ester), a C10-16 fatty acid ester (e.g. C10-16 alkyl fatty acid ester), or C12-14 fatty acid ester (e.g. C12-14 alkyl fatty acid ester). Preferably, the fatty acid moiety is a C12 fatty acid ester (e.g. C12 alkyl fatty acid ester, such as lauric acid ester). Most preferably, the first surfactant comprises polyethylene glycol sorbitan monolaurate.

[0146] The term “alkyl” as used herein refers to both straight and branched chain radicals of up to twenty carbons. For example, an alkyl group may contain 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19 or 20 carbon atoms. Non-limiting examples of C1-C12 alkyl groups include methyl, ethyl, propyl, isopropyl, butyl, sec-butyl, tert-butyl, 3-pentyl, hexyl and octyl groups.

[0147] The term “alkyl” as used herein refers to both straight and branched chain radicals of up to twenty carbons. For example, an alkyl group may contain 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19 or 20 carbon atoms. Non-limiting examples of C1-C12 alkyl groups include methyl, ethyl, propyl, isopropyl, butyl, sec-butyl, tert-butyl, 3-pentyl, hexyl and octyl groups.

[0148] The number of PEG linkages (that is to say, the number of PEG units attached to sorbitan) in the PEGylated sorbitan or fatty acid ester thereof may vary. In some embodiments, the PEGylated sorbitan or fatty acid thereof comprises 2 to 50, 5 to 40, 10 to 30, 15 to 25 PEG linkages. Preferably, the PEGylated sorbitan or fatty acid thereof comprises 20 PEG linkages. More preferably, the PEGylated sorbitan comprises a single C12 fatty acid ester (e.g. C12 alkyl fatty acid ester, such as lauric acid ester), and 20 PEG linkages (denoted by Formulae Illa and lllb below). This compound is commercially available under the tradename Tween™ 20.

[0149] The number of PEG linkages may be varied according to the desired properties of the first surfactant. Generally, the greater the number of PEG linkages in the PEGylated sorbitan or fatty acid ester thereof, the more water-soluble the compound will be.

[0150] Therefore, in samples with a low water content (for example, the water content of jet fuel may vary from 5 mg to 30 mg per kg fuel), it may be desirable to provide only 15 to 25 PEG linkages to enable efficient extraction of an analyte. In other samples with a higher water content, the number of PEG linkages may be increased to achieve efficient extraction of an analyte. The number of PEG linkages may also affect the aggregation properties of the surfactant such that a greater number of PEG linkages may be associated with a reduced propensity for aggregation.

[0151] Having regard to the above and in accordance with the invention, the PEGylated sorbitan may be represented structurally by Formula IVa or Formula IVb as follows:

[0152] Formula IVa:

[0153] Formula IVb: wherein R is hydrogen or C(=O)Ra; and Rais C5-C19 alkyl.

[0154] In accordance with the embodiments mentioned above:

[0155] C(=O)Rarepresents a fatty acid ester moiety. In preferred embodiments, Rais C9-15 alkyl (such that the fatty acid ester is a C10-16 alkyl fatty acid ester), or Cn-13 alkyl (such that the fatty acid ester is a C12-14 alkyl fatty acid ester). In further preferred embodiments, Rais C11 alkyl (corresponding to a C12 alkyl fatty acid ester (e.g. lauric acid ester)).

[0156] Preferably the number of R groups where R is C(=O)Rais between 1 to 4 or from 1 or 2. More preferably, the number of R groups where R is C(=O)Rais 1 (that is to say, the PEGylated sorbitan comprises a single fatty acid ester moiety.) In this preferred embodiment, the PEGylated sorbitan may be represented structurally by Formula Va or Formula Vb as follows, where Rais as defined above:

[0157] Formula Va Formula Vb

[0158] In Formulae IV and V, the sum of w, x, y and z represents the total number of PEG linkages. Thus, w+x+y+z may be from 2 to 50, from 5 to 40, from 10 to 30, or from 15 to 25. Preferably, w+x+y+z is 20.

[0159] More preferably, in Formulae Va and Vb, Rais On alkyl and w+x+y+z is 20. This compound is commercially available as Tween™ 20.

[0160] The second surfactant comprises a PEGylated alcohol. The PEGylated alcohol may comprise a PEGylated C1-C20 alcohol which may be represented structurally by Formula

[0161] VI below:

[0162] Formula VI: wherein R is C1-C20 alkyl; and x is 1 , 2 or greater than 2. Preferably, the PEGylated alcohol comprises a PEGylated Ce-Ci6 alcohol (R in Formula 3 is Ce-Ci6 alkyl).

[0163] More preferably, the PEGylated alcohol comprises a first PEGylated alcohol and a second PEGylated alcohol, wherein the first PEGylated alcohol comprises a PEGylated C6-C12 alcohol (R in Formula VI is C6-C12 alkyl) and the second PEGylated alcohol comprises a PEGylated C10-C16 alcohol (R in Formula VI is C10-C16 alkyl). An example of a preferred second surfactant is Ecosurf™ SA-9 (Dow).

[0164] In the above embodiments, the PEGylated alcohol may further comprise a polypropylene glycol (PPG) linkage. If the PEGylated alcohol comprises a first PEGylated alcohol and a second PEGylated alcohol, the first PEGylated alcohol and / or the second PEGylated alcohol may further comprise a PPG linkage. In these embodiments, the PEGylated alcohol comprising a PPG linkage may be represented structurally by Formula Vila and Formula VI lb below:

[0165] Formula Vila: wherein x is 1 , 2 or greater than 2; and y is 1 , 2 or greater than 2. In one embodiment, the PEGylated alcohol comprising the PPG linkage (or the first PEGylated alcohol comprising the PPG linkage and / or second PEGylated alcohol comprising the PPG linkage) is a block co-polymer or co-oligomer comprising at least one PEG linkage and at least one PPG linkage. In another embodiment, the PEGylated alcohol comprising the PPG linkage (or the first PEGylated alcohol comprising the PPG linkage and / or second PEGylated alcohol comprising the PPG linkage) is a random copolymer or co-oligomer comprising at least one PEG linkage and at least one PPG linkage.

[0166] The first surfactant and second surfactant are mixed with the sample in order to extract the analyte from the sample, and facilitate subsequent interaction with the lectin-label conjugate. By “mixing”, it is meant that the sample is brought into contact with the first surfactant and second surfactant. Mixing may further involve agitation which may be conducted manually or with an automated shaker. Preferably, the mixing is conducted at ambient temperature (for example from 20°C to 25°C). The duration of mixing is not particularly limited and may range from 30 seconds to 5 minutes. Preferably, the duration of mixing is from 30 seconds to 1 minute. The duration of mixing may be adjusted depending on the suspected level of analyte in a sample.

[0167] In some embodiments, the first and second surfactants are provided in an extraction composition comprising at least one additional component which is mixed with the test sample. Preferably, the extraction composition comprising at least one additional component is an aqueous composition comprising more than 50 wt.% water, for example, at least 60wt.%, at least 70 wt.%, at least 80 wt.%, at least 90 wt.% or at least 95 wt.% water. The at least one additional component may be selected from a buffering agent (for example, Tris base, PBS etc.), a salt (for example, calcium chloride), a dye (for example, a food dye such as Patent Blue Dye E131), and a preservative (for example, 2-methyl-4-isothiazolin-3-one (MIT)) which is commercially available as Proclin™ 950). Preferably, the salt is a calcium salt. Preferably, the extraction composition comprising the first and second surfactants comprises a calcium salt and a dye. As discussed above, the presence of calcium ions in the treated sample which is subsequently applied to a detection system such as the lateral flow test device described herein, may facilitate binding of certain lectins to the analyte. As discussed below, the presence of a dye in the composition may facilitate subsequent separation of a free water phase containing the extracted analyte from another phase which comprises substantially no analyte in the treated sample.

[0168] In a particular embodiment, the aqueous extraction composition comprises 10 to 20mM Tris base, 300 to 400 mM calcium salt, a preservative and a dye, in addition to the first and second surfactants. In a further embodiment, the aqueous extraction composition comprises 10 to 15 mM Tris base, 350 to 400 mM calcium salt, a preservative and a dye, in addition to the first and second surfactants In a preferred embodiment, the aqueous extraction composition comprises 10 to 15 mM Tris base, 350 to 400 mM calcium salt, 0.05 to 0.1% (v / v) ProclinTM950, and 0.5 to 1 .5% (w / v) Patent blue dye E131 , in addition to the first and second surfactants. The calcium salt may comprise calcium chloride.

[0169] The first surfactant and the second surfactant may each be present in the aqueous composition in an amount of from 0.01 to 0.05 wt.%, or from 0.02 to 0.04 wt.%, from 0.030 to 0.035 wt.%. The combined amount of the first and second surfactants may be from 0.02 to 0.1 wt.%, or from 0.05 to 0.07 wt.% or from 0.060 to 0.065 wt.%. Preferably, the first surfactant and the second surfactant are provided in the aqueous extraction composition in equal amounts by weight of the composition. In some embodiments, the combined amount of the first and second surfactants in the aqueous composition is 0.0625 wt.%.

[0170] In alternative embodiments, the first and second surfactants are provided in separate aqueous extraction compositions, and may be added to the sample sequentially prior to mixing.

[0171] When the test sample containing the analyte to be detected is non-aqueous or comprises a low amount of water (for example, less than 2 wt.% or less than 1 wt.%) such as a “fuel only” sample, or a separated fuel phase, as described above (see Figure 11), it is preferred that the first and second surfactants are provided within an aqueous composition (or separate aqueous extraction compositions, as mentioned above). As described below, this may facilitate extraction of the analyte into a free water phase, and subsequent detection of the analyte using the lateral flow test device as described herein or other systems such as ELISAs. The aqueous extraction composition comprising the first and second surfactants may be mixed with the sample with the sample by vigorous shaking, and allowed to settle for 1 to 10 minutes, or 2 to 6 minutes or 5 minutes. When the test sample comprises a low amount of water (for example, less than 2 wt.% or less than 1 wt.%) such as “fuel only” sample, or a separated fuel phase, the sample may fractionate into a free water phase which is enriched with the extracted analyte, and a non-aqueous phase which does not comprise the analyte or comprises negligible amounts of the analyte (by gravity separation). The different phases may be visible as layers in a vessel containing the sample. The free water phase enriched with the extracted analyte will typically form a lower layer due to its higher density and the non-aqueous phase which does not comprise the analyte will typically form an upper layer due to the lower density. In preferred embodiments, the aqueous extraction composition comprising the first and second surfactants comprises a dye to enhance visibility of the free water phase (or layer) comprising the extracted analyte. The free water phase (or layer) enriched with the extracted analyte may then be separated or isolated for subsequent detection of the analyte (see Figure 11). In some embodiments, a syringe, pipette or the like may be used to isolate the phase (or layer) enriched with the extracted analyte for subsequent application to the lateral flow test device of the invention, and more specifically, to the sample-receiving region of the lateral flow test device. In other embodiments, the sample may be treated with the first and second surfactants in a vessel which is configured to dispense the free water phase (or layer) enriched with the extracted analyte onto the sample-receiving region of the lateral flow test device through a dropper or the like.

[0172] In these embodiments, the aqueous extraction composition comprising the first and second surfactants becomes the running assay buffer of the lateral flow test device. However, other methods of detection of the extracted analyte are also envisaged by the invention including ELISA systems. As the aqueous extraction composition comprising the first and second surfactants may become the running assay buffer for the lateral flow test device and other detection systems, the inclusion of additional components in the aqueous composition may facilitate the detection of the analyte. For example, a calcium salt may facilitate the interaction of a calcium-dependent lectin such as MBL with the analyte. A buffer component may maintain the optimum pH (and thus optimum protein configurations) for binding of the lectin to the analyte. The aqueous extraction composition comprising the first and second surfactants may be mixed with the test sample containing the analyte for the time periods described above. Typically, the aqueous composition comprising the first and second surfactants is mixed with the test sample in an aqueous extraction composition : test sample ratio of 1 :100 to 1 :200, or from 1 :130 to 1 :160, or 1 :150 by volume. The aforementioned volume ratios are particularly preferred when the test sample is non-aqueous and comprises a low amount of water (for example, less than 2 wt.% or less than 1 wt.%) such as a “fuel only” sample (which does not separate into a fuel phase and a water phase after settling), or a separated fuel phase. Possible amounts of the first and second surfactants in the aqueous composition are defined above and are compatible with the aforementioned volume ratios.

[0173] A test sample containing an analyte to be tested which is aqueous or comprises a high amount of water (for example, more than 80 wt.% or more than 90 wt.% or more than 95 wt.%), such as a separated water phase obtained from a fuel sample or any other waterbased sample, may also be mixed with an aqueous extraction composition comprising the first and second surfactants. However, in these embodiments, there may be no separation of phases on mixing the test sample with the aqueous extraction composition comprising the first and second surfactants. Therefore, in these embodiments, the extracted analyte is expected to be distributed throughout the test sample. As such, no separation step is required before applying the treated sample to the sample-receiving region of lateral flow test device or allowing the treated sample to contact the lectin-label conjugate in another detection system (see Figure 11). In these embodiments, the aqueous extraction composition : test sample ratio may be from 1 :3 to 1 :1 , or from 1 :2 to 1 :1 , or 1 :1 , by volume. Possible amounts of the first and second surfactants in the aqueous composition are defined above and are compatible with the aforementioned volume ratios. In alternative embodiments, and as described above, if a test sample containing an analyte to be tested is aqueous or comprises a high amount of water as described above, the test sample may be mixed with the first and second surfactants directly (that is to say without providing the surfactants as an aqueous composition).

[0174] In some embodiments, the first and second surfactants may be mixed directly with the test sample (that is to say, without providing the surfactants in an aqueous extraction composition). In these embodiments, the test sample may comprise a high amount of water (for example, more than 80 wt.%, more than 90 wt.% of more than 95 wt.% water) such as the free water phase of a fuel sample. In these embodiments, one or more components of the aqueous extraction composition mentioned above (for example, buffer, calcium salt and preservative) may be added to the lateral flow test device. For example, the sample-receiving region may be pre-treated with one or more of the aforementioned components and dried, such that on contact with the sample, the components are released into the flow of sample.

[0175] In other embodiments, the first and second surfactants may themselves be contained within the lateral flow test device. For example, the sample-receiving region and / or conjugate pad may be pre-treated with the first and second surfactants and optionally, one or more of the other components of the aqueous extraction composition mentioned above (for example, buffer, calcium salt and preservative) and dried, such that on contact with the sample, the first and second surfactants and other components of the extraction composition, if present, are released into the flow of sample. In these embodiments, a free water phase derived from a fuel sample (for example, by gravity separation) may be added directly to the lateral flow test device without any prior treatment. Alternatively, in these embodiments, a “fuel only” sample or a fuel phase isolated from a fuel sample may be mixed with water to obtain a free water phase which can be added directly to the lateral flow test device which comprises the first and second surfactants and optionally, one or more of the other components of the aqueous extraction composition mentioned above (for example, buffer, calcium salt and preservative).

[0176] In some embodiments, the amount of each of the first and second surfactants when added to the test sample (whether added in the form of an aqueous composition or directly) may be less than 0.03 wt.%, less than 0.02 wt.%, less than 0.01 wt.% or less than 0.005 wt.%.

[0177] Detection of analyte

[0178] According to the method of the invention, the treated sample (that is to say, the sample that has been tmixed with the first and second surfactants) is allowed to contact a lectin- label conjugate. The lectin-label conjugate comprises a conjugate of a lectin and a first detectable label as described above. In some embodiments, the lectin-label conjugate is deposited on a solid support and provided in dry form. The solid support may be a component of a lateral flow device, such as a conjugate pad. The lectin-label conjugate binds to the analyte in order to form a first complex comprising the analyte and lectin- label conjugate, as illustrated in Figure 3.

[0179] In preferred embodiments, a quantity of the treated sample is applied to the samplereceiving region of lateral flow test device as described above. Typically, the sample is applied as 2 to 5 drops, preferably 4 drops, with each drop comprising approximately 50pl. With reference to Figure 3, the sample migrates by capillary action to the conjugate pad where deposited lectin-label conjugate becomes mobilised on contact with the sample. As described above, the analyte in the sample, if present, binds to mobilised lectin-label conjugate to form a first complex comprising the analyte and lectin-label conjugate. The sample containing the first complex subsequently migrates to the first detection region comprising an immobilised capture molecule. The capture molecule binds to the analyte from the first complex to form a second complex comprising the capture molecule, analyte and lectin-label conjugate. The second complex is immobilised at the first detection region, and a detectable signal is thus produced at the first detection region. The capture molecule and the detection of the signal at the first detection region may be as described above.

[0180] The method of detecting the analyte using the lateral flow test device described above may further comprise allowing the treated sample to contact a primary antibody conjugated to a second detectable label (primary antibody-label conjugate), wherein the primary antibody does not bind to the analyte. In this embodiment, the primary antibodylabel conjugate is deposited on the conjugate pad. On contact with the sample, the primary antibody-label conjugate becomes mobilised, migrates to the second detection region, and binds to a secondary antibody which has binding affinity for the primary antibody, to form a third complex. The third complex is immobilised at the second detection region, and a detectable signal is thus produced at the second detection region. The primary antibody-label conjugate, secondary antibody and detection of the signal at the second detection region essentially provide a control system to ensure correct functioning of the assay, and may be as described above. However, molecules of the control system are not particularly limited. Therefore, another control molecule which does not bind the analye may be used in place of the primary antibody. The secondary antibody may accordingly bind specifically to the control molecule. It will be understood that whilst the method of detecting an analyte as described herein is suitably performed using the lateral flow test device described herein, other systems for detection are also envisaged, for example, ELISAs. Thus, for example, the sample treated with the first and second surfactant may be contacted with the lectin-label conjugate in solution, and subsequently applied to a microtitre plate which is coated with the capture molecule such that the capture molecule is immobilised onto the microtitre plate. If the treated sample contains the target analyte, the first complex comprising the analyte and lectin-label conjugate will bind to the capture molecule. The label may then be detected using routine methods known to a person skilled in the art, as described above. The detectable label may be modified according to the detection system that is used. Thus, in an ELISA system, it may be desirable to use an enzyme such as alkaline phosphatase or horseradish peroxidase as the first detection label which provides a coIometric range on addition of the appropriate substrate.

[0181] Analyte

[0182] In preferred embodiments, the device and methods described herein are used for detecting a microorganism in a test sample. The microorganism may comprise a bacterium and / or a fungus. In some embodiments, the test sample comprises a bacterium and a fungus. In some embodiments, the test sample comprises a plurality of microorganisms.

[0183] In the above embodiments, the bacterium may comprise one or more of: Acinetobacter venetianus RAG-1 (ATCC 31012), Alcaligenes species, Bacillus licheniformis, Nocardiodes luteus, Pseudomonas aerigunosa (ATCC 27853), Pseudomonas putida (ATCC 12633), Pseudomonas fluorescens, Kocuria kristinae (ATCC 27570), Brevundimonas vesicularis (ATCC 11426), and Rhodococcus erythropolis.

[0184] In the above embodiments, the fungus may comprise one or more yeast selected from Candida guilliermondii (ATCC 6260), Rhodotorula mucilaginosa (ATCC 2510), Candida kersosiniea and Yarrowia lipolytica (ATCC 9773).

[0185] In other embodiments, the fungus may comprise one or more filamentous fungi selected from: Hormoconis. resinae (ATCC 20495), Aspergillus niger (ATCC 9642) and Penicillium coryphilum. In preferred embodiments, the microorganism comprises Hormoconis. resinae (ATCC 20495).

[0186] Kit

[0187] In a further aspect, the present invention also provides a kit comprising the lateral flow test device described above, a first surfactant comprising a PEGylated sorbitan or fatty acid ester thereof and a second surfactant comprising a PEGylated alcohol. The first and second surfactants may be provided within a composition, preferably an aqueous composition. The first and second surfactants and composition comprising the surfactants may be as described herein. The kit may also comprise instructions for using the lateral flow test device, and / or further components for carrying out the method of the present invention. The further components may be, for example, a test bottle containing the composition to which the fuel sample may be added. The test bottle may be configured to enable dispensing of the free water phase containing the extracted analyte to the sample-receiving region by means of a dropper or the like. Other components may include one or more syringes for separating a free water phase from a fuel phase in a fuel sample before treatment of the sample. The further components may be packaged separately from the lateral flow test device within the kit, such as in individual bottles or wrappers. In addition, a device for measuring optical density as described above for measuring the signal intensity at each detection region may be included with the kit. Alternatively, or in addition, the kit may comprise instructions for downloading and / or using software on a user device (e.g. a smartphone) for measuring the signal intensity at each detection region and quantifying the amount of analyte in the test sample.

[0188] Examples

[0189] Embodiments of the invention will be described by the following Example.

[0190] Example 1 - specification of lateral flow test device

[0191] A lateral flow test device was constructed according to the following specification and methods:

[0192] Sample Pad - Ahlstrom, grade 1281 (cotton / rayon blend); Width: 15mm.

[0193] Conjugate Pad- Ahlstrom, polyester pad, grade 661; Width: 15mm. The pad was pretreated withlOOmM Tris, 0.25% Tween 20, 0.5% BSA, pH 8.0.

[0194] Conjugates: i) Recombinant human MBL (Bio-Techne, 9086-MB) was conjugated with 40nm gold colloid, GC40). The concentration of the gold particles was OD 1. The MBL was coated at a concentration of 4.5pg / ml. The conjugation was performed in 25mM boric acid, 25mM sodium borate, pH 7. The conjugated particles were resuspended in 4mM Sodium tetraborate, 2% BSA, 0.2% Sodium azide, 0.2% Tween20 (v / v), 20% Trehalose, pH 9.0) at a final OD30 for application to the conjugate pad. ii) Chicken IgY (Lampire) was conjugated with 40nm gold colloid, GNS40OD10. The concentration of the gold particles was OD 10, and diluted to OD 1 prior to conjugation. The antibody was coated at a concentration of 2.5pg / ml. The conjugation was performed in 25mM boric acid, 25mM sodium borate, pH 7. The conjugated particles were resuspended in 4mM Sodium tetraborate, 2% BSA, 0.2% Sodium azide, 0.2% Tween20 (v / v), 20% Trehalose, pH 9.0) at a final OD25 for application to the conjugate pad.

[0195] The MBL-gold and IgY-gold conjugate preparations were mixed together prior to spray application to the conjugate pad.

[0196] Nitrocellulose membrane - Sartorius, Unisart CN 95, 25mm.

[0197] Striping of test line: The MBL coating concentration was 0.15 mg / mL (variable, used as steering lever). MBL (Bio-Techne, 9086-MB) was dispensed in 10mM sodium tetraborate, 300mM Sodium Chloride, 0.125% Tween20, pH8.0). The dispense rate was 0.1 |jL / mm; Striping of control line: The coating concentration of goat anti-chicken IgY (Lampire) was 0.5 mg / mL. Goat anti-chicken IgY was dispensed in 50 mM sodium borate, 1.5M NaCI, 10% Sucrose, pH8.0). The dispense rate was 0.1 L / mm.

[0198] After dispensing, the membrane was dried at 37°C for 16 to 24 hours and cured at 45°C in a desiccated foil bag for 44 to 52 hours.

[0199] Absorbent pad - Alhstrom, grade 222 (dried); Width: 27 mm.

[0200] Backing card - Kenosha, KN-V1075.60, 75 x 350 mm.

[0201] Test size - Length: 75 mm; Width: 4.0 mm

[0202] Assembly: Conidia Fuelstat™ ONE cassette

[0203] Calibration standard - Mannan (Sigma M7504) dissolved in UP water & subsequently diluted in extraction buffer (12.5mM Tris base, 375mM Calcium chloride, 0.03125% Tween™20, 0.03125% Ecosurf™SA9 , 0.6mL / L Proclin 950, 12.6mg / L Patent blue dye E131 , pH7.0).

[0204] Extraction buffer / assay buffer: 12.5mM Tris, 375mM CaCh, 0.03125 wt.% Tween™ 20, 0.03125 wt.% Ecosurf™ SA-9 surfactant, 0.6 ml / litre Proclin 950, and 12.5mg / litre Patent Blue dye E131.

[0205] Example 2 - Analyte detection using different surfactants

[0206] A microbial consortium (a laboratory-grown mixture of microorganisms derived from various contaminated jet fuel samples) was diluted with clean jet fuel in a ratio of 1 :20 by volume. Extraction of microorganisms was performed by mixing the contaminated jet fuel with an extraction buffer comprising 12.5mM Tris, 375mM CaCh, pH7, and 0.0625 wt.% surfactant. Specifically, the extraction buffer was mixed with the artificially contaminated jet fuel in a buffer to fuel ratio of 1 :150 (333pl buffer in 50ml fuel) by agitation for 30 seconds or 60 seconds. After the specified time periods, the sample was allowed to settle into a free water phase and a non-aqueous phase (by gravity separation). Four drops of the free water phase were applied to a lateral flow test (LFT) strip comprising MBL-gold deposited on a conjugate pad and immobilised MBL on a nitrocellulose membrane as described in Example 1 with the exception that MBL was immobilised onto the nitrocellulose membrane in a spot format rather than as a test line. The resulting colour change of the indicator spot was then scored by eye using a score chart, and optical density of the spot measured using a cube reader (Chembio Diagnostics GmbH). The results are illustrated in Table 1 below.

[0207] Table 1 - Results of analyte detection using different surfactants Table 1 illustrates that Tween™ 20 exhibited effective analyte detection. Ecosurf™ SA-

[0208] 9 enabled an improved detection of analyte relative to Tween™ 20, particularly after 30 seconds of mixing. Other surfactants such as TMN10 and Ecosurf™ EH9 appeared to perform less effectively, relative to Tween™ 20. Therefore, Tween™ 20 and Ecosurf™ SA-9 were selected for further investigation.

[0209] Example 3 - comparison of Tween™ 20 and Ecosurf™ SA-9 in field diesel samples with 30 and 60 seconds extraction time.

[0210] Three different diesel samples (contaminated field samples) (diesel samples 1 to 3, respectively) were subjected to analyte extraction using the protocol and extraction buffer detailed in Example 2. Analyte was subsequently detected using an LFT strip and cube reader as detailed in Example 2. The results are illustrated in Figures 4 to 6. In these Figures, “T” represents Tween™ 20 (referred to hereinafter as Tween 20) and “S” represents Ecosurf™ SA-9 (referred to hereinafter as Ecosurf SA-9).

[0211] Figure 4 (diesel sample 1) illustrates that Tween 20 performs better than Ecosurf SA-9 with 30 seconds mixing and 60 seconds mixing (that is to say, a higher amount of analyte was detected using Tween 20 as compared to Ecosurf SA-9) . Figure 5 (diesel sample 2) illustrates that Tween 20 and Ecosurf SA-9 perform comparably. Figure 6 (diesel sample 3) illustrates that Ecosurf SA-9 performs betters than Tween 20 with 30 seconds mixing and 60 seconds mixing. Thus, we may conclude that there is variability in the relative performance of Tween 20 and Ecosurf SA-9 across different fuel samples.

[0212] Example 4 - effect of combining Tween 20 and Ecosurf SA-9 on analyte extraction (1)

[0213] The method of Example 3 was repeated with diesel sample 1 with some modifications. Briefly, an extraction buffer comprising 12.5mM Tris, 375mM CaCh, and 0.0625 wt.% Tween 20, pH7 was added to the diesel sample. The mixing time was 30 seconds prior to applying to an LFT strip. Additionally, an extraction buffer comprising 12.5mM Tris, 375mM CaCh, 0.03125 wt.% Tween 20 and 0.03125 wt.% Ecosurf SA-9, pH7 was tested (referred to as “TSA”). (In the TSA buffer, the total amount of surfactants was maintained at 0.0625 wt.%). Analyte was detected by performing optical density measurements on the test spot of the LFT device using a Cube reader as above.

[0214] Figure 7 illustrates that TSA performs similarly to Tween in diesel sample 1 at the same total detergent concentration. The results of the extraction buffer comprising Tween 20 as the only surfactant are consistent with Example 3. Thus, it may be concluded that both TSA and Tween 20 perform better than EcoSurf SA-9 in diesel sample 1. (The extraction buffer alone (that is to say, without diesel sample) acted as a negative control.)

[0215] Example 5 - effect of combining Tween 20 and Ecosurf SA-9 on analyte extraction (2)

[0216] The method of Example 3 was conducted on new diesel sample 4 with some modifications. Analogously to Example 3, an extraction buffer comprising 12.5mM Tris, 375mM CaCh, and 0.0625 wt.% Tween 20, pH7 was mixed with the sample for 60 seconds prior to applying to an LFT strip. Additionally, the amount of surfactant in the extraction buffer was varied as follows:

[0217] Buffer 1 : 0.03125 wt.% Tween 20 and 0.03125 wt.% Ecosurf SA-9;

[0218] Buffer 2: 0.0625 wt.% Tween 20 and 0.0625 wt.% Ecosurf SA-9; and Buffer 3: 0.125 wt.% Tween 20 and 0.125 wt.% Ecosurf SA-9

[0219] Analyte was detected by performing optical density measurements on the test spot of the LFT device using a Cube reader as above. The total amount of surfactants was maintained in extraction buffer 1 and thus provided the most appropriate comparison with the extraction buffer comprising Tween 20 as the only surfactant. Figure 8 illustrates that the combination of Tween 20 and Ecosurf SA-9 in a total amount of 0.0625 wt.% enabled a more effective analyte detection than Tween 20 in the same amount of 0.0625 wt.%.

[0220] Example 6 - effect of combining Tween 20 and Ecosurf SA-9 on analyte extraction (4)

[0221] Three buffers were prepared containing: 12.5mM Tris, 375mM CaCh, 0.6ml / litre Proclin 950, 12.5mg / litre Patent Blue dye E131 and 0.0625% total surfactant. The surfactant was varied as follows: i) 0.0625 wt.% Tween 20 (“T20”); ii) 0.0625 wt.% Ecosurf SA-9 (“SA9”) iii) 0.03125 wt.% Tween 20 and 0.03125 wt.% Ecosurf SA-9 (“TSA”)

[0222] As in the above Examples, 333pl of each of buffers i) to iii) were mixed with 50ml aliquots of contaminated jet fuel field samples CB23 / 4 / 7, CB23 / 48, CB23 / 413 and CB23 / 415 and shaken for 60 seconds to extract the analyte. After allowing the samples to settle, a free water phase became visible at the bottom of the sample. Four drops of the free water phase were applied to an LFT strip as described in the above Examples, and analyte detection was carried out by visual inspection and scoring of the indicator spot on the LFT strip, and by taking optical density measurements using a Cube reader as above.

[0223] Figure 9A which represents the visual scoring illustrates that in each of samples CB23 / 4 / 7, CB23 / 48, and CB23 / 415, the TSA extraction buffer (comprising the combination of Tween 20 and Ecosurf SA-9) enabled an improved analyte detection over the buffer containing Tween 20 alone or Ecosurf SA-9 alone.

[0224] Figure 10 represents the optical density results of the test spot and similarly illustrates that in each of samples CB23 / 4 / 7, CB23 / 48, and CB23 / 415, the TSA extraction buffer (comprising the combination of Tween 20 and Ecosurf SA-9) enabled an improved analyte detection over the buffer containing Tween 20 alone or Ecosurf SA-9 alone.

[0225] Although in sample CB23 / 4 / 13 there was an observed increased in extraction efficiency with the TSA extraction buffer as compared to the buffer comprising Tween 20 alone, there did not appear to be any significant increase in extraction efficiency over Ecosurf SA-9 alone.

[0226] Summary of Examples

[0227] The data presented herein demonstrate that detection of the presence of microorganisms in fuel samples using a lateral flow test device may be improved when the fuel samples undergo a treatment to extract the microorganisms and microbial products before application to the lateral flow test device. In particular, the data demonstrate that extracting microorganisms and microbial products using Tween 20 or Ecosurf SA-9 as the only surfactant may produce variable results across different samples. For example, it has been demonstrated that in some fuel samples Tween 20 allows a more efficient detection of microbial status than EcoSurf SA-9, whilst in other fuel samples, EcoSurf SA-9 outperforms Tween 20. However, when Tween 20 and Ecosurf SA-9 are provided in combination, there is an unexpected improvement in extraction of microorganisms and and microbial products and their subsequent detection by lateral flow testing. The improvement is observed across different samples indicating that extraction using the combination of Tween 20 and Ecosurf SA-9 is unexpectedly not only more effective than the individual surfactants, but additionally, the improved efficiency of extraction is consistent despite the inevitable variation across different samples. It is further demonstrated that the lateral flow test devices used in the above examples which comprise MBL-gold in the conjugate pad as the target analyte-binding molecule, and MBL in the test area to capture MBL-gold which has bound to microorganisms, rather than conventional antibodies, are effective in detecting a broad range of microorganisms and microbial products in fuel samples.

Claims

CLAIMS:

1. A lateral flow test device for detecting an analyte in a test sample, the device comprising a solid support structure, the solid support structure comprising a samplereceiving region, a conjugate pad, and at least one detection region, wherein the at least one detection region comprises a first detection region, wherein the solid support is configured to permit liquid to flow sequentially from the sample-receiving region to the first detection region via the conjugate pad, wherein: i) the conjugate pad comprises a lectin conjugated to a detectable label (lectin-label conjugate), wherein the lectin-label conjugate is capable of mobilisation on contact with the sample, wherein the lectin moiety of the lectin-label conjugate is capable of binding to the analyte to form a first complex comprising the analyte and lectin-label conjugate; ii) the first detection region comprises an immobilised capture molecule which is capable of binding to the analyte in the first complex to immobilise the first complex and to form a second complex comprising the capture molecule, analyte and lectin-label conjugate; and iii) the detectable label of the lectin-label conjugate in the second complex is capable of producing a detectable signal in the first detection region.

2. The lateral flow test device according to claim 1 , wherein the lectin conjugated to the detectable label comprises mannan-binding lectin, and / or wherein the detectable label of the lectin-label conjugate comprises a nanoparticle, preferably a gold nanoparticle.

3. The lateral flow test device according to claim 1 or claim 2, wherein the immobilised capture molecule comprises a lectin, preferably mannan-binding lectin.

4. The lateral flow test device according to any one of claims 1 to 3, wherein the conjugate pad further comprises a primary antibody conjugated to a detectable label (primary antibody-label conjugate), wherein the primary antibody-label conjugate is capable of mobilisation on contact with the sample, and wherein the primary antibody does not bind to the analyte, andwherein the at least one detection region comprises a second detection region, wherein the second detection region comprises an immobilised secondary antibody which is capable of binding to the primary antibody to immobilise the primary antibody-label conjugate and form a third complex, and wherein the detectable label of the primary antibody-label conjugate in the third complex is capable of producing a detectable signal in the second detection region, optionally wherein the detectable label of the primary antibody-label conjugate comprises a nanoparticle, preferably a gold nanoparticle.

5. A method for detecting an analyte in a test sample, the method comprising the steps of: i) mixing the test sample with at least a first surfactant and a second surfactant, wherein the first surfactant comprises a PEGylated sorbitan or fatty acid ester thereof, and wherein the second surfactant comprises a PEGylated alcohol, to obtain a treated sample; ii) allowing the treated sample to contact a lectin conjugated to a detectable label (lectin-label conjugate), wherein the lectin is capable of binding to the analyte, wherein the lectin-label conjugate binds to the analyte in order to form a first complex comprising the analyte and lectin-label conjugate; iii) allowing the first complex obtained in step ii) to contact a capture molecule, wherein the capture molecule is capable of binding to the analyte in the first complex, wherein the capture molecule binds to the analyte in the first complex in order to form a second complex comprising the capture molecule, analyte and lectin-label conjugate; and iv) detecting a signal from the detectable label of the lectin-label conjugate in the second complex formed in step iii), wherein a detected signal indicates the presence of analyte in the test sample.

6. The method according to claim 5, wherein the test sample comprises a fuel sample, preferably a hydrocarbon fuel sample or a biodiesel fuel sample, optionally wherein the method comprises separating the fuel sample into a fuel phase and a free water phase, and in step i), the test sample comprising the separated fuel phase or theseparated free water phase is mixed with the at least first surfactant and the second surfactant to obtain the treated sample, or wherein the test sample comprises a lubrication fluid sample.

7. The method according to claim 5 or claim 6, wherein in step i), the test sample is mixed with an aqueous composition comprising the first and second surfactants, optionally, wherein the aqueous composition further comprises one or more components selected from a buffer, a salt, a dye and a preservative.

8. The method according to claim 7, wherein in step i), after the test sample is mixed with an aqueous composition comprising the first and second surfactants, the test sample is separated into a free water phase enriched with analyte and at least one other phase, and wherein the treated sample comprises the separated free water phase enriched with analyte.

9. The method according to claim 7 or claim 8, wherein each of the first surfactant and second surfactant is present in the aqueous composition in an amount of from 0.01 to 0.05 wt.%, or from 0.02 to 0.04 wt.%, or from 0.030 to 0.035 wt.% by total weight of the composition, and optionally, wherein the test sample is non-aqueous and in step i), the ratio of aqueous composition to test sample is from 1 :100 to 1 :200, or from 1 :130 to 1 :160, or 1 :150 by volume, or wherein the test sample is aqueous and in step i), the ratio of aqueous composition to test sample is from 1 :3 to 1 :1 , or from 1 :2 to 1 :1 , or 1 :1 , by volume.

10. The method according to any one of claims 5 to 9, wherein the lectin conjugated to the first detectable label comprises mannan-binding lectin, and / or wherein the capture molecule comprises a lectin, preferably, mannan-binding lectin.

11. The method according to any one of claims 5 to 10, wherein the lectin-label conjugate is deposited on a solid support, wherein the lectin-label conjugate is mobilisedwhen brought into contact with the sample comprising the analyte in step ii), and optionally, wherein the capture molecule is immobilised on a solid support, and immobilises the first complex on binding to the analyte in the first complex.

12. The method according to any one of claims 5 to 11 , wherein the first surfactant comprises a fatty acid ester of PEGylated sorbitan, preferably wherein the PEGylated sorbitan comprises one to four fatty acid esters, or one to two fatty acid esters, or one fatty acid ester, and optionally, wherein the fatty acid ester is a C6-20 fatty acid ester, C10-16 fatty acid ester, C12-14 fatty acid ester, or a C12 fatty acid ester.

13. The method according to claim 12, wherein the first surfactant comprises polyethylene glycol sorbitan monolaurate.

14. The method according to any one of claims 5 to 13, wherein the PEGylated sorbitan or fatty acid ester thereof comprises 2 to 50, 5 to 40, 10 to 30, 15 to 25, or 20 PEG linkages.

15. The method according to any one of claims 5 to 14, wherein the PEGylated alcohol comprises a PEGylated C1-C20 alcohol, preferably wherein the PEGylated alcohol comprises a PEGylated Ce-Cie alcohol, and more preferably, wherein the PEGylated alcohol further comprises a polypropylene glycol (PPG) linkage.

16. The method according to any one of claims 5 to 15, wherein the PEGylated alcohol comprises a first PEGylated alcohol and a second PEGylated alcohol, the first PEGylated alcohol comprises a PEGylated C6-C12 alcohol and the first PEGylated alcohol comprises a PEGylated C10-C16 alcohol, preferably,wherein the first PEGylated alcohol and the second PEGylated alcohol each comprise a polypropylene glycol (PPG) linkage.

17. The method according to any one of claims 5 to 16, wherein the method further comprises allowing the treated sample to contact a primary antibody conjugated to a detectable label (primary antibody-label conjugate), wherein the antibody moiety of the primary antibody-label conjugate does not bind to the analyte, and wherein the method further comprises contacting the primary antibody-label conjugate with a secondary antibody which binds to the primary antibody to form a third complex comprising the primary antibody-label conjugate and secondary antibody bound thereto, and detecting a signal from the detectable label in the third complex, preferably wherein the primary antibody-label conjugate is provided on a solid support, and wherein the primary-label conjugate is mobilised when brought into contact with the sample obtained from step i), and more preferably, wherein the secondary antibody is immobilised on a solid support, and immobilises the third complex on binding to the primary antibody in the third complex.

18. The method according to any one of claims 5 to 17, wherein the detectable label of the lectin-label conjugate comprises a nanoparticle, preferably a gold nanoparticle, and / or the method according to claim 17, wherein the detectable label of the primary antibody-label conjugate comprises a nanoparticle, preferably a gold nanoparticle.

19. The method according to any one of claims 5 to 18, wherein the analyte comprises a microorganism and / or a microbial product which is optionally a microbial polysaccharide, wherein the microorganism comprises a bacterium and / or fungus, and preferably, wherein the bacterium comprises one or more of: Acinetobacter venetianus RAG-1 (ATCC 31012), Alcaligenes species, Bacillus licheniformis, Nocardiodes luteus, Pseudomonas aerigunosa (ATCC 27853), Pseudomonas putida (ATCC 12633),Pseudomonas fluorescens, Kocuria kristinae (ATCC 27570), Brevundimonas vesicularis (ATCC 11426), and Rhodococcus erythropolis', wherein the fungus comprises one or more yeast selected from Candida guilliermondii (ATCC 6260), Rhodotorula mucilaginosa (ATCC 2510), Candida kersosiniea and Yarrowia lipolytica (ATCC 9773); and / or wherein the fungus comprises one or more filamentous fungi selected from: Hormoconis. resinae (ATCC 20495), Aspergillus niger (ATCC 9642) and Penicillium coryphilum.

20. The method according to any one of claims 5 to 19, wherein the method comprises using the lateral flow test device of any one of claims 1 to 4.

21. A kit comprising: i) a lateral flow test device according to any one of claims 1 to 4; and ii) a first surfactant comprising a PEGylated sorbitan or fatty acid ester thereof and a second surfactant comprising a PEGylated alcohol.

22. The kit according to claim 21 , wherein the first surfactant is as defined in any one of claims 12 to 14, the second surfactant is as defined in claim 15 or claim 16, and optionally, wherein the first surfactant and the second surfactant are provided within an aqueous composition, preferably, wherein the aqueous composition is as defined in claim 7 or claim 9.

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