Lateral flow device

Abstract

The invention provides lateral flow membranes, lateral flow devices, and methods of producing lateral flow membranes. Exemplary lateral flow membranes include a fluid flow path, wherein the fluid flow path includes a detection zone and one or more regions of higher density than another region of the fluid flow path, wherein the one or more regions of higher density extend across the entire thickness of the lateral flow membrane, and wherein the one or more regions of higher density are at least located within and / or adjacent to the detection zone.

Description

[0001] Lateral Flow Device

[0002] Field of the Invention

[0003] The present invention relates to a lateral flow membrane, a lateral flow device comprising said membrane, and a method of producing said lateral flow membrane.

[0004] Background to the Invention

[0005] As a commonly used embodiment of the diagnostic immunoassay, lateral flow devices (LFDs) approximate many ideal features of a diagnostic device. These comprise stability, low cost, scalability, few handling steps, ease of use and a clear indication of results. With regard to conducting the diagnostic read-out of the assay, it has no need for trained staff or specialised equipment.

[0006] Known for their well-known use in home pregnancy tests, LFDs have demonstrated their utility in both a point-of-care, clinical and home diagnostic setting at an unprecedented scale and with indisputable public health benefits within the context of the SARS nCOV2 pandemic. To date, LFDs remain in widespread clinical use in healthcare settings, despite many advances in diagnosis.

[0007] Lateral flow tests typically utilise a single capillary device or porous carrier that contains some (or preferably all) of the reagents necessary for the performance of an assay. These reagents are typically contained within discrete zones of the device, such that as fluid flows along the device by capillary flow, the various reactions occur sequentially, and a signal is generated at a detection zone that is indicative of the presence and / or amount of analyte in the sample. The devices can be a single membrane with reagents deposited at specific sites or be composed of a series of discrete pads or membranes (each containing none, one or more reactants) arranged such that their edges are in liquid contact with one another. A typical lateral flow device will comprise a sample receiving zone (which may optionally contain buffers and chemicals necessary for the test), a label zone (which contains an analyte-specific binding reagent, such as an antibody, releasably bound to the membrane), a capture and detection zone (which contains an analyte-binding reagent immovably immobilised on the membrane), and an absorption sink of sufficient capacity to enable unbound labelled reagent to wash out of the detection zone. The pads or membranes are typically attached to an impervious backing, and the pads or membranes are in liquid contact with one another (usually achieved by overlapping the edges and the use of an adhesive or a lamination layer). Optionally, the device is encased in a protective housing with defined apertures for sample application and visualisation of the result. Examples of such devices include those disclosed in US 5,656,503, US 5,622,871, US 5,602,040, and US 4,861,711.

[0008] The SARS nCOV-2 context has highlighted the issue of low and insufficient sensitivity of current approaches in LFDs. This presented a major concern in the SARS nCOV-2 pandemic, specifically the carriage of lower levels of the virus by asymptomatic or pre- symptomatic individuals, which were not detected by LFDs. Whilst being highly specific, the low sensitivity of these assays is a major drawback of the technology and leads to an increased sense of confidence provided by a false-negative result. This was highlighted by group and population-level studies (Deeks et al., 2022), as well as highlighting the significant delay post-infection for visible detection thresholds to be reached (Cobb et al., 2022).

[0009] A problem with LFDs is the insufficient or absent optical read-out, as generated on the test region of the strip. Many factors have been shown to affect the signal generation on a true positive LFD, such as speed and composition of the flow through the porous membrane, antibody concentration and its affinity / avidity to the antigen, as well as buffer choices. A number of factors have been implicated in assay performance and potential improvement in sensitivity and specificity. However, these are principally targeted at sample pre-treatment (e.g., isothermal amplification and sample enrichment) or signal amplification through additional steps to generate a higher signal read-out.

[0010] Previous studies have attempted to improve LFD read-out sensitivity through signal amplification, such as laser-based detection systems or secondary reporters. These may be based on peroxidases, such as luciferase or horseradish peroxidase with luminogenic or chromogenic substrates. However, whilst delivering improved sensitivity, these approaches negate some intrinsic aspects of the simplicity of LFDs. The steps may introduce the need for specialised light sources and detectors, or introduce further steps, increasing background signal, cost and complexity of the aforementioned tests.

[0011] There is, therefore, a need to provide LFDs with improved sensitivity, which does not require additional processing steps or specialised light sources or detectors.

[0012] Summary of the Invention

[0013] According to a first aspect, there is provided a lateral flow membrane comprising a fluid flow path, wherein the fluid flow path comprises a detection zone and one or more regions of higher density than another region of the fluid flow path, wherein the one or more regions of higher density extend across the entire thickness of the lateral flow membrane, and wherein the one or more regions of higher density are at least located within and / or adjacent to the detection zone.

[0014] The inventors have developed an improved lateral flow membrane with different regions of density within the fluid flow path. One or more regions of higher density are located at least within and / or adjacent to the detection zone, which, without wishing to be bound by theory, are considered to alter the speed and / or the flow path of the fluid flow to increase the deposition of signal in the detection zone. This means that signal is more likely to be detected (e.g., by the naked eye or using a detection means), thus improving the sensitivity of the lateral flow assay and increasing the confidence of a positive result when the concentration of analyte is low. Accordingly, the number of false negative readouts is reduced. In addition, the membrane can be easily integrated into current manufacturing and assay methodologies in a cost-effective manner without the introduction of further steps, analytes, or detection methods required for assay read-out.

[0015] The term "lateral flow", as used herein, refers to capillary flow through a material, often in a horizontal direction, but will be understood to apply to the flow of a liquid from the point of application of the liquid to another lateral position, even if, for example, the device is vertical or on an incline. Lateral flow depends upon the properties of the liquid / substrate interaction (surface wetting or wicking action) and does not require or involve the application of outside forces, e.g., vacuum or pressure applications by the user. The lateral flow membrane may be any suitable membrane capable of providing lateral flow and that can be modified to provide the one or more regions of higher density. The term "lateral flow membrane" used herein is intended to mean the membrane comprising reagents (e.g., antibodies) through which the sample migrates. Typically, the lateral flow membrane comprises one or more porous carrier materials, including any natural or synthetic polymer that is capable of providing lateral flow. Various lateral flow membranes are known in the art and include membranes formed from or comprising nitrocellulose, nitrocellulose blends with polyester and / or cellulose, cellulose, cellulose acetate, untreated paper, porous paper, rayon, glass fiber, acrylonitrile copolymer, polyvinylidene fluoride (PVDF), cross-linked dextran, charge-modified nylon, polyethersulfone (PES), polydimethylsiloxane (PDMS), and other porous polymers. In some embodiments, the lateral flow membrane is a cellulose-based membrane. In some embodiments, the lateral flow membrane is a nitrocellulose membrane. Nitrocellulose membranes used for lateral flow assays are well known in the art (e.g., Millipore HF75, HF90, HF120, HF135, HF180; Sartorius CN95, CN140, CN150; Whatman / GE AE98, AE99, FF120HP, FF170HP; MDI NC 15 pm, NC 8pm, NC 5 pm).

[0016] Any suitable pore size or average pore size may be used for the lateral flow membrane. The term “pore size”, as used herein, refers to the diameter of the pore in the filtration direction. The average pore size may be used, referring to the average (mean) diameter of the pores in the filtration direction. The skilled person will appreciate that pore size or average pore size may be adjusted to modify the capillary flow time. In some embodiments, the pore size or average pore size is 1 to 20 pm.

[0017] The lateral flow membrane has a length (i.e., in the longitudinal direction of the lateral fluid flow path), a width (i.e., perpendicular to the length and in the plane of the membrane) and a thickness (i.e., the height of the membrane). The lateral flow membrane may be any suitable size or shape provided the basic functional requirements are met for a lateral flow membrane, which are known to the person skilled in the art. Typically, the lateral flow membrane is rectangular. Any suitable membrane thickness can be selected for the lateral flow membrane. The skilled person will appreciate that membrane thickness impacts the bed volume, and thus critical parameters in routine use of LFDs, such as flow rates and background signal, in addition to readout times and sensitivity. In some embodiments, the membrane thickness (at the unmodified regions and / or at a region which is not the one or more regions of higher density) is 50 to 250 gm. In some embodiments, the membrane thickness is 75 to 225 gm. In some embodiments, the membrane thickness is 100 to 200 gm. In some embodiments, the membrane thickness is 125 to 175 pm. In some embodiments, the membrane thickness is 150 to 250 gm. In some embodiments, the membrane thickness is 160 to 240 gm. In some embodiments, the membrane thickness is 170 to 230 gm. In some embodiments, the membrane thickness is 180 to 220 pm. In some embodiments, the membrane thickness is 190 to 210 gm. In some embodiments, the membrane thickness is 100 to 120 gm. In some embodiments, the membrane thickness is about 200 gm.

[0018] In some embodiments, the membrane thickness at the one or more regions of higher density is 10 to 150 gm. In some embodiments, the membrane thickness at the one or more regions of higher density is 20 to 125 gm. In some embodiments, the membrane thickness at the one or more regions of higher density is 30 to 100 gm. In some embodiments, the membrane thickness at the one or more regions of higher density is 40 to 75 pm. In some embodiments, the membrane thickness at the one or more regions of higher density is 20 to 75 gm. In some embodiments, the membrane thickness at the one or more regions of higher density is 20 to 70 gm.

[0019] Any combination of the above ranges is possible. For example, in some embodiments, the membrane thickness at the one or more regions of higher density is 10 to 150 pm and the membrane thickness at the other region is 160 to 250 pm. As a further example, in some embodiments, the membrane thickness at the one or more regions of higher density is 20 to 70 pm and the membrane thickness at the other region is 100-120 pm.

[0020] In some embodiments, the lateral flow membrane is not folded or bent out of plane.

[0021] In some embodiments, a surface film, foil or coating is applied to the lateral flow membrane. Various films, foils and coatings are known in the art and may be applied, for example, to improve analyte binding and / or to modify the hydrophobicity. In some embodiments, the film, foil or coating is a nanofibre coating, for example, a nitrocellulose fibre coating.

[0022] The term “fluid flow path”, as used herein, refers to the portion of the lateral flow membrane through which reagents and analytes flow. This is typically through the thickness of the membrane along the length of the membrane. Typically, the fluid flow path comprises a sample-receiving zone (i.e., where sample and conjugate is applied or wicked onto the membrane; typically in contact with a conjugate release pad), a detection zone (i.e., where the analyte-conjugate complexes are detected) and an absorbent zone (typically in contact with an absorbent pad). The detection zone typically includes a test region for detecting the presence of a target analyte in a fluid sample and a control region for indicating the successful completion of an assay test. The test region typically comprises analyte-specific molecules which are able to bind to and immobilize the analyte-conjugate complex, resulting in a visible coloured line. The control region typically does not contain analyte-specific molecules but is able to fix non-bound conjugate-containing particles. The formation of a coloured line at the control region indicates that the sample has flowed past the test region. The colour intensity observed at the test region is directly proportional to the analyte concentration in the sample and therefore enables semi-quantitative interpretation of the test result. The control region is typically downstream of the test region (i.e., the control region is after the test region in the fluid flow path; the control region is a greater distance from the sample-receiving zone compared to the test region).

[0023] In a typical lateral flow test, a fluid sample to be tested is loaded onto a sample application pad. This may also involve the separation of analyte constituents. For instance, for whole blood or capillary blood samples, separation of blood cells and plasma takes place on the sample pad. The liquid fraction of the sample then moves through a conjugate release pad onto which a conjugate has been dried. The conjugate consists of detection molecules specifically directed against the analyte of interest and indicator particles, such as colloidal gold or gold sol. Upon contact with the liquid sample, the conjugate redissolves and specifically binds to any analyte present in the sample to form an analyte-conjugate complex. This complex flows through a lateral flow membrane, on which test and control reagents have been immobilized at the test and control regions, respectively. Analyte- conjugate complexes are immobilised at the test region and non-bound conjugatecontaining particles are immobilised at the control region. An absorbent pad collects the fluid flowing through the lateral flow membrane and prevents any backflow of fluid.

[0024] The term “one or more regions of higher density than another region”, as used herein, refers to a difference in density of different portions of the fluid flow path. The terms “another region” and “the other region” refer to a portion of the fluid flow path that is comparatively less than that the one or more regions of higher density. In some embodiments, the other region is a region of the fluid flow path which has not been embossed and / or debossed.

[0025] In some embodiments, the one or more regions of higher density are 1.3-10 x denser than the other region. In some embodiments, the one or more regions of higher density are 1.5- 9 x denser than the other region. In some embodiments, the one or more regions of higher density are 1.7-8 x denser than the other region. In some embodiments, the one or more regions of higher density are 2-7 x denser than the other region. In some embodiments, the one or more regions of higher density are 3-6 x denser than the other region. In some embodiments, the one or more regions of higher density are 4-5 x denser than the other region. In some embodiments, the one or more regions of higher density are at least twice as dense as the other region. In some embodiments, the one or more regions of higher density have a density of 78 to 5100 mg / cm3and the other region has a density of 60 to 510 mg / cm3. The skilled person understands how to calculate density of a portion of a lateral flow membrane. The formula for calculating density is p = m / V, where p is the density, m is the mass and V is the volume.

[0026] In some embodiments, the one or more regions of higher density have a smaller cross- sectional thickness than the other region. In some embodiments, the cross-sectional thickness of the one or more regions of higher density is 25-100 pm less than the cross- sectional area of the other region. In some embodiments, the cross-sectional thickness of the one or more regions of higher density is 25-90% of the cross-sectional thickness of the other region. In some embodiments, the one or more regions of higher density are one or more regions of lower porosity (than the other region). In some embodiments, the one or more regions of higher density have a porosity of 40-65% and the other region has a porosity of 70- 95%. The skilled person understands how to calculate porosity of a portion of a lateral flow membrane, for example, using the weighting method, which is described in detail in Sun et al. (“Microstructural effects on permeability of Nitrocellulose membranes for biomedical applications”, J. Membr. Sci., 595 (2020) 117502), which is hereby incorporated by reference in its entirety.

[0027] The term “the one or more regions of higher density are at least located within and / or adjacent to the detection zone” is intended to mean that the higher density regions are partially or fully present inside the detection zone (i.e., the area of the fluid flow path in which the label is detected) or are next to the detection zone. “Adjacent” or “next to” may be interpreted to mean within 10 mm of the detection zone. “Adjacent” or “next to” may be interpreted to mean within 5 mm of the detection zone. “Adjacent” or “next to” may be interpreted to mean within 2 mm of the detection zone. “Adjacent” or “next to” may be interpreted to mean within 1 mm of the detection zone. In some embodiments, the one or more regions of higher density are at least located within and / or directly adjacent to the detection zone. In some embodiments, the one or more regions of higher density are at least located within and / or overlapping the detection zone. In some embodiments, the one or more regions of higher density are located within the detection zone.

[0028] In some embodiments, the one or more regions of higher density are formed by embossing and / or debossing the lateral flow membrane. Advantageously, this compacts the lateral flow membrane (thus increasing the density at those areas) and reduces the thickness of the fluid flow path, thus improving the signal deposition in the detection zone. In some embodiments, the read-out surface (i.e., the surface of the lateral flow membrane on which the result of the assay is visually or optically discernible) is embossed and / or debossed to form the one or more regions of higher density. In some embodiments, the embossed and / or debossed regions have a membrane thickness of 5-50% of the thickness of the regions which are not embossed and / or debossed. In some embodiments, the embossed and / or debossed regions have a membrane thickness of 10-40% of the thickness of the regions which are not embossed and / or debossed. In some embodiments, the embossed and / or debossed regions have a membrane thickness of 15-30% of the thickness of the regions which are not embossed and / or debossed.

[0029] In some embodiments, the one or more regions of higher density are formed by fabricating the lateral flow membrane with one or more regions of higher density within the fluid flow path. Advantageously, the one or more areas of increased density obstruct the flow path and improve the signal deposition in the detection zone.

[0030] The term “the one or more regions of higher density extend across the entire thickness of the lateral flow membrane” is intended to mean that the regions of higher density are positioned in the fluid flow path and extend across the height of the fluid flow path (i.e., from the top readout surface of the lateral flow membrane through to the bottom surface of the lateral flow membrane).

[0031] In some embodiments, the one or more regions of higher density extend across the entire width of the fluid flow path. The term “extend across the entire width” is interpreted to mean that the one or more regions of higher density are substantially present from one side (of the flow path) to the other side (of the flow path) in a continuous or discontinuous manner. In some embodiments, the one or more regions of higher density extend continuously across the entire width of the fluid flow path.

[0032] In some embodiments, the one or more regions of higher density extend across the entire length of the fluid flow path. The term “extend across the entire length” is interpreted to mean that the one or more regions of higher density are substantially present from one end (of the flow path; e.g., the sample receiving zone) to the other end (of the flow path; e.g., the absorbent zone) in a continuous or discontinuous manner. In some embodiments, the one or more regions of higher density extend continuously across the entire length of the fluid flow path.

[0033] In some embodiments, there is one region of higher density than another region. In some embodiments, there are two or more regions of higher density than another region (i.e., a plurality of regions of higher density). In some embodiments, there are three or more regions of higher density than another region. In some embodiments, there are four or more regions of higher density than another region. In some embodiments, there are five or more regions of higher density than another region. In some embodiments, there are ten or more regions of higher density than another region. In some embodiments, there are 20 or more regions of higher density than another region. In some embodiments, there are 30 or more regions of higher density than another region. In some embodiments, there are 40 or more regions of higher density than another region. In some embodiments, there are 50 or more regions of higher density than another region.

[0034] The density of the one or more regions of higher density is such that when a sample flows along the fluid flow channel, the speed and / or flow path of the flow of the sample is altered (i.e., diverted, restricted or delayed) with respect to the overall direction of flow from the sample-receiving zone to the absorbent zone.

[0035] In some embodiments, the one or more regions of higher density are permeable and porous. This provides the advantage that the fluid still flows through the regions of higher density, but will pass through these regions slower than the areas of lower density. This means that the fluid is more likely to flow through the areas of lower density than the areas of higher density.

[0036] In some embodiments, the fluid flow path and the one or more regions of higher density are formed from the same material. This provides the advantage that no additional materials are required, thus making the lateral flow membrane easier to manufacture. In some embodiments, the material (of the fluid flow path and the one or more regions of higher density) comprises or consists of nitrocellulose, cellulose polyester / nitrocellulose blend, nitrocellulose / cellulose blend, cellulose acetate, untreated paper, porous paper, rayon, glass fiber, acrylonitrile copolymer, polyvinylidene fluoride, cross-linked dextran, charge-modified nylon, polyethersulfone, or combinations thereof. In some embodiments, the material comprises or consists of nitrocellulose, cellulose or polyvinylidene fluoride. In some embodiments, the material comprises or consists of nitrocellulose.

[0037] In some embodiments, the one or more regions of higher density are at least located within and / or adjacent a test region of the detection zone. In some embodiments, the one or more regions of higher density are at least located within a test region of the detection zone. This provides the advantage of increased signal deposition in the test region. In some embodiments, the test region is embossed and / or debossed.

[0038] Any percentage of the test region may comprise the one or more regions of higher density, provided that the structure leads to an increased signal deposition. In some embodiments, at least about 5% of the test region comprises the one or more regions of higher density. In some embodiments, at least about 10% of the test region comprises the one or more regions of higher density. In some embodiments, at least about 15% of the test region comprises the one or more regions of higher density. In some embodiments, at least about 20% of the test region comprises the one or more regions of higher density. In some embodiments, at least about 25% of the test region comprises the one or more regions of higher density. In some embodiments, at least about 30% of the test region comprises the one or more regions of higher density. In some embodiments, at least about 35% of the test region comprises the one or more regions of higher density. In some embodiments, at least about 40% of the test region comprises the one or more regions of higher density. In some embodiments, at least about 45% of the test region comprises the one or more regions of higher density. In some embodiments, at least about 50% of the test region comprises the one or more regions of higher density. In some embodiments, at least about 60% of the test region comprises the one or more regions of higher density. In some embodiments, at least about 70% of the test region comprises the one or more regions of higher density. In some embodiments, at least about 80% of the test region comprises the one or more regions of higher density. In some embodiments, at least about 90% of the test region comprises the one or more regions of higher density. In some embodiments, at least about 95% of the test region comprises the one or more regions of higher density. In some embodiments, at least about 98% of the test region comprises the one or more regions of higher density. In some embodiments, at least about 99% of the test region comprises the one or more regions of higher density. In some embodiments, about 100% of the test region comprises the one or more regions of higher density.

[0039] In some embodiments, the one or more regions of higher density are at least located within and / or adjacent a control region of the detection zone. In some embodiments, the one or more regions of higher density are at least located within a control region of the detection zone. This provides the advantage of increased signal deposition in the control region. In some embodiments, the control region is embossed and / or debossed. The above description in relation to the percentage coverage of the test region is equally applicable to the control region.

[0040] In some embodiments, the one or more regions of higher density are at least located within and / or adjacent a test region and a control region of the detection zone.

[0041] In addition to the one or more regions of higher density being at least located within and / or adjacent to the detection zone, the one or more regions of higher density may additionally be located in any other region or combination of regions, or all of the regions of the lateral flow membrane. For example, the one or more regions of higher density may additionally be located in the sample-receiving zone and / or the labelling zone.

[0042] Any percentage of the fluid flow path may comprise the one or more regions of higher density, provided that the structure leads to an increased signal deposition. In some embodiments, at least about 5% of the fluid flow path comprises the one or more regions of higher density. In some embodiments, at least about 10% of the fluid flow path comprises the one or more regions of higher density. In some embodiments, at least about 15% of the fluid flow path comprises the one or more regions of higher density. In some embodiments, at least about 20% of the fluid flow path comprises the one or more regions of higher density. In some embodiments, at least about 25% of the fluid flow path comprises the one or more regions of higher density. In some embodiments, at least about 30% of the fluid flow path comprises the one or more regions of higher density. In some embodiments, at least about 35% of the fluid flow path comprises the one or more regions of higher density. In some embodiments, at least about 40% of the fluid flow path comprises the one or more regions of higher density. In some embodiments, at least about 45% of the fluid flow path comprises the one or more regions of higher density. In some embodiments, at least about 50% of the fluid flow path comprises the one or more regions of higher density. In some embodiments, at least about 60% of the fluid flow path comprises the one or more regions of higher density. In some embodiments, at least about 70% of the fluid flow path comprises the one or more regions of higher density. In some embodiments, at least about 80% of the fluid flow path comprises the one or more regions of higher density.

[0043] The positioning / design of the one or more regions of higher density is not limited provided the structure results in an increased signal deposition. As the skilled person will appreciate, many designs and patterns are possible and will be suitable for use in the present invention. In some embodiments, the one or more regions of higher density comprise a polygonal, linear, or pillared design or a combination thereof. In some embodiments, the one or more regions of higher density comprise a polygonal, linear, or pillared repeating pattern, or a combination thereof. By “repeating pattern” it is meant a design that has been repeated. In some embodiments, the one or more regions of higher density comprise a polygonal, linear, or pillared array or a combination thereof. By “array”, it is meant an ordered series or arrangement. In some embodiments, the one or more regions of higher density comprise a circular design.

[0044] In some embodiments, one or more regions of higher density comprise a hexagonal design or pattern. The inventors have demonstrated that linear and hexagonal patterns, among others, increase signal deposition.

[0045] In some embodiments, the lateral flow membrane is a nitrocellulose membrane and the one or more regions of higher density are formed of nitrocellulose and comprise a hexagonal design or repeating pattern. Advantageously, this has been shown to increase signal deposition.

[0046] In some embodiments, the one or more regions of higher density comprise a repeating pattern. As an example, the fluid flow path may comprise a pattern of a plurality of regions of higher density. The repeating pattern may be any suitable pattern. In some embodiments, the repeating pattern comprises a tessellating polygonal pattern. In some embodiments, the tessellating polygonal pattern is a tessellating hexagonal pattern.

[0047] The repeating pattern may have any repeating unit size, provided the repeating pattern increases the signal deposition. The term “repeating unit size” as used herein is intended to mean the longest dimension of the design that repeats in the pattern (i.e., the simplest repeating unit). In some embodiments, the repeating pattern has a repeating unit size of 10-1000 pm. In some embodiments, the repeating pattern has a repeating unit size of 10- 750 pm. In some embodiments, the repeating pattern has a repeating unit size of 10-500 pm. In some embodiments, the repeating pattern has a repeating unit size of 25-500 pm. In some embodiments, the pattern has a repeating unit size of 15-400 pm. In some embodiments, the pattern has a repeating unit size of 20-300 pm. In some embodiments, the pattern has a repeating unit size of 25-250 pm. In some embodiments, the pattern has a repeating unit size of 30-200 pm. In some embodiments, the pattern has a repeating unit size of 40-150 pm. In some embodiments, the pattern has a repeating unit size of 50-100 pm. In some embodiments, the repeating pattern has a repeating unit size of 50-400 pm. In some embodiments, the repeating pattern extends across the entire width of the membrane and has a repeating unit size of 1000-3000 pm.

[0048] In some embodiments, the lateral flow membrane is a single unitary membrane (i.e., one element formed of the same material) and the one or more regions of higher density are formed in the single unitary membrane. In some embodiments, the lateral flow membrane is formed of a single material (e.g., nitrocellulose) and is not impregnated with any other material (e.g., wax) other than the necessary reagents for the lateral flow test (e.g., analyte-specific binding reagents, etc).

[0049] According to a second aspect, there is provided a lateral flow device comprising a lateral flow membrane described herein. The above description in relation to the lateral flow membrane is equally applicable to this aspect. Lateral flow devices are well known in the art and are for example described in US 5252496 and US 5591645. The skilled person will appreciate that various features may be included, for example, a backing, a sample application pad, a conjugate release pad, an absorbent pad, a housing, or a combination thereof. These features are known and routinely used in the art. The lateral flow membrane according to the present invention may be used in any suitable lateral flow device known in the art.

[0050] According to a third aspect, there is provided a method of producing a lateral flow membrane comprising a fluid flow path, wherein the fluid flow path comprises a detection zone, wherein the method comprises: forming one or more regions of higher density in the fluid flow path that have a higher density compared to another region in the fluid flow path, wherein the one or more regions of higher density extend across the entire thickness of the lateral flow membrane and are at least located within and / or adjacent to the detection.

[0051] The above description in relation to the lateral flow membrane is equally applicable to this aspect. As an example, the above description in relation to the one or more regions of higher density is equally applicable here. The method of producing may also be referred to as a method of manufacturing or a method of modifying.

[0052] In some embodiments, the one or more regions of higher density are formed by embossing and / or debossing the lateral flow membrane. Methods of embossing / stamping and debossing are well known in the art and are not limited. For example, wet embossing / debossing, heat embossing / debossing, dry embossing / debossing or die cut embossing / debossing may be used. In some embodiments, die cut embossing or debossing is used.

[0053] In some embodiments, the one or more regions of higher density are formed by printing / forming the lateral flow membrane.

[0054] In some embodiments, the one or more regions of higher density are formed at least located within and / or adjacent a test region of the detection zone. In some embodiments, the one or more regions of higher density are formed at least located within and / or adjacent a control region of the detection zone. In some embodiments, the one or more regions of higher density are formed at least located within and / or adjacent a test region and a control region of the detection zone. This provides the advantage of increasing signal deposition in the areas of signal read-out. The description above in relation to the lateral flow membrane aspect is equally applicable here.

[0055] In some embodiments, a patterned template is used emboss and / or deboss the read-out surface. The design of the patterned template is not particularly limited provided the embossing or debossing using the patterned template results in an increase signal deposition of the lateral flow membrane. As the skilled person will appreciate, many patterns are possible and will be suitable for use in the present invention. In some embodiments, the patterned template comprises a polygonal, linear, or pillared pattern, or a combination thereof. In some embodiments, the patterned template comprises a circular pattern. In some embodiments, the patterned template comprises a tessellating pattern. In some embodiments, the tessellating pattern is a tessellating polygonal pattern. In some embodiments, the tessellating pattern is a tessellating hexagonal pattern.

[0056] The patterned template may have any repeating unit size provided the pattern increases the signal deposition of the lateral flow membrane. In some embodiments, the patterned template has a repeating unit size of 10-1000 pm. In some embodiments, the patterned template has a repeating unit size of 10-750 pm. In some embodiments, the patterned template has a repeating unit size of 10-500 pm. In some embodiments, the patterned template has a repeating unit size of 25-500 pm. In some embodiments, the patterned template has a repeating unit size of 15-400 pm. In some embodiments, the patterned template has a repeating unit size of 20-300 pm. In some embodiments, the patterned template has a repeating unit size of 25-250 pm. In some embodiments, the patterned template has a repeating unit size of 30-200 pm. In some embodiments, the patterned template has a repeating unit size of 40-150 pm. In some embodiments, the patterned template has a repeating unit size of 50-100 pm. In preferable embodiments, the patterned template has a repeating unit size of 50-400 pm. In some embodiments, the patterned template has a repeating unit size of 1000-3000 pm.

[0057] The material of the patterned template is not particularly limited, provided the template embosses and / or debosses the lateral flow membrane. In some embodiments, the patterned template is a silicon wafer.

[0058] The lateral flow membranes produced by the method can be processed into different formats well known to those skilled in the art.

[0059] A skilled person will appreciate that all aspects of the invention, whether they relate to, for example, the lateral flow membrane, are equally applicable to all other aspects of the invention. In particular, aspects of the lateral flow membrane may have been described in greater detail than in aspects of the method or the lateral flow device. However, the skilled person will appreciate where more detailed information has been given for a particular aspect of the invention, this information is generally equally applicable to other aspects of the invention. In particular, the skilled person will appreciate that where there is description of the one or more regions of higher density, these regions may be formed by embossing and / or debossing.

[0060] All patent and literature references cited in the present specification are hereby incorporated by reference in their entirety.

[0061] The invention will now be described in detail by way of example only with reference to the figures.

[0062] Brief Description of the Drawings

[0063] Figure 1 (A) shows an exemplary patterned template for embossing lateral flow membranes. Scale bar 100 pm, magnification x350. (B-E) show exemplary lateral flow membranes according to the disclosure. (B) scale bar 100 pm, magnification x200; (C) scale bar 100 pm; (D, E) field width 30 mm, magnification 50x.

[0064] Figure 2 shows successful transfer and demonstration of retention (stability) of embossed micropattems on commercial LFDs using SARS n-COV2 Antigen test (Innova) without affecting signal generation, as exemplified by the comparison between the control (A) and modified (B, C) assay control region of the membrane. The modified LFDs harboured wide-pitch, hexagonal and linear patterns. These were retained after the sample run. The red line (grey in the figures) comprised the respective control band in a negative control (negative sample) with excess detection antibody (free antibody -gold complex). Scale bar 500 pm. (D) shows the cross section of the lateral flow membrane of (C), revealing the ridged profile of continuous lines perpendicular to the direction of flow, which is retained after the sample run; scale bar 10 pm.

[0065] Figure 3 shows LFD modifications result in improved visual detection limits as highlighted by (A) SARS nCOV2 and (B) human chorionic gonadotrophin (hGC) LFDs, highlighting the side-by-side improvements possible. Both analytes were included at levels below the visual detection limit for the unmodified membranes at 10 pfu SARS nCOV2 and 0.7 ng / ml hGC. The arrow indicates the read-out test region. (A) Modified LFD (top), unmodified control LFD (bottom); Scale bar = 1 mm. (B) Modified LFD (top three), unmodified control LFD (bottom); Scale bar = 2 mm.

[0066] Figure 4 shows cross-sections of (A, C) modified and (B, D) non-modified (control) LFDs, including the lateral flow membrane, adhesive and backing (x20 magnification).

[0067] Figure 5 shows leading / trailing of modified (top) and non-modified (control) (bottom) SARS nCOV2 LFD devices 40 seconds after application of the analyte, highlighting the flow of analyte in a direction perpendicular to flow (dashed arrow) along linear patterns resulting in flow-altering troughs along the fluid path (solid arrow). The overall direction of flow is from left to right. Scale bar = 1 mm. The modified LFD had an embossed pattern of parallel lines with a spacing of 150 pm and a recess of 90%, resulting in greater admixture of the flow.

[0068] Figure 6 shows LFD modifications result in improved limits of detection by means of synchrontron X-ray fluorescence, as highlighted by (A&B) SARS nCOV2 Innova LFD and (C&D) human chorionic gonadotrophin LFD (Clear & Simple Pregnancy Test), highlighting the side-by-side improvements possible. Visual results are provided in Figures A and C and corresponding graphical results are provided in Figures B and D. Both analytes were included at levels below the visual detection limit for the unmodified membranes at 10 pfu SARS nCOV2 and 0.7 ng / ml hGC. (A & C) top membrane is unmodified control, second after top membrane is modified Hl (hexagonal embossed pattern, 50 pm pattern size), third after top membrane is modified H2 (hexagonal embossed pattern, 175 pm pattern size), fourth after top membrane is modified H3 (hexagonal embossed pattern, 350 pm pattern size). The visual bar represents the mass fraction of gold nanoparticles deposited at the test field (in ppm), with the corresponding gold quantities plotted along the length immediately before, at the test field and immediately after the test field (units represent mm in the direction of analyte flow). The total test field length in the flow direction was 1.75 and 1.5 mm for the SARS nCOV2 and hCG assay, respectively. Figure 7 exemplifies the positive visual detection for hCG at the test region at lower analyte levels for modified LFDs compared to an unmodified LFD (Control). Innova LFDs were modified with: a raised circular pattern of 170 pm spacing (Circular 170 pm); a raised circular pattern of 120 pm spacing (Circular 120 pm); or a raised hexagonal pattern of 350 pm spacing. The improved detection is exemplified for modified LFDs. Scale bar = 1 mm.

[0069] Figure 8 shows the preferential and higher levels of antigen-antibody gold detection complex deposition around circular features (A) and the retention of features after running a liquid sample in the LFD assay (B). Scale bar 10 pm.

[0070] Detailed Description of the Invention

[0071] The inventors have demonstrated that creating one or more areas of higher density in the fluid flow path of a lateral flow membrane, at least within or adjacent to the detection zone, increases the signal deposition and signal concentration in the detection zone. Without wishing to be bound by any particular theory, the inventors consider that the one or more regions of higher density alter the speed and / or the flow path of the fluid flow to increase the deposition of signal in the detection zone, thus increasing the sensitivity of the signal read-out.

[0072] In the following example, the inventors formed regions of higher density through embossing lateral flow membranes. The embossing created regions of higher density (and lower porosity) in the fluid flow path, and the inventors demonstrated that these regions increased the signal deposition in the detection zone. However, the skilled person will recognise that others methods may alternatively or additionally be used to form the one or more regions of higher density in the fluid flow path.

[0073] An exemplary patterned template used to emboss and / or deboss a lateral flow membrane is shown in Figure 1A. The patterned template is a silicon wafer with a tessellating hexagonal pattern, with a repeating unit spacing of 100 pm. However, the skilled person will appreciate that various shapes, designs and spacings can be used when forming the one or more regions of higher density. As an example, patterned templates (hexagonal, circular, linear) with a repeating unit spacing of 25-3000 pm were tested.

[0074] The patterned template can be used to emboss and / or deboss the read-out surface of a lateral flow membrane. Embossing / debossing techniques are known to the skilled person and the various parameters (e.g., compressive force, application time, etc.) can be selected without undue burden. By way of example only, the inventors embossed the membrane using templates with compressive force between 250 and 800 N for 60 seconds (approximately 25-80 N per cm2embossing area). It will be recognised by the skilled person that other technique, such as applying the pattern with a textured roller, may be used.

[0075] An exemplary lateral flow membrane (prior to running an assay) with an embossed readout surface is shown in Figure IB. The read-out surface, including the areas within and adjacent to the detection zone, have an embossed tessellating hexagonal pattern with a pattern spacing of 350 pm. As can be seen in Figure 1C, when the assay is run on the lateral flow device, there is a greater signal deposition at the test region.

[0076] An exemplary lateral flow membrane is shown in Figures ID and IE, after embossing of the read-out surface and prior to running an assay, highlighting the fidelity of the pattern transfer attained.

[0077] The present invention is further illustrated by the following example, without being limited thereto.

[0078] Example

[0079] The inventors identified that sensitivity shortcomings of LFDs could be targeted by improving the intrinsic sensitivity of LFDs, rather than adjunctive signal amplification. The present invention enables improved read-outs without introducing expensive modifications to the assay or necessitating further tests / manipulation steps by the user.

[0080] By introducing one or more areas of higher density, the inventors demonstrated dramatic improvements in the sensitivity of LFDs. This was attained by the introduction of topographically functionalised zones by embossing the detection zone and the area adjacent to the detection zone of the device, which improves the respective interactions prior to forming detection particle-antigen complexes during the running phase, and concentrates the signal of the gold / antibody-antigen complexes bound to capture antibodies within the test region. The modification provided a dense, visually discernible (more concentrated) signal at the beginning of the test region, thus significantly improving the recognised shortcomings of LFDs. The inventors identified that the increased signal arose from increased overall deposition of material as well as concentration of the read-out at the beginning of the test line area of the device.

[0081] Without wishing the be bound by theory, the inventors consider that the speed and / or flow path of the flow is altered by the differential density in the fluid flow path, causing the flow to preferentially flow through the areas of lower density than the areas of higher density.

[0082] The data confirmed the feasibility to imprint or pattern the nitrocellulose substrate (which forms the wi eking and deposition component) of LFDs by embossing or patterning using micropatterns, including textured surfaces and custom silicon wafers with differential patterns. The approach improved test sensitivity by a factor of 10-100x without the need of adjunctive reagents, readers or procedural changes in how the test is conducted. In addition, the patterning methods can be easily introduced into current manufacturing processes and without further steps in conducting the assay.

[0083] Methods

[0084] To demonstrate the concept of improving the sensitivity of LFD devices stringently, the inventors initially used commercially validated LFDs for diverse target analytes. For instance, these targeted SARS nCOV2 or human chorionic gonadotrophin, validating their approach and benchmarking the sensitivity against industry -leading LFD read-outs. For each analyte, devices from three manufacturers were tested.

[0085] After removal from the cassettes (if present), the membranes were modified by embossing the running and test regions with patterns introduced using high-precision, silicon wafer templates, as exemplified in Figure 1. Read-outs were compared to non-modified and batch-controlled LFDs.

[0086] Templated patterns introduced from the silicon wafers comprised >30 differentially spaced, sized and differentially shaped patterns. For this study, micropatterned hexagonal, linear or pillared structures in the 10-500 pm range of repeating unit size and / or spacing were used. A hexagonal patterned membrane is shown in Figure 2B and a linear patterned membrane is shown in Figure 2C, compared to the unmodified control (Figure 2A). The cross section of the modified membrane of Figure 2C is shown in Figure 2D.

[0087] The inventors next examined the effect of the modified membrane surfaces on performance and limits of detection targeting multiple analytes. To exemplify this, the inventors focused on SARS nCOV2 and human chorionic gonadotrophin (hCG). The assay designs differed between individual assays in that some depended on aggregates of gold / antibody-antigen complexes (multiple identical antigens on the analyte - such as the Innova Spike-protein specific antibodies targeting intact SARS nCOV2 viral particles) and different antigens within a single molecule of the analyte captured (hCG). The modified samples were run concomitantly, followed by visual scoring and quantitative image analysis.

[0088] For the SARS nCOV2 LFDs, the modified LFD revealed a clear read-out at or below levels considered infectious, compared to the unmodified control LFD (Figure 3A). For the hCG LFDs, the modified LFDs (top three membranes) demonstrated the improvements observed by the introduction of differentially sized hexagonal patterns on read-out intensity for the same analyte quantity (Figure 3B). The inventors concluded that the introduction of patterns resulted, relative to the unmodified control assays, in significant improvements in visual read-outs of positive signals.

[0089] In parallel to visual binary scores (5 participants), quantitative pixel analyses were carried out, confirming 24-120 fold improvement in the concentration range where true positive scores were correctly identified in read-outs at the point where control, unmodified LFDs were scored negative. This is highly significant for infections, such as observed in SARS nCOV2, as this likely improves sensitivity to levels that are considered not infectious. Thus, this technology significantly improves the diagnostic sensitivity of lateral flow membranes.

[0090] The inventors demonstrated that patterns were retained after running the assays (Figures 4 and 8). The inventors also demonstrated that the flow of the assay was modified by the micropatterns (Figure 5).

[0091] The inventors further demonstrated, by means of synchrotron X-ray fluorescence, that modification to LFDs resulted in improved limits of detection (Figure 6). The inventors modified Innova LFDs (testing SARS nCOV2; Figures 6A and 6B) and Clear & Simple Pregnancy Test LFDs (testing human chorionic gonadotrophin; Figures 6C and 6D) by embossing the membranes with differentially sized hexagonal patterns (Hl, H2, H3), and ran the assays. The modifications significantly increased the concentration of complex deposition at the beginning of the test region, as depicted visually in Figures 6(A) and 6(C) and graphically in Figures 6(B) and 6(D). This demonstrated a clear improvement in visual detection of the read-out at or below levels considered infectious.

[0092] Positive visual detection at the test region was possible for modified LFDs with various patterns (circular, hexagonal) at lower analyte levels, compared to unmodified control LFDs (Figure 7). The inventors therefore considered that the type and size of the modification pattern is not particularly limiting.

[0093] Although the invention has been described in relation to one or more preferred embodiments, it will be appreciated that various changes or modifications may be made without departing from the scope of the invention as defined in the appended claims.

[0094] References

[0095] Agarwal, S., et al. (2022). "Lateral flow-based nucleic acid detection of SARS-CoV-2 using enzymatic incorporation of biotin-labeled dUTP for POCT use." Anal Bioanal Chem 414(10): 3177-3186.

[0096] Cobb, B. L., et al. (2022). "Clinical Performance of a Lateral Flow SARS-CoV-2 Total Antibody Assay." J Appl Lab Med. 2022 Jun 30;7(4):827-833. doi: 10.1093 / jalm / jfac003 Deeks, J. J., et al. (2022). "SARS-CoV-2 antigen lateral flow tests for detecting infectious people: linked data analysis." BMJ 376:e066871. doi: 10.1136 / bmj-2021-066871. Koller, G., et al. (2021). "More than the Eye Can See: Shedding New Light on SARS- CoV-2 Lateral Flow Device-Based Immunoassays." ACS Appl Mater Interfaces 13(22): 25694-25700.

Claims

Claims1. A lateral flow membrane comprising a fluid flow path, wherein the fluid flow path comprises a detection zone and one or more regions of higher density than another region of the fluid flow path, wherein the one or more regions of higher density extend across the entire thickness of the lateral flow membrane, and wherein the one or more regions of higher density are at least located within and / or adjacent to the detection zone.

2. A lateral flow membrane according to claim 1, wherein the one or more regions of higher density are formed by embossing and / or debossing the lateral flow membrane.

3. A lateral flow membrane according to claim 1, wherein the one or more regions of higher density are formed by fabricating the lateral flow membrane with one or more regions of higher density within the fluid flow path.

4. A lateral flow membrane according to any preceding claim, wherein the one or more regions of higher density extend across the entire width of the fluid flow path.

5. A lateral flow membrane according to any preceding claim, wherein the one or more regions of higher density are permeable and porous.

6. A lateral flow membrane according to any preceding claim, wherein the fluid flow path and the one or more regions of higher density are formed from the same material.

7. A lateral flow membrane according to claim 6, wherein the material comprises or consists of nitrocellulose, cellulose polyester / nitrocellulose blend, nitrocellulose / cellulose blend, cellulose acetate, untreated paper, porous paper, rayon, glass fiber, acrylonitrile copolymer, polyvinylidene fluoride, cross-linkeddextran, charge-modified nylon, polyethersulfone, or combinations thereof; optionally wherein the material comprises or consists of nitrocellulose, cellulose or polyvinylidene fluoride.

8. A lateral flow membrane according to any preceding claim, wherein the one or more regions of higher density have a smaller cross-sectional thickness than the other region.

9. A lateral flow membrane according to any preceding claim, wherein the one or more regions of higher density are at least twice as dense as the other region.

10. A lateral flow membrane according to any preceding claim, wherein the one or more regions of higher density are at least located within and / or adjacent a test region of the detection zone.

11. A lateral flow membrane according to claim 10, wherein at least about 5% of the test region comprises the one or more regions of higher density, optionally wherein at least about 10% of the test region comprises the one or more regions of higher density.

12. A lateral flow membrane according to any preceding claim, wherein the one or more regions of higher density comprise a polygonal, linear, or pillared design or a combination thereof.

13. A lateral flow membrane according to claim 12, wherein the one or more regions of higher density comprise a hexagonal design or repeating pattern.

14. A lateral flow membrane according to any preceding claim, wherein the lateral flow membrane is not folded or bent out of plane.

15. A lateral flow membrane according to any preceding claim, wherein the one or more regions of higher density comprise a repeating pattern.

16. A lateral flow membrane according to claim 15, wherein the pattern comprises a tessellating polygonal pattern, optionally wherein the tessellating polygonal pattern is a tessellating hexagonal pattern.

17. A lateral flow membrane according to claim 15 or claim 16, wherein the pattern has a repeating unit size of 25-500 pm.

18. A lateral flow device comprising a lateral flow membrane according to any preceding claim.

19. A method of producing a lateral flow membrane comprising a fluid flow path, wherein the fluid flow path comprises a detection zone, wherein the method comprises: forming one or more regions of higher density in the fluid flow path that have a higher density compared to another region in the fluid flow path, wherein the one or more regions of higher density extend across the entire thickness of the lateral flow membrane and are at least located within and / or adjacent to the detection zone.

20. A method of producing a lateral flow membrane according to claim 19, wherein the one or more regions of higher density are formed by embossing and / or debossing the lateral flow membrane.

21. A method of producing a lateral flow membrane according to claim 19 or claim 20, wherein the one or more regions of higher density are formed at least located within and / or adjacent a test region of the detection zone.

22. A method of producing a lateral flow membrane according to claim 20, wherein a patterned template is used emboss and / or deboss the read-out surface.

23. A method of producing a lateral flow membrane according to claim 22, wherein the patterned template comprises a tessellating pattern.

24. A method of producing a lateral flow membrane according to claim 23, wherein the tessellating pattern is a tessellating polygonal pattern, optionally wherein the tessellating pattern is a tessellating hexagonal pattern.

25. A method of producing a lateral flow membrane according to any one of claims 22-24, wherein the patterned template is a silicon wafer.

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