WO2013178739A1

WO2013178739A1 - Assay device with indication of sufficient amount of sample

Info

Publication number: WO2013178739A1
Application number: PCT/EP2013/061178
Authority: WO
Inventors: Lee Walker; Kieran Andrew LEHANE; Steven Hart
Original assignee: Spd Swiss Precision Diagnostics Gmbh
Priority date: 2012-05-30
Filing date: 2013-05-30
Publication date: 2013-12-05
Also published as: GB201209664D0

Abstract

The present invention provides an assay device for detecting an analyte in a fluid sample, comprising: a sample receiving member (1), which is porous and fluidically connected to one or more components (2, 3) defining an assay flow path, at least one of which is a detection member (3) comprising an analyte detection zone (31); and a sample detection element (13), which is located outside the assay flow path and spaced apart from the sample receiving member (1) by a gap (7); wherein the device is adapted such that the sample receiving member (1) and the sample detection element (13) are fluidically connectable when the gap (7) is bridged by the fluid sample.

Description

AS SAY DEVICE WITH INDICATION OF SUFFICIENT AMOUNT OF SAMPLE

The present invention relates to an assay device which gives an indication to the user when a sufficient amount of sample has been applied to the device, for the device to function adequately and produce an accurate result.

Various diagnostic products are known which analyse a fluid sample, such as urine or blood, to determine the presence or amount of one or more analytes. These may be small, handheld devices, which are used by applying the biological sample to an absorbent component; the devices are configured to subsequently convey the fluid along a flow path to an assay test zone without needing any significant gravitational encouragement, e.g. by applied pressure, where a reaction or binding event takes place to afford the assay result.

An example of a device of this nature is the lateral flow type assay device described in EP 0,291,194. This document discloses an immunoassay device comprising a type of sample application region known as a wick, which overlaps and is fluidically connected to a porous carrier containing a reagent zone bearing a mobilisable labelled specific binding reagent for an analyte. Downstream, an unlabelled specific binding reagent for the analyte is immobilised in a test zone or analyte detection zone. The user performs the single step of applying liquid sample to the wick, and the sample is subsequently conveyed along the flow path by capillary action. The device is designed to enable the controlled release of the mobilisable labelled reagent by the sample. Any analyte in the sample will then interact with the released labelled specific binding reagent to form a labelled complex, which is carried to the test zone where it forms a "sandwich" complex with the immobilised unlabelled binding reagent. The label, which may be a coloured particle, thereby becomes concentrated and observable in the test zone to indicate the presence and/or amount of analyte in the sample. The test zone can be directly observed by the user to determine the test result in what are known as visually read tests. However in digital tests, the test zone can be measured by an optical or other reading system and the result presented on a display, such as a LCD display.

The correct functioning of these assays is reliant upon the application of a sufficient volume of sample to the device by the user. On using such tests there are incidences where the user

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3282712v1 provides an insufficient volume of liquid sample, for example urine. This can lead to failure of the assay because the test sample does not reach the assay reagents which are positioned downstream of the wick along the flow path. An insufficient volume of sample can influence the flow rate of the sample along the flow path such that improper or uncontrolled mobilisation of the mobilisable labelled reagent (rate and total quantity released) impacts the performance of the test affecting its ability to deliver the correct result. A correct result is defined by the delivery of a positive or negative result, for example Pregnant or Not Pregnant to the user rather than a void result informing the user to re-test due to a failed device. An insufficient sample volume applied to a visually read device can manifest itself in a number of ways, for example as a device which produces no test and control lines, no control line as well as partly formed test and/or control lines. Various test and control line formations may be accompanied by a smear of mobilisable label in the read window. This leads to confusion in interpreting the result as well as user frustration which ultimately leads to a wasted test device since the test has to be repeated. Digital test devices can comprise numerous monitoring means which track events such as wetting on the test device from the onset of applying the urine sample to the test. Clearblue digital tests (Swiss Precision Diagnostics GmbH) for instance have a 'wake up' period defined as the time taken for current to start flowing through the device from the point of application of the urine sample to the sample receiving member and the flow of sample reaching a pair of electrodes which are in contact with the sample receiving member. This event effectively 'wakes' the digital test up from a standby mode. Once woken, such devices continue to monitor the flow of sample along the flow path by measuring the time for the solvent front to reach one or more optical sensors arranged along the flow path. An onboard digital processor has pre-defined limits for the time taken for the flow to reach the sensor(s) allowing the determination of improper flow to indicate a failed device. Such measurement systems monitor the flow of sample along the flow path; they do not necessarily reduce the incidence of an insufficient volume of sample being applied to the device. Wastage of a test device through insufficient sampling can thus still occur.

Accordingly, the user is generally instructed to take care to apply a certain volume of sample e.g. by holding the wick in their urine stream for 5 seconds or by dipping the wick up to a

3282712v1 certain distance in their collected urine for 5 seconds. The sampling time can of course vary for different test devices.

Whilst sampling onto such devices is largely not an issue, often the user is a na^'ive or an infrequent user of this type of assay and is expected to interpret the procedure for performing the assay from reading instructions and observing diagrams which are provided with the assay. Unfortunately, however, some users may not always comply with the instructions, for instance due to misinterpreting them or even not reading them at all in their eagerness to use the device.

The issue of insufficient sampling is particularly relevant to urine sampling. When detecting analyte in a urine sample, the user may prefer to apply their urine to the sample application area directly from their urine stream ("midstream sampling") rather than collect their urine in a container first, but in practice they may experience difficulties in controlling the direction of their urine stream onto the wick and, especially for women (bearing in mind their anatomy), being able to see what they are doing when midstream sampling.

Provision of an insufficient urine sample may therefore be done by the user, for example if the user does not time the sampling. This applies whether the user is dipping the wick of the device in a container of collected urine or is midstream sampling. Trials have shown the urine stream and the position of the wick as it is held within the urine stream can vary, thus influencing the total volume of urine applied to the wick. Also the user may not dip the wick to the required extent in a container of collected urine influencing the volume of urine collected by the wick causing insufficient sampling.

Efforts have been made to assist the users of such assays by providing in the assay device means for indicating that a sufficient amount of sample has been obtained.

For example, a known assay device as described in EP 0,291,194 has a control zone downstream from the test zone, where the formation of a line at the control zone indicates to the user that the sample ought to have fiowed past the test zone and so it is likely that enough sample has been applied. The user is instructed to reject the test as invalid if the control line

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3282712v1 fails to develop. However, because the user must wait the required time to see if the control line forms (typically several minutes), they do not receive any feedback during the sampling event itself, so the test may be wasted if undersampling occurs. Also, the absence of a control line does not inform the user that undersampling must have occurred; it could be due to degradation of reagents during storage of the device.

US 2002/0185385 describes an underfill detection system for a test sensor. The sensor electrochemically measures the concentration of an analyte in a liquid sample, using a reagent and a pair of electrodes (a "working electrode" and a "counter-reference" electrode), all disposed inside a capillary channel through which the liquid sample is conveyed. A signal electrode is disposed outside the capillary channel, but sufficiently close to its terminus to be in fluid communication with the latter. Only when liquid test sample has completely filled the capillary channel will any of the liquid reach the terminus of the channel and contact the signal electrode. Therefore, detection of the presence of liquid at the signal electrode indicates that the channel must be full of liquid and a sufficient sample volume must be present for accurate testing. However, such a system would not act to detect sufficient sample volume if a porous material (like a wick of the type described above) were used as the sample carrier instead of a capillary channel; liquid can reach the terminus of the porous material before the material is saturated. The extent to which a porous material is wet can influence its ability to release the sample to other porous materials along the flow path.

There is therefore a need in the art for an assay device comprising a porous sample receiver that indicates that sufficient sample has been applied, preferably by giving feedback to the user during the sampling event, and for urine sampling preferably in a way that is easily recognised by the woman without having to remove the wick from her urine stream. This feedback is important as it reassures the user that they are following the correct steps in performing the test and that the test is functioning in its intended manner. Users of such devices may be emotional in anticipation of a negative or positive result; hence any reassurance that they are performing the test correctly is of value.

Accordingly, in a first aspect, the present invention provides an assay device for detecting an analyte in a fluid sample, comprising:

3282712v1 a sample receiving member (1), which is porous and fluidically connected to one or more components (2, 3) defining an assay flow path, at least one of which is a detection member (3) comprising an analyte detection zone (31); and

a sample detection element (13), which is located outside the assay flow path and spaced apart from the sample receiving member (1) by a gap (7);

wherein the device is adapted such that the sample receiving member (1) and the sample detection element (13) are fluidically connectable when the gap (7) is bridged by the fluid sample. Embodiments will now be described, by way of example, with reference to the accompanying drawings in which:

Figure 1 illustrates a side view of an assay device in accordance with an embodiment of the invention, in which the sample detection element is present above the downstream end of the sample receiving member.

Figure 2 illustrates a plan view of part of the assay device of Figure 1 , in which the sample detection element is a pair of electrodes which are present above the downstream end of the sample receiving member.

Figure 3 illustrates a side view of the assay device of Figure 1 when in use, where the sample receiving member is supersaturated and a meniscus of sample fluid has formed which contacts the sample detection element. Figure 4 illustrates a side view of part of an assay device in accordance with another embodiment of the invention, in which the sample detection element is an electrode which is present above the downstream end of the sample receiving member; a second electrode is present on the surface of the sample receiving member. Figure 5 illustrates a side view of part of an assay device in accordance with a further embodiment of the invention, corresponding to the embodiment of Figure 4 but wherein the second electrode is present on a different surface of the sample receiving member.

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3282712v1 Figure 6 illustrates a side view of part of an assay device in accordance with a further embodiment of the invention, in which a component, e.g. tape, is used to control the size of the gap between the sample receiving member and the sample detection element, and also to create a physical barrier to any fluid which might flow along the surface of the sample receiving member. In Figure 6, the component, e.g. tape, is wrapped around the sample receiving member.

Figure 7 illustrates a side view of part of an assay device in accordance with a further embodiment of the invention, in which a component, e.g. tape, is used to control the size of the gap between the sample receiving member and the sample detection element, and also to create a physical barrier to any fluid which might flow along the surface of the sample receiving member. In Figure 7, the component, e.g. tape, is only applied to the surface of the sample receiving member.

Figure 8 illustrates a plan view of part of the assay device of Figure 6/7, where the sample detection element is a pair of electrodes and the component, e.g. tape, spans the whole width of the sample receiving member. Figure 9 illustrates a plan view of part of the assay device of Figure 6/7, where the sample detection element is a pair of electrodes and component, e.g. tape, does not span the whole width of the sample receiving member.

Figure 10a illustrates a side view of a part of an assay device in accordance with a further embodiment of the invention, in which a swellable material (for example a xerogel or hydrogel) is present in the gap between the sample receiving member and the sample detection element, but is only initially in contact with the latter. Figure 1 Ob illustrates a side view of the same part of the assay device when in use, where the swellable material has swelled on contact with sample fluid exuded from the surface of the sample receiving member and has bridged the gap between the sample receiving member and the sample detection element. This allows sample fluid to be freely transported to the sample detection element.

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3282712v1 Figure 11 illustrates a side view of a part of an assay device in accordance with a further embodiment of the invention, in which a fluid transfer component is present in fluidic contact with the sample receiving member, and there is a gap between the fluid transfer component and the sample detection element.

Figure 12 shows the construction of the device used in the Examples.

Figure 13 is a boxplot of the sample volume required to start an assay device of the invention ("Modified") versus a control device ("Control").

Figure 14 shows individual values of the volume required to start an assay device of the invention versus a control device.

By "assay flow path" is meant the path along which the sample fluid is intended to flow during performance of the assay. The assay flow path is made up of one or more components. When more than one component is present, the components are fluidically connected. The assay flow path is typically made up of two components, for example two fluidically connected strips. The assay flow path is also referred to herein as the "test strip". When referring to the location of a component in the assay flow path, "upstream" means that the component is closer to the sample receiving member. Conversely, "downstream" means that the component is further away from the sample receiving member.

The sample receiving member is capable of receiving a liquid sample and of transferring the liquid to the assay flow path. The sample receiving member may act as a sample capture means, and may be present in a sample receiving portion of the assay device. The sample receiving member may be an elongate strip. It may project from a housing that encloses the assay flow path. In the present invention, the sample receiving member is made of a porous material. The sample receiving member is typically non-swellable. In an embodiment, the sample receiving member is macroporous. In an embodiment, the device is adapted to transfer the sample from the sample receiving member to the assay flow path without user- applied force. Preferably, the materials of the sample receiving member and component(s) of

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3282712v1 the assay flow path are selected such that the sample is transferable from the sample receiving member to the start of the assay flow path by capillary action only.

The sample receiving member may comprise one or more different porous materials. It may be fibrous or non- fibrous. Suitable porous materials include glass fibre, cellulose, nitrocellulose, paper, silica, porous synthetic polymers such as sintered PET, and material comprising polyester, nylon, cotton, mono-component fibre combinations thereof, or bi- component fibre combinations thereof. The porous material may be a woven or a non-woven material. In one embodiment, the sample receiving member comprises polyester fibres and/or nylon fibres.

In an embodiment, the sample receiving member is a wick. The wick may comprise a material of relatively high capacity and high capillarity through which liquid can flow relatively easily. This may be relative to the other components of the assay flow path. This allows the wick to rapidly absorb a volume of sample liquid that is applied to the device, and also allows sufficient volume of sample liquid to be transferred easily to the assay flow path.

The sample that is applied to the assay device is a fluid. The sample may naturally be a liquid, or may be a solid that has been pre-treated so as to be provided in liquid form before application to the device. For example, a solid sample such as faeces can be dissolved in a suitable solvent before being applied to the device. Alternatively, a liquid sample may be treated with another liquid (such as water or an aqueous solution) to alter its viscosity and/or increase its volume before being applied to the device. The sample can be derived from any source and may be a bodily fluid, including blood, serum, plasma, saliva, sputum, ocular lens liquid, sweat, urine, milk, ascites liquid, mucous, synovial liquid, peritoneal liquid, transdermal exudates, pharyngeal exudates, bronchoalveolar lavage, tracheal aspirations, cerebrospinal liquid, semen, cervical mucus, vaginal or urethral secretions, and amniotic liquid. Depending upon the analyte of interest, other samples may be contemplated such as ones from industrial, environmental or agricultural sources.

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3282712v1 In an embodiment, the sample is aqueous. In an embodiment, the sample has a viscosity of < 2 mPa.s, < 1.5 mPa.s, or < 1 mPa.s at 25 °C. In an embodiment, the sample is urine or a diluted bodily fluid. During use of the device, the sample is conveyed along the assay flow path in which one or more reagents for the assay are found. The reagent(s) will vary according to the type of assay. The reagent(s) may interact with the analyte to form a detectable product, for example via a simple binding reaction to form an analyte-reagent complex, or via a chemical reaction. The assay reagents may comprise mobilisable and immobilised assay reagents; the mobilisable reagents may be pre-deposited on the component(s) defining the assay flow path in a dry form. The "analyte detection zone", where a signal is formed indicating the presence and/or amount of analyte, may contain the immobilised assay reagents, and the mobilisable assay reagents may be initially provided in a zone ("mobilisable reagent zone") upstream from the analyte detection zone. In the case where the assay comprises a sandwich assay, the mobilisable reagent zone may bear a mobilisable labelled binding reagent for the analyte and, downstream from this, the analyte detection zone bears immobilised non-labelled binding reagent for the analyte. Of course, other assay formats such as a competition assay or inhibition assay are also possible, known to the skilled person and included within the scope of the invention. Another example is where the analyte is an enzyme, which is capable of cleaving the assay reagent to produce a cleavage product that is subsequently detected. In some embodiments, the cleavage product may not be directly detectable; instead the cleavage product can be subsequently involved in other reactions which eventually lead to a product(s) that are detected.

The component(s) defining the assay flow path may also contain one or more control reagents, which may be used in the conventional fashion to provide an indication at a "control zone" that the assay has run correctly. Accordingly, in one embodiment the assay flow path further comprises a control zone. For instance, mobilisable labelled control reagents may be provided at an upstream location in the assay flow path {e.g. in the mobilisable reagent zone), with immobilised control reagents in the control zone. The mobilisable control reagents may bind to the immobilised control reagents, and so accumulation of the label in the control zone

3282712v1 affords a signal that the assay has run correctly. The mobilisable labelled test reagents may bind to the immobilised control reagents to form the control zone. Typically, the control zone is located downstream from the analyte detection zone. The assay flow path may be defined by a component, or by a plurality of fiuidically connected components. For instance, the mobilisable reagent zone may be provided on a first material (which may be a so-called "conjugate pad"), and the analyte detection zone may be provided on a downstream, fiuidically connected, second material. The component(s) defining the assay flow path may comprise any material capable of allowing the sample to flow from the sample receiving member to the assay reagent(s). The assay device may be configured as a lateral flow device, and the component(s) may comprise a porous, fibrous or bibulous carrier. In an embodiment, the component(s) comprises a porous carrier, or a plurality of fiuidically connected porous carriers to form a porous carrier. The porous carrier(s) may comprise any material suitable for conveying the sample to the assay reagent(s). The wicking rate of the sample along the assay flow path is preferably slower than the wicking rate of the sample through the sample receiving member.

Examples of the porous carrier material(s) include glass fibre, cellulose, nitrocellulose, paper, silica, porous synthetic polymers such as sintered PET, and material comprising polyester, nylon, cotton, mono-component fibre combinations thereof, or bi-component fibre combinations thereof. The porous carrier material(s) may be a woven or a non-woven material.

In an embodiment, the porous carrier(s) comprise glass fibre and/or nitrocellulose. In an embodiment, the components defining the assay flow path comprise a glass fibre pad on which the mobilisable reagent zone is located, and a nitrocellulose strip on which the analyte detection zone is located, the glass fibre pad being fiuidically connected to and upstream of the nitrocellulose strip.

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3282712v1 At least a portion of the sample receiving member may overlap the porous carrier. Alternatively, they may be in end-to-end contact.

The assay device may further comprise a sink pad located downstream from all the assay reagent(s) and any control reagent(s) present in the assay flow path, typically at the terminus of the assay flow path. The sink pad encourages continued flow of the sample along the assay flow path, by wicking sample from the other, upstream component(s) defining the assay flow path and retaining it within the sink pad. The sink pad may comprise any suitable absorbent or bibulous material as is known in the art, such as cellulose, cotton and/or glass fibre.

The assay device of the invention is adapted such that the sample receiving member and the sample detection element are fluidically connectable when the gap between the sample receiving member and the sample detection element is bridged by the fluid sample. In other words, the device is adapted such that fluidic contact is made between the sample receiving member and the sample detection element when a sufficient amount of sample has been added to the sample receiving member to bridge the gap. In practice, this means that the sample receiving member and the sample detection element are positioned relative to each other such that when a sufficient amount of fluid is applied to the sample receiving member to supersaturate the sample receiving member, fluidic contact is made between the sample receiving member and the sample detection element.

In the present invention, the sample receiving member is porous. The sample receiving member absorbs fluid until it is saturated, and will then become supersaturated (or oversaturated or superwetted) with sample when further sample fluid is added. In the present invention, the sample receiving member is "supersaturated" at the point when the sample fluid forms a meniscus on the surface of the sample receiving member. This allows fluidic contact to be made between the sample receiving member and the sample detection element by bridging the gap between the two. It is this property that is utilized in the present invention in order to give an indication that a sufficient sample volume has been obtained.

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3282712v1 In practice, the device will be designed to ensure that the sample receiving member is supersaturated only when a predetermined amount of sample fluid has been taken up by the device. The device will therefore be designed such that a predetermined amount of sample fluid will cause the sample receiving member to supersaturate and fluidic contact to be made between the sample receiving member and the sample detection element by bridging of the gap between the two. Fluidic contact can be made between the sample receiving member and the sample detection element either directly or indirectly, as described in detail herein. It is within the ability of a person skilled in the art to select the absorbent capacity of the sample receiving member based on the volume of sample fluid sufficient to make fluidic contact between the sample receiving member and the sample detection element allowing the device to perform adequately and deliver an accurate result.

The absorbent capacity of the sample receiving member will generally therefore be selected to be in the range of sample fluid volumes defined by a lower limit which is the minimum amount of sample fluid required to supersaturate the sample receiving member and cause fluidic contact to be made between the sample receiving member and the sample detection element and an upper limit which may be greater than the maximum volume that the components defining the assay flow path (including sink pad if present) can hold. The sample receiving member should be capable of becoming supersaturated with sample for at least a brief period of time, even when in fluidic contact with a component of the assay flow path.

The surface finish of the sample receiving member will have an impact on the extent to which the supersaturation occurs. Whilst the surface of a porous material may seem smooth to the eye, fibres can be present on the surface of the material. Sample fluid may collect at these fibres only when the material is supersaturated, filling the gap by capillary action and/or wetting of the surface fibres. The surface finish on the sample receiving member may not have any fibres or may have very few fibres on its surface, in which case sample fluid can still fill the gap between the sample receiving member and the sample detection element by capillary action.

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3282712v1 The sample receiving member and the sample detection element can be positioned in various configurations in order to achieve the effect described herein, i.e. that when a sufficient amount of fluid is applied to the sample receiving member to supersaturate the sample receiving member, fluidic contact is made between the sample receiving member and the sample detection element.

The sample detection element is located in close proximity to the sample receiving member, for example just above or below the sample receiving member, such that there is a gap between the sample detection element and the sample receiving member. The sample detection element does not directly contact the sample receiving member. The sample detection element is held a small distance away from the sample receiving member such that there is a gap between the sample receiving member and the sample detection element. The gap can be left empty (an air gap) or can be completely or partially filled with suitable materials.

Subject to the requirement that there be a gap between the sample receiving member and the sample detection element, the sample detection element can be located in any position relative to the sample receiving member. In one embodiment, the sample detection element is located at the downstream end of the sample receiving member. In one embodiment, the sample detection element, for example a pair of electrodes, spans the downstream end of the sample receiving member and the upstream end of the assay flow path.

The gap between the sample receiving member and the sample detection element can be formed using any suitable means. For example, the gap can be formed using plastic mouldings to hold the sample receiving member and the sample detection element apart at the appropriate distance. Alternatively, an additional piece of material such as tape, e.g. electrically insulating tape, can be added to at least one surface of the sample receiving member in order to create a raised surface on which the sample detection element, e.g. electrode pair can lie, thus creating a gap between the sample receiving member and the sample detection element. Such a configuration is shown in Figure 6. The means for forming the gap between the sample receiving member and the sample detection element will be the same whether the gap is an air gap or is completely or partially filled with suitable materials.

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3282712v1 In one embodiment, a physical barrier is formed between the sample receiving member and the sample detection element. This acts as a barrier to any fluid which might flow along the surface of the sample receiving member. The physical barrier can contact at least part, but not all, of the surface of the sample detection element. It is necessary for the physical barrier not to be in contact with the whole surface of the sample detection element in order to create the gap between the sample receiving member and the sample detection element. For example, when tape is used to create the gap, a portion of the sample receiving member underneath the sample detection element will be left uncovered by the tape. In other words, part of the sample receiving member is left exposed. This area is denoted by numeral 12 in Figure 8. This means that the physical barrier is only in contact with part of the sample detection element, as shown in Figures 6 to 9. Typically, the physical barrier is in contact with at least the upstream end of the sample detection element. Typically, the physical barrier is not in contact with at least part of the downstream end of the sample detection element. In some embodiments, the physical barrier is not in contact with the sample detection element at all such that there is a gap between the physical barrier and the sample detection element. The dimensions of the physical barrier such as tape can be chosen by a person of skill in the art according to the dimensions of the sample receiving member and the location and size of the sample detection element.

In one embodiment, the gap between the sample receiving member and the sample detection element is an air gap. This embodiment is shown in Figure 1. In this embodiment, fluidic contact is made between the sample receiving member and the sample detection element by means of the sample fluid bridging the gap between the two components, as shown in Figure 3.

In another embodiment, the gap between the sample receiving member and the sample detection element is completely or partially filled with suitable materials. In one embodiment, a swellable material such as a xerogel or hydrogel is present in the gap between the sample receiving member and the sample detection element. Typically, such a swellable material partially fills the gap between the sample receiving member and the sample

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3282712v1 detection element. An example of this embodiment of the invention is illustrated in Figures 10a and 10b. Figure 10a shows the swellable material initially only in contact with the sample detection element and not with the sample receiving member. The swellable material swells on contact with fluid from the sample receiving member when it is supersaturated. Eventually, the swellable material swells sufficiently to bridge the gap between the sample receiving member and the sample detection element, as shown in Figure 10b. In an alternative embodiment to that shown in Figures 10a and 10b, the swellable material is initially only in contact with the sample receiving member and not the sample detection element. Again, the swellable material swells on contact with fluid from the sample receiving member and eventually the gap between the sample receiving member and the sample detection element is bridged. In this embodiment, fluidic contact is therefore made between the sample receiving member and the sample detection element indirectly, by means of swelling of the swellable material that is situated between the sample receiving member and the sample detection element.

In another embodiment, a fluid transfer component is present in the gap between the sample receiving member and the sample detection element. This embodiment of the invention is illustrated in Figure 11. As shown in Figure 11 , the fluid transfer component is present in fluidic contact with only the sample receiving member. Alternatively, the fluid transfer component is present in fluidic contact only with the sample detection element. In another embodiment, the fluid transfer component completely fills the gap between the sample receiving member and the sample detection element. The fluid transfer component can be any component that allows fluid to pass through or across it, for example a permeable membrane or a piece of paper. In this embodiment, fluidic contact is made between the sample receiving member and the sample detection element indirectly, by means of the fluid passing through or across the fluid transfer component that is situated between the sample receiving member and the sample detection element, in fluidic contact with either the sample receiving member or the sample detection element. In the embodiment where the fluid transfer component completely fills the gap between the sample receiving member and the sample detection element, the time taken for the sample fluid to pass through or across the fluid transfer component can be used to create a delay between sample fluid leaving the sample receiving

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3282712v1 member and reaching the sample detection element. The skilled person will be able to choose suitable materials to create the required delay.

In another embodiment, a dissolvable material completely or partially fills the gap between the sample receiving member and the sample detection element. The dissolvable material is a material that dissolves on contact with sample fluid, for example a sugar or a dissolvable polymer, film or membrane. On contact with sample fluid, the dissolvable material gradually dissolves until the gap between the sample receiving member and the sample detection element is bridged. Again, the speed at which the dissolvable material dissolves can be used to create a delay between sample fluid leaving the sample receiving member and reaching the sample detection element. The skilled person will be able to choose suitable materials to create the required delay.

In one embodiment, part of the sample receiving member may be present in the gap between the sample receiving member and the sample detection element. This applies particularly when the sample receiving member is formed of a fibrous material. Fibres present on the surface of the material can therefore be present in the gap between the sample receiving member and the sample detection element, as described herein. In another embodiment, the sample detection element is an electrode and a second electrode is present in the gap between the sample receiving member and the sample detection element, in contact with the sample receiving member. This embodiment is described in detail below and is shown in Figure 4. The gap will be of a suitable size to ensure that fluid from the sample receiving member when it becomes supersaturated makes fluidic contact with the sample detection element either directly or indirectly, as described herein. The appropriate size of the gap will vary depending on several factors including the size of the sample receiving member, the desired sample volume, the hydrophilicity of the sample and the surface characteristics of the sample receiving member (i.e. contact angle of the sample on the surface of the sample receiving member). The skilled person will be able to calculate using routine experiments a suitable gap size based on these considerations. However, the gap will typically be small enough that

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3282712v1 it allows fluidic connection to be made between sample receiving member and the sample detection element when the sample receiving member when is supersaturated. When the gap is an air gap or is completely or partially filled with a material that does not swell, fluidic contact is typically made between the sample detection element and the sample receiving member by capillary action and the gap between the sample detection element and the sample receiving member is typically of a suitable size to allow this. However, when the gap is partially filled with a material that swells to any degree on contact with fluid (such as a xerogel or hydrogel) the gap may be slightly larger, as described herein. The orientation (vertical or horizontal) in which the device is held at sample addition and therefore at which a test using the device is run can influence the dimensions of the gap required between the sample receiving member and the sample detection element. The gap can be optimised alongside the selection of the materials used for the sample receiving member and the sample detection element, with respect to dimensions, capacity, flow rate etc.

As described herein, the gap will typically be small enough that it allows fluidic connection to be made between sample fluid and the sample detection element when a meniscus is formed from sample fluid on the surface of the sample receiving member when it is supersaturated, typically when the device is held horizontally. For example, the gap will typically not be large enough that it would require sample fluid to drip from the surface of the sample receiving member in order to contact the sample detection element, for example when the device is held vertically or otherwise.

The size of the gap used in the test will also be dependent upon the selection of materials used to form the sample receiving member and the sample detection element. For example, when the sample receiving member is formed of a fibrous material, as described herein, fibres on the surface of the sample receiving member may collect sample fluid when the sample receiving member is supersaturated and fluidic contact can then be made between one or more such fibres and the sample detection element, for example by capillary action.

In some embodiments, when the gap is an air gap or is completely or partially filled with a material that does not swell, an appropriate gap size will be in the region of 0.05mm to

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3282712v1 1.5mm, for example 0.1mm to 1.3mm, for example 0.15mm to 1.25mm, for example 0.18mm to 1.2mm, for example 0.2mm to 1mm, for example 0.25mm to 0.9mm, for example 0.3mm to 0.8mm, for example 0.35mm to 0.75mm, for example 0.4mm to 0.7mm, for example 0.45mm to 0.6mm or 0.5mm.

In other embodiments, when the gap is completely or partially filled with another material that swells to any degree on contact with fluid (such as a xerogel or hydrogel), the gap may be slightly larger and an appropriate gap size will be in the region of 0.05mm to 2mm, for example 0.1mm to 1.8mm, for example 0.15mm to 1.5mm, for example 0.18mm to 1.2mm, for example 0.2mm to 1mm, for example 0.25mm to 0.9mm, for example 0.3mm to 0.8mm, for example 0.35mm to 0.75mm, for example 0.4mm to 0.7mm, for example 0.45mm to 0.6mm or 0.5mm. In this embodiment, when the swellable material partially fills the gap, the gap will be of a suitable size that the remaining gap between the material and the sample receiving member or sample detection element will be of a similar size to or the same size as that of the air gap described herein. In addition the properties of the swellable material including its rate of expansion will also be a factor which influences the size of the gap.

The gap size typically corresponds to the thickness of the material (for example tape) used to create the gap.

The sample detection element may be any suitable means of detecting the presence of sample in the location where the element resides; the detection being performed either solely by the element itself or in conjunction with another component of the device. In some embodiments, the sample detection element itself is adapted to indicate to the user that the presence of sample at the sample detection element has been detected. In some embodiments, the presence of sample at the sample detection element is indicative that a sufficient amount of sample has been obtained. In other embodiments, the presence of sample at the sample detection element triggers a further event or series of events which is/are indicative that a sufficient amount of sample has been obtained.

In one embodiment, the sample detection element comprises a visual sensing system. This may be a chemical sensing system which may comprise a reagent which changes colour in

18

3282712v1 response to an inherent property of the sample, e.g. water content, pH, chemical component (other than the analyte which is being tested for), temperature (if the sample is a recently obtained bodily fluid). Such a reagent is referred to herein as a colour change reagent. Compounds which change colour upon hydration are well known to the skilled person and include cobalt(II)chloride, copper(II)sulphate. The reagent may be supported on a substrate e.g. paper. The colour change may be observable visually through a window in the housing of the device. The colour change may be measurable or visible to the eye through a window in the housing of the device. In some embodiments, the sample detection element consists of a chemical sensing system comprising one or more colour change reagents, i.e. no other components are present in the chemical sensing system. For example, the chemical sensing system can comprise one or more colour change reagents, together with one or more buffers, detergents and/or proteins, but no other components. In one embodiment, the sample detection element is a pH indicator, such as pH indicator paper.

In another embodiment, the sample detection element comprises an optical sensing system. For instance, the colour change of the sensing system described above may be detected using appropriate illumination and detection elements such as light emitting diodes and photodetectors. Such detection elements may be used to detect changes in the optical properties of the sample detection element, for example the change in the transmission of light on the sample detection element, for example when going from the dry to the wet state.

In one embodiment, the sample detection element is non-absorbent.

In another embodiment, the sample detection element comprises an electrode. In one embodiment, the sample detection element is a pair of electrodes. In another embodiment, the sample detection element is a single electrode. When the sample detection element comprises an electrode, the aim of the configuration of the assay device of the invention described herein is that the sample fluid, such as urine, acts as an electrolyte bridge to complete an electrical circuit in which the electrode is comprised.

19

3282712v1 Current then flows in the electrical circuit to power an indication system, which provides the user with an indication that sample fluid has been detected at the sample detection element. An indication can also be provided to the user that a sufficient amount of sample has been applied. The indication of sample sufficiency can be provided either directly or indirectly, as described herein. Preferably, the indication method provides immediate feedback to the user during sampling itself, so that they can ensure sufficient sample is applied. This is particularly useful in relation to midstream sampling of urine. Alternatively, the indication method provides feedback to the user after sampling that the test should be rejected because of undersampling. This provides valuable feedback to the user, making them aware that undersampling was the cause of device failure. On testing with another device the user will be more aware of the importance of sampling correctly.

The electrode is part of a pair of electrodes. The pair of electrodes consists of two electrodes, an anode and a cathode. The pair of electrodes typically consists of two elongated strips, but can be of any suitable shape or size.

In the present invention, the sample detection element is spaced apart from the sample receiving member by a gap such that the sample receiving member and the sample detection element are fluidically connectable when the gap is bridged by the sample fluid.

In one embodiment, the sample detection element is an electrode. The electrode is therefore spaced apart from the sample receiving member. In this embodiment, the other electrode of the electrode pair is in fluidic contact with the sample receiving member and is typically present on the surface of the sample receiving member. The second electrode can be present at any location on the sample receiving member. This embodiment is illustrated in Figures 4 and 5.

In another embodiment, the sample detection element is a pair of electrodes and both electrodes are therefore spaced apart from the sample receiving member. In this embodiment, the two electrodes are typically spaced the same distance apart from the sample receiving member. In other words, the gap between the sample receiving member and each electrode is approximately the same. In any case, the largest gap between the sample receiving member

20

3282712v1 and one of the electrodes should be suitable for direct contact to be made between fluid from the sample receiving member and that electrode when the sample receiving member is supersaturated. In one embodiment, the two electrodes are located in close proximity to the same side of the sample receiving member. In this embodiment, the two electrodes can be elongated strips that are approximately in parallel to each other. In another embodiment, one of the electrodes is located in close proximity to one side of the sample receiving member and the other electrode is located in close proximity to another side of the sample receiving member. The electrodes do not necessarily need to be parallel to each other and can be of different shapes and sizes.

When the sample detection element is an electrode or pair of electrodes, the gap between the sample receiving member and the sample detection element is typically created by forming a physical barrier between the sample receiving member and the sample detection element, for example as shown in Figures 6, 7, 8 and 9. It is not necessary for the physical barrier to cover the whole width of the sample receiving member. However, when the sample detection is a pair of electrodes, which are both spaced apart from the sample receiving member, the physical barrier should at least cover the width of the two electrodes, i.e. it should bridge the gap between the two electrodes, thus preventing current from flowing between the two electrodes until the sample receiving member is supersaturated. This embodiment of the invention is shown in Figure 9.

In one embodiment, where the gap is completely or partially filled with a dissolvable material, the electrodes can be spring loaded such that they are physically held away from the sample receiving member until the material dissolves.

The electrodes can be made of any suitable material. Typically, the electrodes are made of a metal, for example copper, silver, lead or zinc. Alternatively, the electrodes are made of a non-metal, for example carbon. Both electrodes may be made from the same material or they may be made from two different respective materials. In some embodiments, the surface of one or both of the electrodes can be treated, for example with a hydrophilic layer, to help wetting and aid bridging of the gap between the sample receiving member and the electrodes.

21

3282712v1 This may be of particular use, for example, in the embodiment of the invention shown in Figure 4, where the electrode 6 is coated with e.g. a hydrophilic layer for this purpose.

The electrodes thus complete an electrical circuit. When sample fluid contacts the electrodes, an electrolyte bridge is formed between the electrodes and current flows from the battery to power an indication system which connects to the electrical circuit and which provides the user with an indication that sample fluid has been detected at the sample detection element. An indication can also be provided to the user that a sufficient amount of sample has been applied. The indication of sample sufficiency can be provided either directly or indirectly, as described herein. The point at which current begins to flow from the battery is referred to herein as "wake up" of the device.

In some embodiments, detection of sample fluid at the electrodes and therefore the flow of current from the battery is itself indicative that a sufficient amount of sample has been applied. In other embodiments, the flow of current from the battery simply "wakes up" the device and then another method is used to detect and/or indicate sample sufficiency.

For example, in one embodiment an assay device of the invention in which the sample detection element is an electrode or electrodes further comprises one or more electrodes that are in contact with or held slightly apart from one or more of the components defining the assay flow path, for example a conjugate pad or nitrocellulose strip. Such additional electrodes can be held apart from the one or more components defining the assay flow path by means of a gap as described herein and having the same features as the gap described herein. Such electrodes detect the presence of sample fluid on one or more of the components defining the assay flow path, as appropriate.

The device can be set up to "wake up" when sample fluid bridges the gap between the sample receiving member and the sample detection element, but not to provide an indication of sample sufficiency until the sample has flowed a predetermined distance along the assay flow path and therefore to one or more of the components defining the assay flow path, as defined by the location of the electrodes on the assay flow path, for example in contact with or held slightly apart from the conjugate pad or nitrocellulose strip. Once sample fluid has reached

22

3282712v1 those electrodes, current flows into a separate circuit which provides an indication that a sufficient amount of sample has been applied.

Alternatively, the device can be set to "wake up" only when sample fluid has flowed a predetermined distance along the assay flow path and therefore to one or more of the components defining the assay flow path, as defined by the location of the electrodes on the assay flow path, for example in contact with or held slightly apart from the conjugate pad or nitrocellulose strip. Once sample fluid has reached those electrodes, current flows and "wakes up" the device. However, the device will only provide an indication of sample sufficiency when the sample receiving member is supersaturated and sample fluid bridges the gap between the sample receiving member and the sample detection element, thus causing current to flow from the battery to power an indication system which connects to the electrical circuit and which provides the user with an indication that a sufficient amount of sample has been applied.

In these embodiments, a timer can be used to monitor the amount of time elapsed between wake up and indication of sample sufficiency, with an indication being given to the user if a sufficient amount of sample has not been obtained in a given time. The aim of such an indication would be to alert the user to be more vigilant about the sampling procedure when carrying out a subsequent assay.

In one embodiment, the assay device of the invention comprises an indication system adapted to indicate that the presence of sample at the sample detection element has been detected. The indication system is typically present when the sample detection element is an electrode or pair of electrodes.

The indication system can indicate that the presence of sample at the sample detection element has been detected by any suitable method known in the art. The indication method could for example be visual (e.g. optical), aural (e.g. using a piezoelectric sounder) or tactile (e.g. vibrations created in the handle of the device). Optical methods include using LEDs or an LCD screen on which an icon or other message appears once a sufficient amount of sample has been applied to the device. Alternatively, a backlit LCD screen could be used which

23

3282712v1 changes colour to indicate to the user that a sufficient amount of sample has been applied to the device. When the indication method includes an LED, the LED is typically located at the downstream (distal) end of the assay device so that the light is easily visible to the user when carrying out the assay.

Indication methods for example indication by sound or vibration (indirect viewing methods) are preferable for use in midstream sampling, since these methods allow the user to continue holding the device in the urine stream until an indication of sample sufficiency is apparent. Conversely, direct viewing methods generally require the user to bring the device out of the urine stream to visually inspect the status of the sample sufficiency indicator. The device may need to be returned to the urine stream if insufficient sample has been applied to the device.

In a second aspect, therefore, the present invention provides the use of an assay device of the invention for indicating sample sufficiency when the device is held in the urine stream of a patient. In a related aspect, the invention also provides a method of alerting a patient to the fact that a sufficient sample has been obtained when mid-stream urine sampling, comprising: (a) holding an assay device of the invention in the urine stream of a patient; and (b) alerting the patient to the fact that a sufficient sample has been obtained, whilst the patient continues to sample from the urine stream. In these aspects of the invention, the assay device comprises an indication system to alert the patient to the fact that a sufficient sample has been obtained. The patient is typically a female patient as the assay device is typically a pregnancy test. In a related aspect, the present invention provides the use of an assay device of the invention for indicating to a user after sampling that the test has failed due to insufficient sampling. The invention is not limited to the detection of any particular analyte. For instance, the analyte may be of a mammalian, especially of a human origin, or of a bacterial or viral origin. More than one analyte may be detected. In the case where the device includes more than one analyte detection zone, at least a portion of the sample receiving member is preferably upstream of, or level with, at least one of the analyte detection zones.

The presence and/or amount of the analyte(s) may be indicative of any clinical, physiological or medical condition, e.g. pregnancy or fertility. The analyte(s) may, for example, be a toxin,

24

3282712v1 pollutant, organic compound, protein, enzyme, peptide, microorganism, bacterium, virus, amino acid, nucleic acid, carbohydrate, hormone, steroid, vitamin or drug. In an embodiment, the analyte(s) is a hormone. In an embodiment, the analyte(s) is human chorionic gonadotropin (hCG), luteinizing hormone (LH), estrone-3-glucuronide (E3G), or a fragment or isoform thereof. These analytes are used to indicate pregnancy or the fertility status of a female.

The assay device may provide a qualitative, semi-quantitative or quantitative detection of the analyte of interest. The result of the assay can be interpreted by the user by viewing the analyte detection zone(s) and the control zone(s) if present measured by an optical or other measuring system and the result can be displayed in any known suitable form, such as via a digital display or an alternative visual signal of the assay result.

The assay device may detect more than one analyte, for instance via the inclusion of a separate detection zone for each analyte in the assay flow path. Alternatively, the assay device may comprise a plurality of separate assay flow paths; each may have its own associated sample receiving member, or a single sample receiving member may be shared between assay flow paths (e.g. if the assay flow paths are arranged side by side). The device may use a plurality of separate assay flow paths in the quantitation or semi-quantitation of a single analyte.

In use, the sample may be applied directly to the device. When the sample liquid is a bodily fluid, the device can be used to collect the liquid sample directly from a subject. For example, the device can be used to collect a mid-stream urine sample.

Alternatively, the sample may be subjected to a liquid pre-treatment step before being exposed to the assay device. The liquid pre-treatment step may comprise one or more of, but not limited to, a dilution, a liquid suspension, an extraction, a binding reaction, a biochemical reaction, a chemical reaction, a lytic reaction, a buffering or a treatment with a surfactant. Thus, as discussed above, the liquid pre-treatment step may be used in order to ensure that the sample is applied to the device in liquid form, applied with the required and controlled

25

3282712v1 viscosity liquid, and/or to ensure that the analyte of interest is presented in a form which will allow the analyte to react or interact with the one or more assay reagents.

The assay device may further comprise a sampling means for obtaining a sample and transferring the sample to the sample receiving member, after any desired pre-treatment steps have been carried out. The sampling means may be adapted to receive a sample of bodily fluid from a subject.

Referring to Figure 1, an assay device in accordance with the invention comprises a sample receiving member 1 fluidically connected to two components 2 and 3 defining an assay flow path. Components 2 and 3 are fluidically connected strips. Component 3 is a detection member comprising an analyte detection zone 31. The assay flow path is enclosed in a housing 4, and the sample receiving member 1 projects from the housing 4. The downstream end of the sample receiving member 1 overlaps the upstream end of strip 2, which is a conjugate pad bearing labelled mobilisable assay reagents 22 in a mobilisable reagent zone 21. The downstream end of the conjugate pad 2 overlaps the upstream end of strip 3, which bears immobilised non-labelled assay reagents 32 in an analyte detection zone 31. A sample detection element 13 is present above the downstream end of the sample receiving member 1 and there is a gap 7 between the sample receiving member 1 and the sample detection element 13.

Figure 2 illustrates a plan view of part of the assay device shown in Figure 1, in which the sample detection element is a pair of electrodes 5 and 6. Figure 3 illustrates a side view of the assay device shown in Figure 1 when in use. Referring to Figure 3, once the sample receiving member 1 has become supersaturated, fluid 8 forms a meniscus on the surface of the sample receiving member 1 and contacts the sample detection element 13, bridging the gap between the sample receiving member 1 and the sample detection element 13.

Figure 4 illustrates a side view of part of an assay device in accordance with another embodiment of the invention, in which the sample detection element is an electrode 5 which

26

3282712v1 is present above the downstream end of the sample receiving member 1 ; a second electrode 6 is present on the surface of the sample receiving member.

Figure 5 illustrates a side view of part of an assay device in accordance with a further embodiment of the invention, corresponding to the embodiment of Figure 4 but wherein the second electrode 6 is present on a different surface of the sample receiving member 1. Again, the sample detection element is an electrode 5 which is present above the downstream end of the sample receiving member 1. Figure 6 illustrates a side view of part of an assay device in accordance with a further embodiment of the invention, in which a component, e.g. tape, is used to control the size of the gap between the sample receiving member and the sample detection element, and also to create a physical barrier to any fluid which might flow along the surface of the sample receiving member. Referring to Figure 6, in one embodiment a component 9 is wrapped around the sample receiving member 1 in order to raise the level of the surface of the sample receiving member 1 on which the sample detection element 13 lies, thus creating a gap 7 between the sample receiving member 1 and the sample detection element 13.

In an alternative embodiment shown in Figure 7, the component 9 is placed only on the surface of the sample receiving member 1 nearest to the sample detection element 13 in order to raise the level of the surface of the sample receiving member 1 on which the sample detection element 13 lies, thus creating a gap 7 between the sample receiving member 1 and the sample detection element 13. In an alternative embodiment to that shown in Figure 6/7, the component 9 is not in contact with the sample detection element 13 and there is therefore a further gap between the component 9 and the sample detection element 13.

Figure 8 illustrates a plan view of part of the assay device of Figure 6/7, where the sample detection element is a pair of electrodes 5 and 6, and where the component 9 spans the whole width of the sample receiving member 1. The component 9 is positioned on the sample

27

3282712v1 receiving member 1 such that there is an exposed area 12 of the sample receiving member 1 underneath part of the electrodes 5 and 6.

Figure 9 illustrates a plan view of part of the assay device of Figure 6/7, where the sample detection element is a pair of electrodes 5 and 6, and where the component 9 does not span the whole width of the sample receiving member 1. The component 9 spans both electrodes 5 and 6.

Referring to Figure 10a, in one embodiment a swellable material 11 is present in the gap between the sample receiving member 1 and the sample detection element 13. The swellable material 11 is initially only in contact with the sample detection element 13. Referring to Figure 10b, the swellable material 11 swells on contact with fluid from the surface of the sample receiving member 1 and bridges the gap between the sample receiving member 1 and the sample detection element 13. In an alternative embodiment (not shown), the swellable material 11 is initially only in contact with the sample receiving member 1 and swells on contact with sample fluid to bridge the gap between the sample receiving member 1 and the sample detection element 13.

Figure 11 illustrates a side view of a part of an assay device in accordance with a further embodiment of the invention, in which a fluid transfer component 10 is present in fluidic contact with the sample receiving member 1, and there is a gap between the fluid transfer component 10 and the sample detection element 13. In an alternative embodiment (not shown), the fluid transfer component 10 is present in fluidic contact with the sample detection element 13, and there is a gap between the fluid transfer component 10 and the sample detection element 13.

The dimensions of the assay devices illustrated in the Figures are exemplary only. The Figures are not drawn to scale. It will be appreciated that the dimensions and materials of the assay device of the invention can be varied as desired.

Unless technically inconsistent or otherwise stated, the embodiments of the invention described herein apply to both aspects of the invention mutatis mutandis.

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3282712v1 The prior art documents mentioned herein are incorporated to the fullest extent permitted by law. The invention will be further described and illustrated in the following non-limiting example.

The following example utilised single step lateral flow pregnancy test devices, of the type described in EP 0,291,194. Example 1: Examination of the volume of sample required to start the assay device.

The devices comprised a nylon / polyester bicomponent fibre wick as a sample receiving member, and the assay flow path was defined by a glass fibre conjugate pad overlapping a nitrocellulose strip. Blue latex-labelled anti-hCG antibody and blue-latex-labelled rabbit IgG antibody were deposited on the conjugate pad; the test zone on the nitrocellulose strip contained immobilised non-labelled anti-hCG antibody and the control zone contained immobilised non-labelled goat anti-rabbit IgG antibody. When urine containing a sufficient amount of hCG to indicate pregnancy is applied to the wick and the assay runs correctly, a blue line is expected to form in the test zone and in the control zone, caused by the immobilisation of the blue latex in those areas via a "sandwich" or other binding reaction.

The assay devices were Clearblue digital devices (Swiss Precision Diagnostics GmbH), in which the sample detection element is a pair of electrodes (also referred to herein as "auto starts" or "auto-start points") and the sample receiving member is a wick. The device also comprises means to monitor the flow along the flow path as a function of time relative to when the device has woken up. These measurements are used to identify improper flow along the flow path either as a result of insufficient sampling or from flooding the device. Flooding on the device occurs when there is excessive wetting of the flow path such that the sample liquid travels over the surface of the porous carrier rather than through the carrier by capillary action. In addition flooding can also manifest itself as premature wetting of the distal (downstream) end of the flow path ahead of the proximal (upstream) end resulting in improper movement of the assay reagents within the device.

29

3282712v1 The devices were used either unmodified, where there is no gap between the sample receiving member and the electrodes (as a control) or modified according to the present invention by using tape to create an air gap between the sample receiving member and the electrodes.

The length of the tape was 13mm, the sample receiving member had 3mm exposed at the downstream end of the wick and the thickness of the tape (and therefore the size of the gap) was 150μιη, as shown in Figure 12. The devices were arranged horizontally and the sample (negative hCG buffer standard) was added at a constant rate of 2ml/min with the use an A TA FPLC pump.

Figure 13 is a boxplot of the sample volume required to start an assay device of the invention ("Modified") with tape applied versus a control device in which tape was not applied, ("Control"). As can be seen from Figure 13, the average volume required to start an assay device of the invention was ~923μ1 compared to ~566μ1 for a control device. As shown in Figure 14, all control devices returned a book error (n=10), however all modified devices ran to completion (n=20) indicating correct device operation by way of a Not Pregnant result. A volume of -566 μΐ of sample in the control device leads to a book error as the flow characteristics along the flow path are inadequate to provide a viable test result due to an insufficient volume of sample applied to the device. This error symbol indicates that the test is void and the test should be repeated with a fresh device. It can be seen that the assay devices of the present invention are advantageous in that they do not activate until sufficient sample has been added to the device to provide the correct flow characteristics along the flow path which will lead to a viable test result shown as Not pregnant or Pregnant.

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3282712v1

Claims

1. An assay device for detecting an analyte in a fluid sample, comprising:

a sample receiving member (1), which is porous and fluidically connected to one or more components (2, 3) defining an assay flow path, at least one of which is a detection member (3) comprising an analyte detection zone (31); and

wherein the device is adapted such that the sample receiving member (1) and the sample detection element (13) are fluidically connectable when the gap (7) is bridged by the fluid sample.

2. The assay device of claim 1, wherein the sample receiving member (1) is a wick.

3. The assay device of claim 1 or 2, wherein a swellable material (11) is present in the gap between the sample receiving member (1) and the sample detection element (13) and is initially in contact with either the sample receiving member (1) or the sample detection element (13).

4. The assay device of claim 3, wherein the swellable material (11) is a xerogel or hydro gel.

5. The assay device of claim 1 or 2, wherein a fluid transfer component (10) is present in the gap between the sample receiving member (1) and the sample detection element (13) and the fluid transfer component (10) is in fluidic contact with either the sample receiving member (1) or the sample detection element (13).

6. The assay device of claim 1 or 2, wherein a fluid transfer component (10) completely fills the gap between the sample receiving member (1) and the sample detection element (13).

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3282712v1

7. The assay device of claim 1 or 2, wherein a dissolvable material completely or partially fills the gap between the sample receiving member (1) and the sample detection element (13)

8. The assay device of any one of the preceding claims, wherein the gap is created by forming a physical barrier between the sample receiving member (1) and at least part of the sample detection element (13).

9. The assay device of any one of the preceding claims, wherein the sample detection element is non-absorbent.

10. The assay device of any one of the preceding claims, wherein the sample detection element (13) is adapted to indicate that the presence of sample at the sample detection element (13) has been detected.

11. The assay device of claim 10, wherein the sample detection element includes a colour change reagent.

12. The assay device of any one of claims 1 to 9, wherein the sample detection element comprises an electrode.

13. The assay device of claim 12, wherein the sample detection element is a pair of electrodes (5, 6).

14. The assay device of claim 12 or 13, further comprising one or more additional electrodes that are in contact with or held slightly apart from one or more components defining the assay flow path.

15. The assay device of claim 14, wherein the electrode or electrodes of the sample detection element wakes up the device and the other set of electrodes indicates sample sufficiency, or vice versa.

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3282712v1

16. The assay device of any one of the preceding claims, further comprising an indication system adapted to indicate that the presence of sample at the sample detection element (13) has been detected.

17. The assay device of claim 16, wherein the indication system is adapted to indicate that the presence of sample at the sample detection element (13) has been detected visually, aurally and/or tactilely.

18. The assay device of claim 17, wherein the indication system includes an LED.

19. The assay device of any one of claims 16 to 18 when dependent on any one of claims 12 to 15, wherein the electrode or electrodes (5, 6) are part of an electrical circuit that is connected to the indication system.

20. The assay device of any one of the preceding claims, wherein the component(s) defining the assay flow path (2, 3) comprise(s) a porous material or a plurality of fluidicially connected porous materials.

21. The assay device of claim 20, wherein the analyte detection zone (31) contains one or more immobilised assay reagents (32) and the component(s) defining the assay flow path (2,

3) bear one or more mobilisable assay reagent(s) (22) in a mobilisable reagent zone (21) upstream from the analyte detection zone (31).

22. The assay device of claim 21, wherein the mobilisable assay reagent(s) (22) are borne by a first porous material (2), and the immobilised assay reagents (32) are borne by a f uidically connected second porous material (3).

23. The assay device of any one of the preceding claims, wherein the assay flow path (2, 3) further comprises a control zone.

24. The assay device of any one of the preceding claims, adapted to detect an analyte in a sample that has a viscosity of < 2 mPa.s, < 1.5 mPa.s, or < 1 mPa.s at 25 °C.

33

3282712v1

25. The assay device of any one of the preceding claims, wherein the sample is urine.

26. An assay device for detecting an analyte in a fluid sample substantially hereinbefore described and as shown in Figures 1 to 11.

27. Use of the assay device of any one of the preceding claims for indicating sample sufficiency when the device is held in the urine stream of a patient.

28. A method of alerting a patient to the fact that a sufficient sample has been obtained when mid-stream urine sampling, comprising:

(a) holding an assay device of any one of claims 1 to 26 in the urine stream of a patient; and

(b) alerting the patient to the fact that a sufficient sample has been obtained, whilst patient continues to sample from the urine stream.

29. Use of the assay device of any one of claims 1 to 26 for indicating to a user after sampling that the test has failed due to insufficient sampling.

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3282712v1