Field

The present invention relates to a fluid control device for a fluidic cartridge and a method for its manufacture, and more particularly to the method of injection-overmoulding a second polymer layer onto a first polymer layer wherein the second polymer layer is fused to the first polymer layer.

Background

Sample preparation and analysis presents many logistical problems. Conventionally, many medical samples (such as blood, saliva, urine and swab eluate) are provided to a doctor, for example a general practitioner doctor (GP) or a principle care physician (PCP), in a local surgery without the equipment necessary to analyse the sample. Hence, the sample must be sent to a laboratory where the sample is analysed. The test results must then be collated and returned to the GP to analyse the results and make a diagnosis. This approach is inadequate. Firstly, there is a significant risk that a sample is lost in transit or mismatched with the wrong patient. Moreover, whilst recent developments in technology have reduced the overall time taken to conduct the test, the delay involved in sending the sample to a laboratory is unsatisfactory.

Nevertheless, analytical systems of the kind found in laboratories are complex and it is often difficult to provide sufficient amounts of pure targets from source samples to reliably perform downstream analytical assays. This typically prohibits local GP surgeries from being able to carry out such tests on site.

However, in recent years efforts have been made to reduce the scale of the analytical systems to make tests faster and simpler to run, and require smaller quantities of sample. For instance, "laboratory on a chip" (LOC) devices (a subset of microfluidic devices) integrate almost all medical tests or diagnostic operations performed in a hospital on a single microfluidic chip. The channels forming such microfluidics devices handle small fluid volumes and are connected together so as to achieve a desired function such as mixing of a sample, moving the sample through the device, reacting the sample with different reagents, and so on. These chips may be inserted into machines to control the performance of a test and measure the results.

However, it has been found that handling a sample in a microfluidics device can be very difficult. In such small channels as are found on a conventional LOC, it is difficult to apply external forces to move the sample from one site to another to perform different actions on the sample. There is also a limit to the complexity of a LOC device which operates purely using capillary action. Furthermore, owing to the small sample sizes of LOC's, the devices have reduced sensitivity and the probability of a target being present in the sample is thus reduced.

An alternative approach is to use a fluidic cartridge. The scale of the components of a fluidic cartridge is larger than for a microfluidic device, and so it becomes possible to move a sample through various different sites to perform different actions on it. This makes it possible to perform more complex tests than may be conducted using typical LOC devices, whilst still providing an analytical system of potential use in a local GP surgery.

Scientific assays useful in medical diagnostics have increasingly involved biochemical procedures, such as the polymerase chain reaction ("PCR"). The PCR assay has provided a powerful method of assaying for the presence of defined segments of nucleic acids. It is therefore desirable to perform a PCR assay on a fluidic cartridge.

Reducing PCR to the microchip level is important for portable detection technologies and high- throughput analytical systems. The method can be used to assay body fluids for the presence of nucleic acid specific for particular pathogens, such as the Chlamydia trachomatis bacterium, HIV or any other pathogenic microbe.

The introduction of commercially available automated DNA amplification assays has allowed more laboratories to introduce these technologies for routine testing of specimens. However, there is a need to improve the fluidic devices used for this purpose.

In the prior art various microfluidic devices have been described. In particular, fluid control devices comprising one or more valves have been described. These devices are typically manufactured from a series of layers extending throughout the device, wherein one or more of the layers acts as a frame for the fluidic device, defining the fluid flow channels and cavities, and a further layer acts as a flexible diaphragm which controls fluid flow through the device.

US patent no. US 4,869,282, discloses a valve assembly produced as a layered sandwich made up of individual wafers, including an actuator layer, a stop layer, a valve seat layer, and a layer which has flow channels receiving gas from the valve seat layer and making the necessary interconnections to provide outlets. The valves are micromachined to make valve passageways and openings in a silicon layer during batch processing utilizing known micromaching techniques, such as photolithography and etching techniques. An organic diaphragm layer may then be positioned between the valve seat layer and the stop layer and functions as a valve diaphragm, selectively sealing the valve seat and preventing gas flow through the port opening. The diaphragm layer is sealed to the silicon layer in a separate process involving fusing, such as glass frit or solder sealing. A permanent bond is achieved using glass frits, particularly when the silicon layers are sandwiched against the diaphragm layer.

International patent publication no. WO 95/08716 also presents a valve assembly, composed of a brittle layer and a second layer of material, wherein a flexible sheet of material is sandwiched between the brittle layer and the second layer of material such that it includes a diaphragm so as to control fluid flow through port openings in the brittle layer. Additional layers are incorporated into the design such as a release material layer, for example gold, that is bonded to the flexible sheet of material so as to resist adhesion of the flexible sheet to the brittle layer in regions adjacent to the port openings. The gold layer is not directly bonded to the flexible sheet, but is instead bonded to a thermoplastic Teflon layer by sputtering, evaporation or electroless plating methods. This thermoplastic layer may already be joined to a flexible organic material and also be bonded to a valve seat. The method of manufacture therefore involves several steps and the build-up of several layers of different materials so as to provide a flexible membrane which is only joined to the brittle layer in well-defined regions (away from the port openings). These additional stages add to the time and cost of the manufacturing process. The multiple stages provide increased risk of contamination, and furthermore, some stages require the application of high temperatures and pressures (page 7, line 1 to page 8, line 9) so that care is needed to avoid deformation of the final product.

Other methods of securing the layers of the device together have been suggested in US patent nos. 4,858,883 and 5,932,799. US patent no. 4,858,883 defines a valve comprising a first body portion, a second body portion and a flexible sheet, wherein the flexible sheet can be secured to the second surface by screws, pressure bonding, chemical bonding or an appropriate adhesive. The use of screws would not necessarily provide a leak-proof seal between layers, and thus stronger bonding is required. However, the chemicals and adhesives used to bond layers can provide undesired contaminant molecules, especially owing to their proximity to the fluid containing channels and may interfere with the accuracy of the assay as discussed in US patent no. 5,932,799.

For many applications, the small amount of leakage or contamination is insignificant and the valves operate satisfactorily. However, in other applications, where sample control is important, the valves do not satisfy the sealing requirements. For example, for fluidic PCR cartridges, even the smallest contamination of the sample can lead to amplification of errors. The use of adhesives can also lead to compromised geometry of the system, in particular, if dry adhesives are used, they tend not to be patternable and can lead to voids along the channel or cavities. These voids are "dead" volumes which destroy the desirable properties of the flow channel and make the behaviour of the system less reproducible from system to system. US patent no. US 5,932,799 uses polymeric layers which can be adhesivelessly bonded together (such as polyimides) to form a layered microfluidics analyser module. The layers of polyimides are heated to high temperatures and subject to high pressures in order to allow adhesiveless bonding. Less pressure is applied to the regions of the laminate which overly the valve areas, in order to avoid these relief areas bonding to the valves. This enables the layer over the relief areas to remain flexible and perform as a valve, but adds additional complexity to the manufacture process and the post-construction heat treatment could lead to deformation of the final product.

The current methods of manufacture of these layered fluidic control structures require the use of bonding techniques that may introduce contaminants to the system or alter the geometry of the channels. Furthermore, the processes can be complicated, involving high pressures and temperatures at specific locations on each layer, or the addition of layers to prevent bonding to certain regions of the structure. The use of sheets of flexible polymer in between layers of rigid polymers can also be expensive and wasteful, and thus the designs of these layered devices require further investigation where cost and space are important considerations.

It is desirable to produce a fluidic control device wherein the multiple layers are bonded together to provide leak-proof seals, but without the risk of sample contamination through the presence of adhesives or chemicals. Furthermore, it would be beneficial to avoid the use of high temperatures and pressures post-fabrication so as to avoid distortion or deformation of the formed product. In addition, so as to keep the cost of production of the device to a minimum, materials wastage should be avoided and time and ease of manufacture should be considered.

Summary of Invention

According to a first aspect of the invention, there is provided a method of forming a fluid control device for a fluidic cartridge, comprising the steps of: a) injection-moulding a first polymer layer by injecting a first polymer into a cavity between a first tool portion and a second tool portion, such that the first polymer layer has a top surface, a bottom surface and a void extending through the first polymer layer from the top surface to the bottom surface; b) separating the second tool portion from the first tool portion and applying a third tool portion to the first tool portion; and c) injection- overmoulding a second polymer layer onto at least a portion of the first polymer layer, such that the second polymer layer provides a membrane across the void in the first polymer layer, wherein the step of injection-overmoulding the second polymer layer comprises injecting a second polymer into a cavity between the first polymer layer and the third tool portion to form a seal between the membrane and the first polymer layer.

Preferably the step of injecting a second polymer into a cavity between the first polymer layer and the third tool portion comprises injecting the second polymer into a cavity before the first polymer has cooled to a setting temperature, thereby fusing the first and second polymer layers together

This method is particularly effective since the first tool it simplifies manufacture of the device, thus the first polymer layer does not need to be moved to a different tool or mould, and the second polymer layer can be overmoulded into the cavity formed by the first polymer layer and the new third tool, which further reduces the risk of contamination of the polymer during change of tools. Furthermore, because the second polymer can be injection-overmoulded onto the first polymer layer before that layer has had a chance to cool to a setting temperature, the second polymer can form a strong bond with the first polymer layer, producing an improved seal when compared with prior art manufacturing techniques.

Preferably, a third polymer layer is formed comprising a top surface and a bottom surface, and the method step comprises arranging the layers such that the second polymer layer is disposed between the first and third polymer layers, and such that the bottom surface of said first polymer layer and the top surface of said third polymer layer are in contacting arrangement around at least a portion of the perimeter of the second polymer layer.

Preferably, the step of forming the third polymer layer comprises pre-forming the third polymer layer, and wherein the step of arranging the layers further comprises joining the bottom surface of said first polymer layer to the top surface of said third polymer layer.

Preferably, the step of forming the third polymer layer comprises injection-moulding the third polymer layer, and wherein the step of arranging the layers further comprises joining the bottom surface of said first polymer layer to the top surface of said third polymer layer.

The step of joining the bottom surface of said first polymer layer to the top surface of said third polymer layer preferably comprises clamping the first and third polymer layers together. Preferably, the step of joining the bottom surface of said first polymer layer to the top surface of said third polymer layer comprises bonding, preferably heat sealing, diffusion bonding, adhesively bonding, ultrasonically welding or laser welding the surfaces together. The bonding of the first polymer layer and third polymer layer provides an additional method of securing the second polymer layer between the first and third layers (in addition to the clamping), which can improve the seal between the polymer layers.

Preferably, the steps of injection-moulding the first and second polymer layers, respectively, comprise: a) injecting the first polymer into the cavity between the first tool portion and the second tool portion, such that the first polymer layer has a first thickness; and b) injecting the second polymer into the cavity between the first polymer layer and the third tool portion, such that the second polymer layer has a second thickness; wherein the thickness of the first polymer layer is greater than the thickness of the second polymer layer. The second polymer layer acts as a membrane across the void in the first polymer layer.

The step of injecting the second polymer into the cavity between the first polymer layer and the third tool portion preferably comprises injecting the second polymer from a location in the third tool portion which is offset from the void in the first polymer layer such that the injection point in the second polymer layer is spaced apart from the membrane.

The advantage of having the injection point spaced apart from the membrane is such that any distortion around the injection point does not affect the sealing of the membrane. Distortion may be due to cooling of the second polymer after injection-moulding.

Preferably, the steps of injection-moulding the first and second polymer layers, respectively, comprise: a) injecting the first polymer into the cavity between the first tool portion and the second tool portion such that the first polymer layer further comprises an annular shelf recessed into its bottom surface, the annular shelf extending around the perimeter of the void, and b) injecting the second polymer into the cavity between the first polymer layer and the third tool portion such that the second polymer layer lies across the annular shelf; such that the second polymer layer is flush with the bottom surface of the first polymer layer. This provides a more compact fluidic control device by negating the need for a separate flexible membrane sandwiched between (and thus entirely separating) the first and third polymer layers, and also simplifies the structure of the third polymer layer, which need not accommodate the shape of the flexible membrane.

Preferably, the steps of injection-moulding the first and second polymer layers, respectively, comprise: a) injecting the first polymer into the cavity between the first tool portion and the second tool portion such that the first polymer layer further comprises a depression recessed into its bottom surface, and b) injecting the second polymer into the cavity between the first polymer layer and the third tool portion such that the second polymer layer occupies at least a portion of the depression. The step of injection-moulding the first polymer layer preferably comprises injecting the first polymer into the cavity between the first tool portion and the second tool portion such that the depression forms an annular ring surrounding the void.

The increased area of the surface onto which the flexible membrane may be formed provides for an increased seal surface area, and increases the strength of the bond between the flexible membrane and the first polymer layer.

Preferably, the step of injection-moulding the first polymer layer comprises injecting the first polymer into the cavity between the first tool portion and the second tool portion such that the annular ring comprises a bevelled edge on its inner periphery. The bevelled edge may facilitate manufacture of the device.

Preferably, the step of injection-moulding the first polymer layer comprises injecting the first polymer into the cavity between the first tool portion and the second tool portion such that the depression is formed in the annular shelf.

Preferably, the step of injection-moulding the first polymer layer comprises injecting the first polymer into the cavity between the first tool portion and the second tool portion such that the cross-section of the void in the first polymer layer is circular in plan. The step of injection-moulding the first polymer layer preferably comprises injecting the first polymer into the cavity between the first tool portion and the second tool portion such that the or a diameter of the void is between 2 and 10 mm, preferably between 3 and 7 mm, more preferably between 4 and 6 mm.

Preferably, the step of injection-moulding the first polymer layer comprises injecting the first polymer into the cavity between the first tool portion and the second tool portion, such that the first polymer layer comprises a fluid passageway having an opening in the void in the first polymer layer. The fluid passageway may enable the transmission of a positive or negative fluid pressure into the void for moving the membrane formed over the void

The step of forming the third polymer layer preferably comprises forming the layer such that it further comprises first and second fluid passageways having openings in the top surface of the third polymer layer, and wherein the step of arranging the layers comprises arranging the third polymer layer such that the openings are located within the perimeter of the void formed in the first polymer layer. Preferably, the openings of the first and second fluid passageways are flush with the second polymer layer once the layers are arranged. Alternatively, the openings of the first and second fluid passageways are spaced apart from the second polymer layer once the layers are arranged.

Fluid may be prevented from flowing between the first and second fluid passageways when the second polymer layer is sealed against the openings. Thus the flexible membrane may act as a valve or pump.

Preferably, the setting temperature is between 10 °C and 100°C, preferably between 20°C and 80°C, preferably between 30°C and 70°C, preferably between 40°C and 60°C. The setting temperature may be a property of the first polymer layer.

Preferably, the method of forming a fluid control device comprises the first polymer forming a rigid polymer layer and the second polymer forming a flexible polymer layer. The first polymer may preferably be a thermoplastic selected from one of a) polycarbonate, or b) polypropylene. The second polymer may preferably be a thermoplastic, said thermoplastic is preferably polypropylene. The second polymer may preferably be a thermoplastic elastomer, selected from one or more of: styrenic block copolymers, polyolefin blends, elastomeric alloys, thermoplastic polyurethanes, thermoplastic copolyesters and thermoplastic polyamides.

Preferably, when the first polymer is polycarbonate the second polymer is a thermoplastic elastomer. Preferably, when the first polymer is polypropylene the second polymer is a thermoplastic elastomer.

Polypropylene provides an inert material for fluid contact and also enables heat sealing between the first and third polymer layers, as well as to the second thermoplastic elastomer layer.

According to a second aspect of the invention, there is provided a fluid control device for a fluidic cartridge, comprising; a first polymer layer having a top surface, a bottom surface and a void extending through the first polymer layer and having an opening in at least the bottom surface; a second polymer layer forming a membrane across the void in the first polymer layer, the second polymer layer being fused to the first polymer layer to form a seal between the membrane and the first polymer layer; and a third polymer layer having a top surface and a bottom surface, the second polymer layer disposed between the first and third polymer layers; wherein the bottom surface of the first polymer layer and the top surface of the third polymer layer are in contacting arrangement around at least a portion of the perimeter of the second polymer layer. Preferably, the bottom surface of the first polymer layer and the top surface of the third polymer layer are in contacting arrangement around the entire perimeter of the second polymer layer.

The bottom surface of the first polymer layer and the top surface of the third polymer layer are preferably joined together. Preferably, the bottom surface of the first polymer layer and the top surface of the third polymer layer are mechanically joined together, preferably clamped. Preferably, the bottom surface of the first polymer layer and the top surface of the third polymer layer are bonded together, preferably heat sealed, diffusion bonded, adhesively bonded, ultrasonically welded or laser welded together. The joining of the first and third polymer layers together seals the layers, and reduces the risk of fluid leakage.

Preferably, the first polymer layer has a first thickness and the second polymer layer has a second thickness; and wherein the thickness of the first polymer layer is greater than the thickness of the second polymer layer. This enables the second polymer layer to act as a flexible membrane across the void in the first polymer layer.

Preferably, the second polymer layer is an injection-moulded layer, and the injection point in the second polymer layer is spaced apart from the membrane. As discussed above, any distortion around the injection point does not affect the sealing of the membrane.

Preferably, the first polymer layer further comprises an annular shelf recessed into its bottom surface, wherein the annular shelf extends around the perimeter of the void; and wherein the second polymer layer is flush with the bottom surface of the first polymer layer.

Preferably, the first polymer layer further comprises a depression recessed into its bottom surface; and wherein the second polymer layer occupies at least a portion of the depression. The depression is preferably an annular ring surrounding the void. Preferably, the annular ring comprises a bevelled edge on its inner periphery. Preferably, the depression is formed in the annular shelf. These features of the first polymer layer provide an increased surface onto which the flexible membrane may be formed, and thus increases the strength of the bond between the flexible membrane and the first polymer layer, and facilitate manufacture.

Preferably, the void extending through the first polymer layer has a circular cross-section in plan. The or a diameter of the void is preferably between 2 and 10 mm, preferably between 3 and 7 mm and more preferably 4 and 6 mm. Preferably, the first polymer layer further comprises a fluid passageway having an opening in the void in the first polymer layer to enable transmission of a positive or negative fluid pressure to the void for moving the membrane. The fluid passageway is preferably coupled to a fluid interface for connecting to a source of positive or negative fluid pressure. Preferably, the fluid interface is a pneumatic interface for connecting to a source of positive or gauge gas pressure.

Preferably, the third polymer layer comprises first and second fluid passageways having openings in the top surface of the third polymer layer, wherein the openings are located within the perimeter of the void formed in the first polymer layer. In this configuration the membrane can act as a valve, such that it can seal against the openings in a closed position.

Preferably, the membrane is movable between a closed position when a positive pressure is applied to the void in which the membrane is sealed against the openings of the first and second fluid passageways to prevent fluid communication between the first and second passageways and an open position when a vacuum is applied to the void in which the membrane is spaced apart from the openings of the first and second fluid passageways to permit fluid communication between the first and second passageways.

The fluid control device is preferably operable as a valve or bellows pump. Valves can be used to control fluid flow in the fluidic cartridge and bellows pumps can be used to bump fluid around the cartridge.

When operable as a bellows pump, the fluid control device preferably comprises a cavity between the third polymer layer and the membrane, and wherein the membrane is movable to vary the volume of the cavity. This enables the variation of the volume of fluid that is pumped around the device, in particular, to maximise the volume of fluid that can be pumped.

When operable as a valve, the fluid control device preferably comprises an abutment within the void, wherein the abutment restricts movement of the membrane in its open position. Preferably, the abutment may comprise one or more of a protrusion, a cage, a lip or a cross structure. This abutment can be used to limit the volume of the valve when the membrane is in its open position, thus minimising the dead volume within the valve.

Preferably, the first polymer is a rigid polymer and the second polymer is a flexible polymer. The first polymer is preferably a thermoplastic selected from one of a) polycarbonate, or b) polypropylene. The second polymer is preferably a thermoplastic. Preferably, the thermoplastic is polypropylene. Preferably, the thermoplastic can be a thermoplastic elastomer, preferably selected from one or more of: styrenic block copolymers, polyolefin blends, elastomeric alloys, thermoplastic polyurethanes, thermoplastic copolyesters or thermoplastic polyamides.

When the first polymer is polypropylene the second polymer is preferably a thermoplastic elastomer.

Brief Description of the Figures

Figure 1 is a schematic diagram of an exemplary fluidic cartridge in which the invention may be provided.

Figure 2 is a top view of an exemplary fluidic cartridge in which the invention may be provided.

Figure 3 is an exploded view of the exemplary fluidic cartridge of figure 2.

Figure 4 is a perspective view of the housing of the exemplary fluidic cartridge of figure 2.

Figure 5 is a perspective view of the blister sub-assembly of the exemplary fluidic cartridge of figure 2.

Figure 6A is a top view of the pneumatic layer of the exemplary fluidic cartridge of figure 2.

Figure 6B is a bottom view of the pneumatic layer of the exemplary fluidic cartridge of figure 2.

Figure 7 is a top view of the pneumatic foil of the exemplary fluidic cartridge of figure 2.

Figure 8A is a top view of the fluidic layer of the exemplary fluidic cartridge of figure 2.

Figure 8B is a bottom view of the fluidic layer of the exemplary fluidic cartridge of figure 2.

Figure 9 is a top view of the fluidic foil of the exemplary fluidic cartridge of figure 2.

Figure 10 is a top view of the electrode layer of the exemplary fluidic cartridge of figure 2.

Figure 1 1 is a section view of an advantageous valve arrangement which may form an isolated inventive aspect. Figure 12 is a section view of another advantageous valve arrangement which may form an isolated inventive aspect.

Figure 13a is a section view of an advantageous inlet port arrangement which may form an isolated inventive aspect.

Figure 13b is a perspective section view of the inlet port arrangement of figure 13a.

Figure 14a is a section view of an advantageous capture column arrangement which may form an isolated inventive aspect.

Figure 14b is a perspective section view of a portion of the capture column arrangement of figure 14a.

Figure 15a is a section view of an advantageous waste chamber arrangement which may form an isolated inventive aspect.

Figure 15b is a perspective section view of the waste chamber arrangement of figure 15a.

Figure 16a is a cross section of a first embodiment of a fluidic control device according to the present invention.

Figure 16b is a cross section of a second embodiment of a fluidic control device according to the present invention.

Figure 17 is a cross section of a portion of the fluidic control device of figure 11 a illustrating preferred dimensions of components therein.

Figure 18a is a cross section of a third embodiment of a fluidic control device according to the present invention.

Figure 18b is a cross section of a fourth embodiment of a fluidic control device according to the present invention.

Figure 18c is a cross section of a fifth embodiment of a fluidic control device according to the present invention. Figure 18d is a cross section of a sixth embodiment of a fluidic control device according to the present invention.

Figure 19 is a cross section of a portion of the exemplary cartridge in which the embodiment of figure 18d is disposed.

Figure 20a is a cross section of the embodiment of figure 19 in a closed position.

Figure 20b is a cross section of the embodiment of figure 19 in an open position.

Figure 21 is a cross section of a portion of the exemplary cartridge in which a sixth embodiment of a fluidic control device is disposed.

Figure 22 is a cross section of a portion of the exemplary cartridge in which a seventh embodiment of a fluidic control device is disposed.

Figure 23 is a diagrammatic flow diagram of a first embodiment of a method according to the present invention.

Figure 24 is a diagrammatic flow diagram of a second embodiment of a method according to the present invention.

Figure 25 is a diagrammatic flow diagram of a third embodiment of a method according to the present invention.

Figure 26 is a diagrammatic flow diagram of a fourth embodiment of a method according to the present invention.

Figure 27 is a cross section of an eighth embodiment of a fluidic control device of the invention which also serves to illustrate a preferred method for manufacturing the device.

Detailed Description

Embodiments of the invention will now be described in the context of an exemplary fluid cartridge in which the invention is implemented. Whilst not necessary to understand the present invention, it is beneficial to provide general description of the principles of the structure, manufacture, function and use of the fluidic cartridge and associated methods for performing a test. The exemplary fluidic cartridge and associated methods chosen to illustrate the present invention are for the detection of Chlamydia trachomatis bacterium using PCR amplification and electrochemical detection. However, the skilled person would understand that the invention is not limited to the exemplary fluidic cartridge and associated methods, and is suitable for use in with various different cartridges for a wide variety of sample analysis techniques or biological assays; for example, assays of target nucleic acid sequences in a liquid sample.

Those skilled in the art will understand that the devices and methods of the invention described herein and illustrated in the accompanying drawings are non-limiting exemplary embodiments and that the scope of the present invention is defined solely by the claims. The features illustrated or described in connection with one exemplary embodiment may be combined with features of other embodiments. Such modifications and variations are included within the scope of the present disclosures.

The exemplary cartridge comprises: a fluidic portion through which the sample flows and in which nucleic acid amplification and detection take place; a pneumatic portion which controls flow through the fluidic portion; and at least two electrodes which provide a potential difference for the detection of an amplified nucleic acid of interest. The fluidic portion and pneumatic portion may be constructed of a fluidic layer, a fluidic foil, a pneumatic layer and a pneumatic foil such as those described in relation to the exemplary cartridge below. However, the fluidic portion does not necessarily consist only of a fluidic layer and a fluidic foil and the pneumatic portion does not necessarily consist only of a pneumatic layer and a pneumatic foil. Rather, the layers may interact to produce the fluidic portion and the pneumatic portion such that parts of all or some of the layers make up each portion. Rather than referring to the particular layers of the cartridge, the fluidic portion refers to the particular areas of the cartridge which provide the function of allowing controlled sample flow, and the pneumatic portion refers to the particular areas of the cartridge which provide the function of controlling the flow through the fluidic portion.

The housing, fluidic portion and pneumatic portion are made of plastic. By plastic is meant a synthetic or natural organic material that may be shaped when soft and then hardened, including resins, resinoids, polymers, cellulose derivatives, casein materials, and protein plastics. Examples of plastics from which the cartridge may be constructed include, but are not limited to thermoplastics, for example polycarbonate, polyethylene terephthalate, cyclic olefin copolymers such as Topaz, acrylonitrile butadiene styrene, and thermoplastic elastomers, for example polypropylene. Plastic housings, fluidic portions and pneumatic portions can include components which are not made of plastic (e.g. blisters made from metal foil, metallic inserts at the sample inlet), but they are formed primarily from plastic. The use of plastic materials facilitates economical manufacture of the cartridges.

Whilst the pneumatic and fluidic foils may be made from a metal foil, the preferred materials are plastic including those mentioned above. In particular, it is preferred that foils are a polyethylene terephthalate / polypropylene composite.

The target nucleic acid sequence is any nucleic acid to be detected in a sample. The target nucleic acid(s) to be amplified and detected in the cartridge will usually be DNA, but it is also possible to amplify and detect RNA. In some embodiments a cartridge may permit amplification and/or detection of both DNA and RNA targets.

The liquid sample is the composition which is introduced into the cartridge in order to determine whether the target nucleic acid(s) of interest is/are present. The sample may be a composition in which the nucleic acid to be detected is suspected to be present (e.g. for clinical diagnosis), or may be a composition in which the nucleic acid to be detected is potentially present (e.g. for contamination testing).

The liquid sample can have various sources. For instance, it can be material obtained from an animal or plant (e.g. for diagnosis of infections or for genotyping). Such samples may be obtained with minimal invasiveness or non-invasively, e.g., the sample may be obtained from an animal using a swab, or may be a bodily fluid. As an alternative, the sample may be material obtained from food or water (e.g. for contamination testing). The sample will usually include cells, and the target nucleic acid (if present) can be extracted from these cells within the cartridge. One skilled in the art will appreciate that samples can be diluted or otherwise treated prior to being introduced into the cartridge, but it is preferred that the cartridge can handle material which has not been pre- treated in this way.

An animal from whom the sample is obtained may be a vertebrate or non-vertebrate animal. Vertebrate animals may be mammals. Examples of mammals include but are not limited to mouse, rat, pig, dog, cat, rabbit, primates or the like. The animal may be a primate, and is preferably a human. Thus the cartridge can be used for clinical diagnosis of human samples.

In addition to analysing a sample, the cartridge can analyse a positive and/or negative control to provide confirmation that the cartridge is functioning as expected. The control(s) can be introduced into the cartridge by a user, or can be included within a cartridge before use. The inclusion of an internal positive control nucleic acid allows a user to identify whether a negative result for the sample has been obtained because the nucleic acid amplification has been unsuccessful (false negative). If the positive control nucleic acid fails to be detected in the detection chamber, despite its presence in an amplification chamber, the user will be able to identify the test as a potential false negative result, and can perform another test.

The inclusion of an internal negative control allows a user to identify whether a positive result has been falsely obtained because of the presence of contamination. A negative control can involve performing PCR in a chamber in which no nucleic acid is provided, or in which a sample undergoes an amplification reaction without necessary components e.g. PCR without primers. If nucleic acid is nevertheless detected in the detection chamber, despite its intended absence in an amplification chamber, the user will be able to identify the test as a potential false positive result, and can perform another test.

A positive control nucleic acid may be any nucleic acid that will not be found in a sample used in the cartridge. The internal control DNA may be taken from a bacterium that is not pathogenic to animals and which contains a nucleic acid that is highly specific to the bacterium. One example of a possible bacterium from which the control nucleic acid may be taken for an animal sample is Pectobacterium atrosepticum, although any control nucleic acid may be used that will not be present in a sample.

The fluidic portion of the cartridge comprises channels and chambers through which sample flows. The flow of sample through the cartridge is controlled in two ways. Firstly, the fluidic portion has a gas inlet. The gas inlet is connected to a gas supply, and injection of gas into the fluidic portion via this inlet allows the sample to be pushed downstream through the cartridge, towards the detection chamber. The gas supply may be provided by the reader. As an alternative, the gas supply may be an on-board gas supply. Preferably, the gas supply is provided by an external source and the gas inlet is connected to a pneumatic circuit such that the gas supply is provided via a pneumatic inlet on the cartridge. Secondly, at least one pneumatically controlled valve controls local movement of the sample through the fluidic portion. The pneumatically controlled valve(s) may be controlled independently of other pneumatically controlled valves and may be controlled independently of the gas supply that generally causes downstream movement of the sample via the gas inlet. The gas inlet and the pneumatically controlled valve(s) also permit sample to be flushed through the fluidic portion e.g. to exclude excess volumes of material. The fluidic portion also has an exhaust which allows air and waste material to exit the channels and chambers of the fluidic portion without a build-up of pressure occurring in the cartridge. Preferably, the exhaust comprises a waste chamber and/or a waste vent. The fluidic portion of the cartridge includes reagents and/or physical components for cell lysis and nucleic acid separation. These may be any reagents or physical components that are capable of lysing cells and separating nucleic acids from cell debris and other cellular components. For instance, they may comprise (i) a lysis buffer which is capable of causing lysis of target cells which may be present in the sample e.g. buffers including a detergent such as nonyl phenoxypolyethoxylethanol (available as NP-40) or t-octylphenoxypolyethoxyethanol, (available as Triton X 100), or including guanidine thiocyanate, and/or (ii) a capture support or column which specifically binds nucleic acids but does not bind other undesired cellular components (e.g. proteins and lipids). The capture column comprises a capture filter and may additionally comprise a depth filter. The filters may be made of glass fibres (available as Whatman filters), or may be made of silica, although any column or support which is capable of separating nucleic acids from other cellular components may be used. Elution using a wash buffer to remove cell debris and other cellular components, followed by elution using an elution buffer to elute the separated nucleic acids from the capture support or column can be undertaken such that the capture column can separate nucleic acids from cell debris and other cellular components.

A channel through which the sample flows fluidly connects the sample inlet to at least one amplification chamber where nucleic acid amplification can take place. The purpose of the amplification chamber(s) is to permit amplification of any target nucleic acid of interest that is present in the sample (and, where present, any positive control nucleic acid). Any nucleic acid amplification method may be used and these are described in more detail below in relation to an exemplary cartridge. The different nucleic acid amplification reagents that are required for different nucleic acid amplification methods are well known in the art. These reagents are provided in or upstream of the amplification chamber(s) such that the sample (and any positive control) includes all necessary reagents for nucleic acid amplification once it reaches the amplification chamber. Adaptation of a nucleic acid amplification method according to the target nucleic acid to be detected is also well known in the art (e.g. design of primers). The skilled person would therefore be able to adapt the reagents for nucleic acid amplification accordingly. The term "chamber" does not denote any particular size or geometry, but instead it means a region within the fluidic portion which is designed to permit nucleic acid amplification to occur. Thus, for instance, it could be a region in which the sample can be fluidically isolated (e.g. via the use of pneumatically controlled valves) while the steps required for nucleic acid amplification (e.g. thermocycling, etc.) occur, and it can be located within the cartridge so that it is in the proximity of any external resources that are needed (e.g. next to a heat source within a cartridge reader, thereby permitting thermal cycling to occur). Multiple test amplification channels and/or chambers may be included in the cartridge. The different test amplification channels and/or chambers may include reagents required to amplify different nucleic acids of interest. Therefore using multiple amplification test channels and/or chambers allows multiple tests to be performed on a single cartridge, simultaneously (including any controls). As an alternative, reagents for amplification of multiple different nucleic acids may be present in a single amplification chamber, and the different nucleic acids (whether multiple target nucleic acids, or a target nucleic acid and a control nucleic acid) may be amplified simultaneously in the same amplification chamber.

A further channel through which the sample flows after nucleic acid amplification fluidly connects the at least one amplification chamber to at least one detection chamber where the results of nucleic acid amplification can be detected. In or upstream of the detection chamber are reagents for nucleic acid detection such that the sample includes all necessary reagents for the detection once it reaches the detection chamber. The reagents for nucleic acid detection may be specific for the particular target nucleic acid, i.e. they may allow for detection of the presence of the specific nucleic acid sequence. As an alternative, the reagents for nucleic acid detection may be generic reagents to detect the presence of any nucleic acids. Such generic reagents may be used if all nucleic acids other than the target nucleic acid are removed prior to detection. For example, this may be achieved by providing a nuclease that is capable of hydrolysing all nucleic acids present in the sample other than the target nucleic. The amplified target nucleic acid can be protected from hydrolysis, for example by inclusion of chemical modifications in the primers which are incorporated into the amplified product and which cannot be hydrolysed. Reagents for nucleic acid detection are described below in relation to an exemplary cartridge but usually comprise a probe including a label. The probe is capable of hybridising to the amplified nucleic acid which has been amplified in the amplification chamber(s). Following hybridisation of the probe to the amplified nucleic acid, the detection of the nucleic acid may occur via a detectable change in the signal from the label. In some embodiments the change may be caused by hydrolysis of the probe. Where the probe is hydrolysed, hydrolysis is usually achieved using a double strand specific nuclease, which can be an exonuclease or an endonuclease. Preferably, the nuclease is T7 endonuclease. The signal from the label is capable of undergoing a change following hydrolysis of the probe. This is due to a change in the environment of the label when it moves from being bound to the rest of the probe to being free from the rest of the probe or bound to a single nucleotide or a short part of the probe. Further details of the types of probes and detection methods that may be used can be found in Hillier et al. Bioelectrochemistry, 63 (2004), 307-310. As an alternative, methods for causing a detectable change in the signal from the label which do not rely on hydrolysis of the probe may be used e.g. see lhara et al. Nucleic Acids Research, 1996, Vol. 24, No. 21 4273-4280. This change in environment of the label leads to a change in the signal from the label. The change in signal from the label can be detected in order to detect the presence of the nucleic acid of interest.

Where a positive control nucleic acid is used, the reagents for nucleic acid detection will additionally include a positive control probe including a label. The positive control probe is capable of hybridising to the amplified control nucleic acid. The signal provided by the labels of the positive control and target probes may be the same, but present in separate detection chambers such that the signals corresponding to the control and test nucleic acids can be distinguished. As an alternative, the signal provided by the labels of the control and target probes may be different, such that the signals are distinguishable from one another, even if the probes are present in the same detection chamber.

Multiple test detection channels and/or chambers may be included in the cartridge. The different test detection channels and/or chambers may include reagents required to detect different nucleic acids of interest. Therefore using multiple detection test channels and/or chambers allows multiple tests to be performed on a single cartridge, simultaneously. As an alternative, reagents for detection of multiple different nucleic acids may be present in a single detection chamber, and the different nucleic acids (whether multiple target nucleic acids or a target nucleic acid and a control nucleic acid) may be detected simultaneously in the same detection chamber.

The label is detectable by use of the cartridge's electrodes, and so the label will usually be an electrochemical label, such as a ferrocene. Examples of labels which may be used can be found in WO03/074731 , WO2012/085591 and PCT/GB2013/051643. Signal emitted by the label can be detected by a cartridge reader.

The pneumatic portion of the cartridge comprises at least one pneumatic circuit which each control at least one pneumatically controlled valve. The pneumatic portion controls sample flow through the cartridge by the opening and closing of pneumatically controlled valves. The opening and closing of the valves is controlled by changes in pneumatic pressure in the pneumatic circuit that is applied through a pneumatic pressure inlet. Usually, the cartridge contains many pneumatically controlled valves. The pneumatically controlled valves may be controlled by separate pneumatic pressure inlets. These valves can be used to prevent downstream movement of sample through the fluidic portion until necessary steps have been performed and/or to prevent unwanted reverse movement of sample upstream. For example, a valve may be provided upstream of the at least one amplification chamber in order to prevent downstream movement into the at least one amplification chamber until cell lysis and nucleic acid separation has taken place. Following cell lysis and nucleic acid separation the valve upstream of the at least one amplification chamber may be opened in order to allow downstream flow. It can then be closed again, to prevent backflow out of the chamber back towards the sample inlet.

The cartridge comprises at least two electrodes which can provide a potential difference across the at least one detection chamber. The potential difference causes current to flow through the at least one detection chamber, thereby permitting the detection of signal from electrochemically active labels.

An exemplary cartridge which operates according to the above description will now be described with reference to the accompanying drawings.

1. The exemplary cartridge

1.1 Overview

The exemplary cartridge described below is intended to be a single-use, disposable cartridge for performing a test on a sample introduced into the cartridge. The exemplary cartridge is a fluidic cartridge with channels of an appropriate scale (as detailed hereafter). However, the invention may be performed on a microfluidic device, or an LOC. Once the test has been run, it is preferred that the cartridge is disposed of. However, if desired, the cartridge may be sent for re-processing to enable it to be used again.

It is preferred that the cartridge comprises all of the biological agents necessary for conducting the test of choice. For example, the exemplary cartridge is used for detecting the presence, absence or amount of a pathogen of interest. Any pathogen may be detected. Examples of pathogens which may be detected by the cartridge are Chlamydia trachomatis, Trichomonas vaginalis, Neisseria gonorrhoea, Mycoplasma genitalium and methicillin resistant Staphylococcus aureus. To that end the cartridge comprises reagents for nucleic acid amplification. Nucleic acid amplification may be performed using any nucleic acid amplification method. The nucleic acid amplification method may be a thermocycling method in which the temperature at which the method is performed is varied such that different steps of the amplification are able to take place at different temperatures within the cycle. For example melting, annealing of primers and extension may each be performed at different temperatures. By cycling through the temperatures, the timing of each of the steps of the method can be controlled. As an alternative, the nucleic acid amplification may be an isothermal method in which the temperature is kept constant. In both the thermocycling and the isothermal nucleic acid amplification methods, the temperature is controlled during nucleic acid amplification. Examples of nucleic acid amplification methods are the polymerase chain reaction (PCR), the ligase chain reaction (LCR), strand displacement amplification (SDA), transcription mediated amplification, nucleic acid sequence-based amplification (NASBA), helicase-dependent amplification and loop-mediated isothermal amplification. The reagents for nucleic acid amplification will vary depending of the nucleic acid amplification method used but include a polymerase and nucleotide triphosphates.

As explained below, the cartridge also comprises detection reagents which are capable of detecting the presence or absence of amplified nucleic acids which are the product of the nucleic acid amplification method. The reagents for nucleic acid detection comprise a probe which is capable of hybridising to the amplified nucleic acid. The probe includes a ferrocene label. Following hybridisation of the probe to the amplified nucleic acid, the detection of the nucleic acid occurs via a detectable change in the signal from the label. The change is caused by hydrolysis of the probe, which is achieved using a double strand specific nuclease. The nuclease is a T7 endonuclease. The ferrocene gives different electrochemical signals when it is part of a probe or when it is attached only to a single nucleotide, and so hydrolysis is easily detected. Thus, the change in signal from the label permits detection of the presence of the nucleic acid of interest.

The electrodes allow the detectable change in the signal from the label, which occurs in the presence of the target nucleic acid, to be detected.

The cartridge is configured for use with a cartridge reader (not shown). The cartridge comprises a number of pneumatic, mechanical, thermal and electrical interfaces (described in more detail below) through which the reader interacts with the cartridge to perform the test. Hence, in use, the cartridge would be inserted into the reader, and the reader would be activated to begin interacting with the cartridge via the interfaces to perform the test. For the purposes of understanding the present invention, it is not necessary to describe exactly how the cartridge interacts with the reader to conduct a particular test and provide the test results, but an overview of an exemplary operation of a cartridge is provided hereafter.

1.2 Schematic diagram of the exemplary cartridge

Before explaining the structure and arrangement of the components of an exemplary fluid cartridge in detail, it is helpful to describe the layout of the exemplary cartridge at a high level with reference to the schematic shown in figure 1. It is convenient to consider the overall layout of the cartridge in terms of the flow of liquids, including the liquid sample, through the cartridge. Unless otherwise specified hereafter, the passage of liquids including the liquid sample and the liquid buffers is referred to as the 'fluid pathway' which has an upstream end and a downstream end. Unless otherwise specified hereafter, 'downstream' generally refers to the direction of flow of the liquids and 'upstream' refers to the direction opposite the direction of flow. The fluid pathway in the exemplary cartridge may have different branches (and thus form different fluid pathways), but all pathways have a recognisable direction of flow which permit a skilled person to identify the upstream and downstream directions. However, there is an exception to this general definition, which is when the liquid sample is pumped between the mixing chamber 10 and the bellows 20. In this case, fluid is intermittently pumped back upstream in the opposite direction to its general direction of fluid flow, which is downstream. This mixing serves to mix the lysis and sample and to rehydrate the internal control.

The liquid sample is introduced into the cartridge at a sample mixing chamber 10 through an entry port. A particular arrangement of a preferred entry port may itself form an isolated inventive aspect of the cartridge, as described further in section 3, below. A sample indicator 12 is fluidly coupled to the sample mixing chamber 10 such that a sample introduced into the sample mixing chamber 10 is visible in the sample indicator 12. Also connected to the sample mixing chamber 10 is a blister 14 containing a lysis buffer. The lysis buffer comprises guanidine thiocyanate. Once the sample has been introduced into the sample mixing chamber 10, and a test is started, the lysis blister 14 is collapsed so as to expel the lysis buffer into the sample mixing chamber 10 where it mixes with the liquid sample introduced therein.

Downstream of the sample mixing chamber 10, along a main channel 16, is a coarse filter 18. The coarse filter 18 filters out any large debris in the liquid sample, such as skin or bodily hair, as the liquid sample passes through main channel 16.

Downstream of the coarse filter 18, along the main channel 16, is a bellows 20 having an upstream bellows valve 22a and a downstream bellows valve 22b. As described in more detail below, the bellows 20, together with its upstream and downstream valves 22a-b, is capable of pumping the liquid sample from the upstream end of the fluid pathway (i.e. from the sample mixing chamber 10) to the downstream end. In summary, this is achieved by virtue of flexible membranes within the bellows 20 and the upstream and downstream bellows valves 22a-b which actuate to create local pressure differentials to, on the one hand, draw in the liquid sample from the sample mixing chamber 10 into the bellows 20 and, on the other hand, from the bellows 20 further downstream through the main channel 16. This is achieved by carefully choreographed pneumatic actuation of the flexible membranes in the valves. Particular arrangements of a preferred valve may themselves form isolated inventive aspects of the cartridge, as described further in section 3, below.

Downstream of the bellows along the main channel 16 is a capture column 24. The purpose of the capture column 24 is to separate nucleic acids from cell debris and other cellular components. The capture column comprises a capture filter and a depth filter both made of glass fibres. A particular arrangement of a preferred capture column may itself form an isolated inventive aspect of the cartridge, as described further in section 3, below.

Two branch channels 26, 28 join the main channel 16 between the downstream bellows valve 22b and the capture column 24. The purpose of the branch channels is to introduce liquid buffers necessary for performing the desired test. For example, with the test conducted by the exemplary cartridge, it is necessary to introduce an elution buffer and a wash buffer into the main channel once the sample has passed through. The wash buffer is contained in a wash buffer blister 30 and the elution buffer is contained in an elution buffer blister 32. The introduction of the wash buffer and elution buffer into the main channel 16 is controlled by wash buffer valve 34 and elution buffer valve 36, respectively. At the appropriate point in the test, the wash and elution buffer blisters 30, 32 are collapsed so as to expel the wash and elution buffers into the branch channels 26, 28 and thence into the main channel 16 through the wash and elution buffer valves 34, 36.

Downstream of the capture column 24, along a waste branch 16a of the main channel 16, is a waste chamber 38. A particular arrangement of a preferred waste chamber may itself form an isolated inventive aspect of the cartridge, as described further in section 3, below. The purpose of the waste chamber 38 is to collect the cell debris and cellular components other than nucleic acids and contain them, thereby preventing them from entering the test channel 54a or the control channel 54b. The waste chamber 38 is vented to atmosphere through a waste vent 40, and an aerosol impactor 42 is provided between the waste chamber 38 and the waste vent 40 to prevent particulate matter from escaping from the waste chamber 38 into the atmosphere. A waste chamber valve 44 in the main channel waste branch 16a of the main channel 16 permits and prevents fluids passing into the waste chamber 38 at appropriate points during the test.

Downstream of the capture column 24, along an elution branch 16b of the main channel 16, is an elution chamber 46. The purpose of the elution chamber 46 is to allow the sample preparation to settle and for bubbles to disperse before the sample enters the amplification chambers. An elution chamber valve 48 in the elution branch 16b of the main channel 16 permits and prevents fluids passing into the elution chamber 46 at appropriate points during the test. Downstream of the elution chamber 46 is a convoluted mixing channel 52. Here the prepared sample is mixed prior to passing through the isolation valve 50.

In the present application, the components upstream of the isolation valve 50 are referred to as being comprised in the 'front end' of the cartridge, whilst the components downstream of the isolation valve 50 are referred to as being comprised in the 'back end' of the cartridge. Broadly speaking, the liquid sample is prepared for analysing in the front end of the cartridge, and the analysis is carried out on the sample in the back end of the cartridge.

The isolation valve 50 is open to permit the prepared liquid sample to pass from the front end to the back end of the cartridge. At an appropriate point in the test, after the liquid sample has been prepared and is within the back end of the cartridge for analysis, the isolation valve 50 is closed to prevent any of the sample from re-entering the front end. Once the isolation valve 50 is closed, it cannot be opened again. The isolation valve 50 also acts as a safeguard in case of a power failure, wherein the reader closes the isolation valve 50 to prevent leakage.

Downstream of the isolation valve 50, the fluid pathway splits into an amplification test channel 54a and an amplification control channel 54b. Each of the amplification channels 54a-b comprises an amplification chamber 56a-b having an amplification chamber inlet valve 58a-b and an amplification chamber outlet valve 60a-b. Any nucleic acid amplification method may be performed in the nucleic acid amplification chamber. If PCR is used, the nucleic acid amplification chambers contain a thermostable DNA polymerase, dNTPs, a pair of primers which are capable of hybridising to the nucleic acid to be amplified. Optionally, the nucleic acid amplification chambers may additionally contain buffer salts, MgCI ₂ , passivation agents, uracil N-glycosylase and dUTP. An example of a thermostable DNA polymerase that may be used is Taq polymerase from Thermus aquaticus.

Each of the nucleic acid amplification chambers in the exemplary cartridge comprises reagent containment features in the form of first and second shallow wells formed in the fluidic layer. The reagents to be used in the cartridge are spotted in the wells. In the exemplary cartridge, the test- specific reagents and the generic reagents are isolated from each other by spotting each in a different well. Hence, the test-specific reagents are spotted in a first well in the chamber and the generic reagents are spotted in a second well in the chamber. By spotting the reagents separately, it is easier to swap the test-specific reagents during manufacture for a different set of test-specific reagents, so as to perform a different test, whilst keeping the generic reagents as they are. In the exemplary cartridge, the ratio of nucleic acid amplification chambers to detection chambers is 1 :2. The prepared sample enters the back end of the cartridge at the isolation valve 50 and is split into two nucleic acid amplification chambers. After processing, the each of the two processed measures of sample from the nucleic acid amplification chamber is split into two detection chambers. Therefore, for each sample introduced into the exemplary cartridge, four detection chambers may be filled from two nucleic acid amplification chambers, thus facilitating duplex amplification and 4-plex detection.

However, it will be appreciated that one or three or more nucleic acid amplification chambers may be provided to provide any level of multiplexing desired, and that the number of the detection chambers provided may be adjusted accordingly to maintain a 1 :2 ratio of nucleic acid amplification chambers to detection chambers.

The ratio 1 :2 is preferred for the exemplary cartridge because such a ratio allows twice the number of target nucleic acids to be assayed compared to the number of different labels required for detection in the detection chambers. However, it will be appreciated that the ratio may be changed depending on the number of labels and PCR targets for the liquid sample. For instance, the ratio may be 1 : 1 , 1 :3 or V.n such that there are n detection chambers branching from the main channel of each fluid pathway when there are n times as many multiplexed PCR targets for the number of labels.

PCR primers specific for Chlamydia trachomatis are dried down in the amplification chamber in the amplification test channel together with the other reagents required for nucleic acid amplification. PCR primers specific for a positive control nucleic acid are dried down in the amplification chamber in the amplification control channel together with the other reagents required for nucleic acid amplification. A positive control nucleic acid is also provided in the amplification chamber in the amplification control channel, taken from Pectobacterium atrosepticum. The dried down reagents are reconstituted when the liquid sample reaches them.

Downstream of the amplification chamber outlet valves 60a-b each of the amplification channels 54a-b splits into two further detection channels, leading to two detection chambers for each amplification chamber, giving a total of four detection chambers 62a-d in total. The reagents for nucleic acid detection, including the target probe, are dried down in the detection chambers 62a-d downstream of the test amplification chamber 56a or 56b. The reagents for nucleic acid detection including the control probe are dried down in the detection chambers downstream of the control amplification chamber 56a or 56b (whichever is not the test chamber mentioned above). Each detection chamber 62a-d is provided with its own gas spring 64a-d which forms a dead end at the downstream end of the fluid pathway.

Reagents for nucleic acid detection are provided in detection chambers. The reagents for nucleic acid detection include probes having a ferrocene label. These probes are capable of hybridising to the amplified nucleic acids. Following hybridisation of the probes to the amplified nucleic acids, the probes are hydrolysed by a double strand specific nuclease which causes the label to be freed from the rest of the probe. As explained above, freeing of the label from the rest of the probe causes a detectable change in the signal from the label. The control probe is provided in separate detection chambers to the target probe and detection of the target nucleic acid and the control nucleic acid take place in different detection chambers, such that the signals are distinguishable from one another.

Downstream of the amplification outlet valves 60a-b, but upstream of the forks creating the four detection channels, two bypass channels 66a-b respectively join the two amplification channels 54a-b. The purpose of the bypass channels 66a-b is to remove excess liquid sample within the amplification channels 54a-b before the liquid sample enters the detection chambers 62a-d. The bypass channels 66a-b connect to a bypass valve 68, which is also fluidly coupled to the elution chamber branch 16b of the main channel 16, downstream of the isolation valve 50, before the channel splits into amplification channels 54a and 54b.

A particular arrangement of a preferred chamber in the cartridge, such as the first and second amplification chambers or the first to fourth detection chambers, may itself form an isolated inventive aspect of the cartridge, as described further in section 3, below.

It will be appreciated that the number of amplification chambers, and the number of detection chambers in the exemplary cartridge may vary depending on the preferred implementation. Moreover, other configurations of channels, chambers, valves and so on are possible without departing from the scope of the invention, as defined by the claims.

The physical structure and operation of the various components of the exemplary cartridge introduced above will now be explained with reference to figures 2 to 10.

1.3 Physical structure of an exemplary cartridge

1.3.1 Overview and external features of the exemplary cartridge An exemplary cartridge is shown in figure 2. As described above, the reader interacts with the cartridge through a plurality of interfaces. The interfaces shown in the exemplary cartridge 100 are: a pneumatic interface 101 ; an electrical interface 102; a bypass valve interface 103; and an isolation valve interface 104. Each of these interfaces is described in more detail below. It will be appreciated that more or fewer interfaces could be provided, depending on the preferred implementation.

Also provided in the cartridge, but not shown, is a thermal interface. The thermal interface allows the temperature of the amplification chambers to be regulated to allow nucleic acid amplification to take place.

The exemplary cartridge 100 shown in figure 2 comprises an insertion end 105 for insertion into the reader, and a non-insertion end 106. Proximate the non-insertion end 106 is a sample inlet 107 for introducing a sample into the sample mixing chamber 10. In the exemplary cartridge, the sample will usually include cells, and the target nucleic acid (if present) can be extracted from these cells, but other fluid samples such as swab eluate, urine, semen, blood, saliva, stool sweat and tears could be used in other implementations. The sample may be introduced into the sample mixing chamber 10 through the sample inlet 107 using a pipette, for example.

The exemplary cartridge 100 and reader are configured such that when the cartridge is inserted into the reader, all of the aforementioned interfaces are actuatable by the reader. On the other hand, the sample inlet 107 remains external to the reader such that a sample may be introduced into the sample mixing chamber 10 whilst the cartridge is inserted into the reader.

The exemplary cartridge 100 shown in figure 2 further comprises a sample indicator window 109, through which the sample indicator 12 is visible to determine whether a sample has been introduced into the sample mixing chamber 10.

All of the pneumatic, mechanical and electrical interfaces in the exemplary cartridge 100 are located on the same face of the cartridge, in this case the top face 1 10. The thermal interface (not shown) is provided on the bottom face of the cartridge. This simplifies the design of the reader, which may this provide the associated pneumatic, mechanical and electrical parts which interact with those interfaces in the same region of the reader, thereby making best use of space. It also enables the thermal part of the reader to be provided away from the pneumatic, mechanical and electrical parts.

Internal components of cartridge The exemplary cartridge 100 shown in figure 2 is formed from various components which shall now be described. Figure 3 shows an exploded view of the exemplary cartridge 100 of figure 2. The cartridge 100 comprises, from top to bottom, a housing 11 1 , a blister sub-assembly 1 12, a pneumatic foil 1 13, a pneumatic layer 1 14, a fluid layer 1 15 and a fluidic foil 1 16. Also shown in figure 3 is an electrode layer 1 17, two filters 1 18 and a plurality of absorbent pads 1 19, which will be described in more detail below.

The housing 1 11 is manufactured from acrylonitrile butadiene styrene. The pneumatic and fluidic foils 113, 116 are manufactured from a polyethylene terephthalate / polypropylene composite. The pneumatic and fluidic layers 1 14, 1 15 are manufacture from polypropylene.

With the exception of the housing 1 11 , filters 1 18 and pads 1 19, each of the components mentioned in the previous paragraph is adhered to its adjacent component or components. Hence, the blister sub-assembly 1 12 is adhered to the pneumatic foil 1 13, which is adhered to the pneumatic layer 114, which is adhered to the fluidic layer 115, which is adhered to the fluidic foil 1 16. The electrode layer 117 is adhered to fluidic layer 115 also.

The adhesion of the layers to each other provides a series of fluid-tight channels in the cartridge, together with associated chambers, valves, pumps, bellows and other components. The channels passing a liquid sample therethrough are liquid-tight and the channels passing a gas therethrough are gas-tight. Optionally, all components are both liquid tight and gas-tight. For example, recesses and openings formed in one or both sides of the pneumatic and fluidic layers create, when sandwiched together and adhered to the pneumatic and fluidic foils, respectively, the shapes necessary to provide the aforesaid channels, chambers, valves, pumps, bellows and other components.

Each of the components referred to above in figure 3 will now be described in more detail. 1.3.3 Housing 11 1

Figure 4 shows housing 1 11 in more detail. As shown, housing 1 11 comprises a generally rectangular upper surface 120 and walls 121 depending therefrom on all four sides (two of which are visible in figure 4). A principal purpose of the housing 1 1 1 is to protect certain components of the cartridge, most notably the blister sub-assembly 1 12 and the isolation valve interface 104. It will therefore be noted that the housing 1 11 is shorter than the pneumatic and fluidic layers 114, 1 15 such that it overlies only a portion of those layers when the cartridge 100 is assembled. In the exemplary cartridge 100, the pneumatic interface 101 , electronic interface 102, and bypass valve interface 103 are not covered by the housing 1 11 to provide ease of access by the reader.

The upper surface 120 of the housing 1 11 has three apertures 122a-c therein, each having walls depending from the peripheries of the apertures to form, when the cartridge is assembled, three recesses. The purpose of the recesses is to house the blisters of the blister sub-assembly 1 12 such that the blisters may be accessed and pressed by the reader, but are otherwise protected from accidental impact. Naturally, since the exemplary cartridge comprises three blisters, the housing 11 1 comprises three corresponding apertures 122a-c forming three corresponding recesses. It will be appreciated that more or fewer blisters, apertures and recesses may be provided, depending on the preferred implementation. Alternatively, the housing 11 1 could comprise a single aperture forming a single recess housing all available blisters.

The side walls 121 of the housing 1 1 1 which run along the length of the housing 1 1 1 between the insertion end 105 and the non-insertion end 106 of the cartridge 100 comprise flanges 123 along at least a portion of their lower edges. The purpose of the flanges 123 is two-fold. Firstly, they comprise one or more windows 124a-b for receiving a corresponding number of tabs formed in the pneumatic layer 1 14 to hold the cartridge 100 together. Secondly, the flanges 123 are dimensioned so as to protrude beyond the lower surface of the fluidic foil 1 16 when the cartridge is assembled, such that the fluidic foil 1 16 is suspended above a flat surface on which the cartridge 100 is placed. This prevents accidental damage to the fluidic foil 1 16 which could otherwise result.

Although in the exemplary cartridge depicted in figure 4 flanges 123 are provided along substantially the length of two opposing sides of the cartridge, it will be appreciated that flanges may be provided along three or four edges of the cartridge and still suspend the foil above a flat surface on which the cartridge is placed. Similarly, although the cartridge depicted in figure 4 shows flanges 123 extending along substantially the entire length of the edge, a flange which extends only partially along an edge may be provided, or multiple flanges may be provided along each edge.

The housing 11 1 further comprises, at the non-insertion end 106, a grip 125 to facilitate insertion of the cartridge into and removal of the cartridge 100 from the reader by hand. The grip 125 comprises a series of ridges and grooves formed in the housing 1 11 , but alternative structures to increase friction, such as knurls, are also possible.

The housing 11 1 further comprises a sample inlet aperture 126 through which a sample may be introduced into the sample mixing chamber 10 of the cartridge 100 using a pipette, for example. Surrounding the inlet aperture 126 for a given diameter is a basin 127 recessed into the upper surface 120 of the housing 1 1 1 to accommodate a certain amount of spillage of the liquid sample. Whilst the basin 127 of the exemplary embodiment is substantially flat, it may be sloped toward the inlet aperture 126, such that any spillage drains through the inlet aperture 126.

The exemplary housing 1 1 1 further comprises a plurality of cut-outs: a first cut-out 128 forming the sample window 109, and a second cut-out 129 to provide access to the isolation valve interface 104. As with the recesses which protect the blisters, by providing access to the isolation valve interface 104 only through a cut-out 129 in the housing 11 1 , the isolation valve interface 104 is protected to some extent from accidental impact, which could actuate the isolation valve and render the cartridge inoperable.

1.3.4 Blister sub-assembly 1 12

Figure 5 shows the blister sub-assembly 112 in more detail. The blister sub-assembly 1 12 may be manufactured separately, during which the blisters are pre-filled with the liquid reagents necessary for conducting the preferred test, and subsequently adhered to the pneumatic foil 1 13.

Blister sub-assemblies (or 'blister packs') are familiar to a skilled person. A blister is a collapsible chamber for containing a liquid, which may be expelled from the blister by pressing on the blister and thereby collapsing it. In typical blister packs, the chamber of a blister is sealed by a foil or other frangible layer which ruptures once the pressure inside the chamber reaches a particular magnitude as the blister is collapsed.

In the exemplary cartridge, the blister sub-assembly 1 12 comprises three blisters 130a-c. These contain, respectively, the lysis buffer which comprises reagents capable of performing cell lysis, the wash buffer and the elution buffer.

The exemplary blister sub-assembly 1 12 comprises a substrate 131 onto which the aforementioned blisters 130a-c are formed by a deformable polymeric layer which is shaped to provide the chambers. Three apertures 132a-c, corresponding to the three blisters 130a-c, pass through the substrate 132. Each of the apertures is covered by the deformable polymeric layer forming the chamber, which thereby connects the aperture to the chamber but for a seal 133a-c between the respective apertures 132a-c and chambers. Upon application of a suitable pressure on the blister 130a-c, the seal 133a-c breaks, thereby causing the liquid contents of the blister to be ejected from the blister and to flow through the aperture 132a-c in the substrate 131 out of the blister sub-assembly. As shown, the seals 133a-c at least partially surround the periphery of the chambers, where they meet the substrate 131. At the point in each seal 133a-c which is designed to break (thereby forming the liquid passageway between the aperture 132a-c and chamber), the seal 133a-c may be weaker than the rest of the periphery. This ensures that the correct part of the seal 133a-c breaks when the suitable pressure is applied.

The blisters may be collapsed by the reader when the cartridge is inserted therein. One or more mechanical actuators (such as a foot) may be applied by the reader into the recess so as to collapse the blister.

The blister sub-assembly 1 12 further comprises two reference holes 134a-b configured to permit an assembly fixture to provide a reference to facilitate positioning of the assembly during manufacture.

1.3.5 Pneumatic layer 114

Figures 6A and 6B show the pneumatic layer 114 in more detail. Figure 6A is a top view of the pneumatic layer and Figure 6B is a bottom view. The pneumatic layer 114 is comprised of a rigid plastic layer 135 which, in certain places, is overmoulded with a plurality of flexible membranes to form certain components when the cartridge is assembled. The flexible membranes are manufactured from a thermoplastic elastomer.

The rigid plastic layer 135 has a plurality of differently-shaped recesses therein and apertures therethrough. In combination with the fluidic layer 1 15, certain recesses within, and/or apertures through, the rigid plastic layer 135 form a number of components, including: the sample mixing chamber 136; the waste chamber 137; the capture column 138; the elution chamber 139; the first and second amplification chambers 140a-b; and the first to fourth detection chambers 141 a-d. An aperture 142 is also provided to give access to the electrode layer 117.

In combination with the overmoulded flexible membranes and the pneumatic foil 113, certain other apertures through the rigid plastic layer form a number of other components, including: the upstream bellows valve 142; the bellows 143; a pneumatic interface 144; the downstream bellows valve 145; the wash buffer inlet valve 146; the wash buffer air inlet valve 146a; the elution buffer inlet valve 147; the elution buffer air inlet valve 147a; the waste chamber valve 148; the elution chamber valve 149; the isolation valve 150; the first and second amplification chamber inlet valves 151 a-b; and first and second amplification chamber outlet valves 152a-b. A further aperture, in combination with an overmoulded flexible membrane (but not the pneumatic foil) forms a bypass valve 153.

With the exception of the isolation valve 150 and the bypass valve 153, the valves formed in the pneumatic layer are pneumatically-operable valves. That is, each valve is operable to open and close a fluidic channel in which the valve is located, and this valve is actuated by applying a particular pressure to a pneumatic control line coupled to the valve. The pneumatic control lines are coupled to the pneumatic interface 144, to which the reader has access when the cartridge 100 is inserted therein. Hence, to actuate a given pneumatic valve, the reader merely applies an appropriate pressure to the pneumatic control line associated with that valve to open or close the valve.

The isolation valve 150 and the bypass valve 153 are also actuated by the reader, but mechanically. Again, each valve is operable to open and close a fluidic channel in which the valve is located, but the valve is actuated by applying one or more mechanical actuators (such as a foot) to the valve.

The pneumatic layer further comprises two reference holes 154a-b configured to permit an assembly fixture to provide a reference to facilitate positioning of the layer during manufacture. When the cartridge is assembled, the reference holes 154a-b in the pneumatic layer align with the reference holes 134a-b in the blister sub-assembly.

The pneumatic layer further comprises apertures 155a-c which, when the cartridge is assembled, line up with apertures 132a-c passing through the substrate 131 of the blister sub-assembly (through the pneumatic foil, as described below).

1.3.6 Pneumatic foil 113

Figure 7 shows the pneumatic foil 1 13 in more detail. As explained above, the pneumatic foil 113 is adhered to the upper surface of the pneumatic layer 1 14, thereby fluidly sealing channels, chambers, valves, pumps, bellows and other components formed therein. Thus, for the most part, the pneumatic foil 113 is a generally rectangular and planar foil sheet so as to provide an effective seal. Beneficially, the pneumatic foil 1 13 is inert such that is does not react with the reagents which move through the pneumatic layer 114.

However, the pneumatic foil 1 13 does not overlie the entire pneumatic layer 1 14. In particular, the pneumatic foil 1 13 does not overlie the sample mixing chamber 136 or the waste chamber 137 at the non-insertion end 106 of the cartridge 100, or the bypass valve 153 at the insertion end 105. Moreover, the pneumatic foil 1 13 comprises cut-outs 156, 157, such that it does not overlie the isolation valve 150 or the pneumatic interface 144, respectively.

The pneumatic foil 1 13 further comprises three apertures 158a-c which, when the cartridge 100 is assembled, line up with apertures 132a-c passing through the substrate 131 of the blister subassembly and 155a-c passing through the pneumatic layer 1 14. The apertures 158a-c permit the liquid reagents within the blisters to pass to the pneumatic layer 114, and thence to the fluidic layer 1 15 through apertures 155a-c.

The pneumatic foil 1 13 comprises two reference holes 159a-b configured to permit an assembly fixture to provide a reference to facilitate positioning of the layer during manufacture. When the cartridge is assembled, the reference holes 159a-b in the pneumatic foil align with the reference holes in the other layers.

The pneumatic foil is a composite foil manufactured from a layer of polyethylene terephthalate, to provide strength, with a layer of polypropylene on top to provide an inert material for contacting the liquid sample and buffers, and also to enable the foil to be heat sealed to the pneumatic layer (also manufactured from polypropylene.

1.3.7 Fluidic layer 115

Figures 8A and 8B show the fluidic layer 1 15 in more detail. Figure 8A is a top view of the pneumatic layer and Figure 8B is a bottom view. The fluidic layer 1 15 is comprised of a rigid plastic layer 160. As explained previously, the top side of the fluidic layer 1 15 (not shown) is adhered to the bottom side of the pneumatic layer 1 13 (see figure 5B) such that the various channels, chambers, valves, pumps, bellows and other components formed by a combination of the pneumatic and fluidic layers are aligned.

As with the rigid plastic layer 135 of the pneumatic layer 113, the rigid plastic layer 160 of the fluidic layer 1 15 has a plurality of differently-shaped recesses therein and apertures therethrough. In combination with the pneumatic layer 113 and the fluidic foil 116, certain recesses within, and/or apertures through, the rigid plastic layer 160 forms certain components, including: the sample inlet chamber 136; the capture column 138; the elution chamber 139; the first and second amplification chambers 140a-b; and the first to fourth detection chambers 141 a-d. the upstream bellows valve 142; the bellows 143; the pneumatic interface 144; the downstream bellows valve 145; the wash buffer inlet valve 146; the wash buffer air inlet valve 146a; the elution buffer inlet valve 147; the elution buffer air inlet valve 147a; the waste chamber valve 148; the elution chamber valve 149; the isolation valve 150; the first and second amplification chamber inlet valves 151 a-b; and first and second amplification chamber outlet valves 152a-b. An aperture 161 is also provided to give access to the electrode layer 117.

Moreover, in combination with the fluidic foil 1 16 (but not the pneumatic layer 1 14), recesses in the fluidic layer 1 15 also provides the coarse filter 162, the convoluted mixing channel 163, and a plurality of channels which, when the cartridge is assembled, connect the aforementioned components together to enable passage of the liquid sample and liquid reagents through the cartridge, and facilitate pneumatic actuation of the valves, pumps, bellows and other components.

The fluidic layer comprises two reference holes 164a-b configured to permit an assembly fixture to provide a reference to facilitate positioning of the layer during manufacture. When the cartridge is assembled, the reference holes 164a-b in the fluidic layer align with the reference holes in the other layers.

As mentioned above, channels are formed between the pneumatic interface and the various valve and bellows described above. In the exemplary cartridge, the pneumatic interface comprises 11 ports which are connected to the various components as follows.

Port 1 : bellows

Port 2: upstream bellows valve

first and second amplification chamber inlet valves

first and second amplification chamber outlet valves

Port 3: downstream bellows valve

Port 4: wash buffer inlet valve

Port 5: wash buffer air inlet

Port 6: wash buffer air inlet valve

elution buffer air inlet valve

Port 7: elution buffer air inlet

Port 8: elution buffer inlet valve

Port 9: reference pressure line

Port 10 elution chamber valve

Port 11 waste chamber valve

It will be understood that whilst various inventive aspects of the exemplary cartridge may be implemented using specific ones of the connections listed above (in particular, the first and second amplification chamber inlet and outlet valves being connected to a single port; and the wash and elution buffer air inlets being connected to a single port); the precise configuration listed above is not essential.

1.3.8 Fluidic Foil

Figure 9 shows the fluidic foil 1 16 in more detail. As explained above, the fluidic foil 1 16 is adhered to the lower surface of the fluidic layer 1 15, thereby fluidly sealing channels, chambers, valves, pumps, bellows and other components formed therein. Thus, for the most part, the fluidic foil 1 16 is a generally rectangular and planar foil sheet so as to provide an effective seal. Beneficially, the foil 1 16 is inert such that is does not react with the reagents which move in the pneumatic layer.

However, the fluidic foil 1 16 does not overlie the entire fluidic layer 115. In particular, the fluidic foil 116 does not overlie the detection chambers 141 a-d at the insertion end 105.

The fluidic foil 116 comprises two reference holes 165a-b configured to permit an assembly fixture to provide a reference to facilitate positioning of the layer during manufacture. When the cartridge is assembled, the reference holes 165a-b in the fluidic foil align with the reference holes in the other layers.

The fluidic foil is a composite foil manufactured from a layer of polyethylene terephthalate, to provide strength, with a layer of polypropylene on top to provide an inert material for contacting the liquid sample and buffers, and also to enable the foil to be heat sealed to the fluidic layer (also manufactured from polypropylene.

1.3.9 Electrode layer 117

Finally, figure 10 shows the electrode layer 1 17 in more detail. As explained above, the electrode layer 117 is adhered to the fluidic layer 115. The electrode layer 117 comprises four sets of detection electrodes 166a-d. Each set of detection electrodes 166a-d comprises first to third electrical contacts 168a-d which couple with corresponding electrical contacts in the reader when the cartridge is inserted therein. Preferably, the electrical contacts are made of silver to optimise the electrical connection. Preferably electrodes which are silver plated with silver chloride are used to ensure a the optimal galvanic behaviour. Each set of detection electrodes 166a-d comprises a working electrode 169a-d; a counter electrode 170a-d and a reference electrode 171 a-d. Each of the electrodes is coupled to a respective electrical contact. Each set of detection electrodes 166a-d also comprises a dielectric 172a-d covering the interface between the electrodes and the respective electrical contacts.

A skilled person understands that electrochemical signalling may be used to indicate the presence of genetic or immuno targets. In the exemplary cartridge this process is performed in the first to fourth detections chambers 141 a-d which are optimised to provide the electrochemical test interface.

The electrodes 166a-d are arranged such that a liquid sample within the first to fourth detection chambers 141 a-d comes into contact with the first to fourth sets of electrodes 166a-d. In the detection chambers, some compounds in the fluid sample (referred to as the 'electrolyte') have a natural tendency to migrate to electrodes and swap electrons. This galvanic effect is how batteries work.

All combinations of soluble compounds have some electrochemical activity, and the rate at which this activity occurs (i.e. the amount of charge exchanged) is determined by exactly what those compounds are. Hence, it is possible to measure the presence of different analytes in the liquid sample, by searching for characteristic features of their redox electrochemistry.

In the exemplary cartridge, the current required to maintain a given redox state in the detection chambers 141 a-d is monitored at different redox states. Current is supplied through the electrolyte from the working electrodes 169a-d to counter electrodes 170a-d.

The reference electrodes 171 a-d also contact the electrolyte. Voltages are declared with respect to this reference electrode because its voltage is largely independent of the redox conditions and this therefore means that it is only the redox state of the chemistry at the control electrode that is being measured.

A voltage sweep is applied between the working electrodes 169a-d and counter electrodes 170a-d by the reader, which generates the characteristic range of redox conditions. The current passing between the working electrodes 169a-d and the counter electrodes 170a-d is then measured to obtain the test results. The voltage sweep is a slowly incrementing set of voltages applied between the electrodes. Preferably the sweep is from about -0.7 volts to about +1 volts relative to the reference electrode. The voltage is applied in consecutive incrementing pulses having a pulse modulation amplitude of between 30 and 80 millivolts (preferably between 40 and 60 millivolts; more preferably 50 millivolts). Preferably the step increment from one pulse to the next is between 1 and 5 millivolts (preferably between 2 and 4 millivolts; more preferably 3 millivolts). By applying these voltages across the electrodes, current measurements in the scale of 100s of nano amps may be obtained.

The particular arrangement of detection electrodes illustrated in figure 10 may itself form an isolated inventive aspect of the cartridge. Conventionally, the counter electrode in a potentiostat is larger than the working electrode to provide an ample supply of surplus electrons. However, it has been found that reversing this convention surprisingly offers better results for the exemplary cartridge. For the electrochemistry performed on the liquid sample described above in the exemplary cartridge, it is found that having a working electrode which is larger than the counter electrode provides larger signals and improved results by way of increased sensitivity. In other words, having a current flow from a relatively large working electrode to a relatively small counter electrode offers improvements over the conventional arrangement.

Preferably each working electrodes 169a-d is formed in a U-shape and each counter electrode 170a-d is formed in a straight elongate shape between the two prongs of the respective U-shaped working electrode.

The method operation of the exemplary cartridge introduced above will now be briefly explained. 1.4 Method of operation of the exemplary cartridge 1.4.1 The front end

As described above, a fluid sample (such as a urine sample) is introduced into the sample mixing chamber 10 using a pipette. A portion of the sample passes to the sample indicator 12 to show that a sample is present in the sample mixing chamber.

Once the cartridge 100 with a sample in the mixing chamber 10 is inserted into a reader, and the reader is activated, the test may commence. Firstly, the reader will apply a mechanical actuator (such as a foot) to collapse the lysis buffer blister 14. In doing so, the lysis buffer will be expelled into the sample mixing chamber 10 where it will mix with the sample.

The bellows 20 and its valves 22a-b then moves the liquid sample and lysis buffer back and forth into the sample mixing chamber 10 so as to mix the lysis and sample and to rehydrate the internal control. Following the mixing step, incubation of the sample and lysis buffer occurs to allow cell lysis to take place.

The bellows 20 and its valves 22a-b will then commence operation to pump the sample from the sample mixing chamber 10, into the main channel 16, through the coarse filter 18 and toward the capture column 24. Within the capture column 24 nucleic acids are specifically bound to a filter in the capture column on the basis of their size and charge. The unwanted liquid sample passes through to the waste chamber 38.

Once the unwanted liquid sample has passed to the waste chamber 38, leaving the nucleic acids bound to the capture column 24, the reader applies a mechanical actuator (such as a foot) to collapse the wash buffer blister 30. In doing so, the wash buffer will be expelled into the first branch channel 26, and thence into the main channel 16. Again, the bellows 20 and its valves 22a-b will commence operation to pump the wash buffer through the main channel 16 and through the capture column 24 to wash any remaining unwanted cell debris and other cellular components out of the capture column with the wash buffer through to the waste chamber 38, or else the wash buffer will be flushed into the waste chambers using air from the wash and/or elution buffer air inlets.

Once the wash sample has passed to the waste chamber 38, leaving only the bound and purified nucleic acids in the capture column 24, the reader applies a mechanical actuator (such as a foot) to collapse the elution buffer blister 32. In doing so, the elution buffer will be expelled into the second branch channel 28, and thence into the main channel 16. Again, the bellows 20 and its valves 22a-b will commence operation to pump the elution buffer through the main channel 16 and through the capture column 24 to elute the nucleic acids from the capture column, or else the elution buffer will be flushed into the capture column using air from the wash and/or elution buffer air inlets. The prepared liquid sample then passes through to the elution chamber 46; again, either by being pumped or flushed as described above.

The sample settles in the elution chamber 46 allowing bubbles to disperse before entering the amplification chambers.

1.4.2 The back end

The bellows 20 and its valves 22a-b will then commence operation to pump the liquid sample from the elution chamber 46, through the isolation valve 59, through the mixing channel 52 and into the amplification chambers 56a-b, or else the sample will be flushed into the amplification chambers using air from the wash and/or elution buffer air inlets. In the nucleic acid amplification chambers 56a-d the nucleic acid of interest, if present, is amplified such that it is present at a detectable level. The control nucleic acid is also amplified such that it is present at a detectable level. As mentioned above, any nucleic acid amplification method may be used. Where PCR is used, primers specifically hybridise to the nucleic acid of interest and are extended by a thermostable polymerase such as Taq polymerase via the addition of dNTPs to the 3' end of each of the primers. Any excess liquid sample may be removed from the fluid pathway through the bypass channels 68.

The bellows 20 and its valves 22a-b will then commence operation to pump the liquid sample from the amplification chambers 56a-b and into the detection chambers 62a-d, or else the sample will be flushed into the detection chambers using air from the wash and/or elution buffer air inlets. In the detection chambers, the target probe specifically hybridises to the target amplified nucleic acid of interest and the control probe specifically hybridises to the amplified control nucleic acid. The nuclease hydrolyses the target and control probes following hybridisation of the probes to the amplified nucleic acid. The hydrolysis of the target and control probes frees the labels from the probes causing a detectable change in the signal from the labels to occur.

Once the liquid sample occupies the detection chambers, the reader applies a mechanical actuator to the isolation valve 50 to close the valve and isolate the liquid sample in the back end of the device.

The electrodes provide a potential difference across the at least one detection chamber. Depending on the state of the label (i.e. whether it is attached to the full length probe or the probe has been hydrolysed and it is free or attached to a single nucleotide or short part of the probe), the current that is able to flow through the detection chamber will differ. The electrodes therefore allow detection by the reader of the change in the signal from the label which results from hydrolysis of hybridised probe.

The present invention will now be described with reference to figures 16 to 27. 2 Fluidic control devices, and their manufacture

The following section describes the present invention in more detail with reference to figures 16 to 27. The invention may be implemented in the exemplary fluidic cartridge described above, specifically to form the valves, bellows and other structures in the pneumatic layer 1 14. However, it will be appreciated that the invention is applicable in other devices, depending on the preferred implementation. 2.1 The structure of a fluidic control device

Figures 16a-b illustrate a fluid control device A100 according to an embodiment of the present invention. The fluidic control device A100 could be a valve such as: the upstream and downstream bellows valves 22a, 22b; the wash and elution buffer valves 34, 36; the waste and elution chambers valves 44, 48; the isolation valve 50; the amplification chamber inlet and outlet valves 58a-b, 60a-b; and the bypass valve. The fluidic control device A100 could also be the bellows 20.

Irrespective of the particular implementation, the fluidic control device of the present invention includes the following principal features. As shown in Figure 16a-b, embodiments of a fluidic control device A100 include a first polymer layer A101 , a second polymer layer A102 and a third polymer layer A103. The first polymer layer A101 has a top surface A101a, a bottom surface A101 b and a void A101c extending through the first polymer layer A101. The void A101c has an opening in at least the bottom surface of the first polymer layer. Figure 16a illustrates an embodiment in which the void has an opening in the bottom surface and the top surface of the first polymer layer, whereas figure 16b illustrates an alternative embodiment, in which the void has an opening only in the bottom surface of the first polymer layer.

In the embodiments disclosed herein, the first and third polymer layers A101 and A103 form rigid plastic layers, such as the pneumatic layer 114 and the fluidic layer 115 described above. The second polymer layer A102 forms a flexible membrane A102a across the void A101 c in the first polymer layer, and is fused to the first polymer layer to form a seal A102b between the membrane and the first polymer layer. The third polymer layer A103 has a top surface A103a and a bottom surface A103b, and the second polymer layer is disposed between the first and third polymer layers.

The first polymer layer A101 and third polymer layer A103 may be formed from a rigid polymer. The rigid polymer may include a crosslinked polymer, a thermoplastic, or a thermoplastic elastomer. The thermoplastic may be selected from one of polycarbonate, polypropylene, acrylonitrile butadiene styrene, polyethylene terephthalate or cyclic olefin copolymers. The rigid polymer may be suitable for injection moulding.

The second polymer layer A102 may be a formed from flexible polymer materials, for example thermoplastics, including polypropylene or thermoplastic elastomers. The thermoplastic elastomer may be selected from one or more of: styrenic block copolymers, polyolefin blends, elastomeric alloys, thermoplastic polyurethanes, thermoplastic copolyesters or thermoplastic polyamides. Preferably, the thermoplastic elastomer is a styrene ethylene butylene styrene block copolymer, for example RTP2740S-40AZ (from RTP Company, US) with a hardness of about 40 Shore A. When the first polymer is polycarbonate the second polymer is preferably a thermoplastic elastomer. When the first polymer is polypropylene the second polymer is preferably a thermoplastic elastomer.

The bottom surface of the first polymer layer A101 b and the top surface of the third polymer layer A103a are in contacting arrangement around at least a portion, preferably all of the perimeter of the second polymer layer. In other words, the perimeter of the second polymer layer A102a - that is, the flexible membrane - is substantially, preferably entirely enclosed by the first and third polymer layers.

The bottom surface of the first polymer layer A101 b and the top surface of the third polymer layer A103a may be joined together using a technique known to those skilled in the art, such as, but not limited to, mechanical joining methods, for example clamping. Alternatively, the surfaces may be bonded together using a technique known to those skilled in the art, such as, but not limited to, chemical bonding, heat sealing, diffusion bonding, adhesive bonding, ultrasonic welding or laser welding.

As shown in figure 17, the first polymer layer A101 of the fluid control device A100 has a first thickness, ti , and the second polymer layer A102 has a second thickness, t ₂ . Figure 17 shows that the thickness of the first polymer layer, ti , is greater than the thickness of the second polymer layer, t ₂ . The thickness of the second polymer layer, t ₂ may be between 0.1 and 1.5 mm, preferably between 0.2 and 1.3 mm and more preferably between 0.4 and 0.9 mm.

Figure 18a-d illustrates various alternative embodiments of the fluidic control device according to the present invention. The embodiments principally differ in terms of the geometry of the flexible membrane A102 and the first polymer layer A101 onto which it is fused, as discussed in more detail below. It should be understood that these embodiments are merely by way of example, and that the flexible membrane A102 and the first polymer layer A101 can assume further geometries not illustrated in the figures. Furthermore, the features illustrated or described in connection with one embodiment may be combined with features of any other embodiment.

In the embodiments shown in figures 18a-d, the bottom surface of the second polymer layer A102 is flush with the bottom surface of the first polymer layer A101 b. This is preferred, since it provides a more compact fluidic control device by negating the need for a separate flexible membrane sandwiched between (and thus entirely separating) the first and third polymer layers, and also simplifies the structure of the third polymer layer, which need not accommodate the shape of the flexible membrane.

In the embodiment of figure 18a, the first polymer layer A101 includes an annular shelf A104 recessed into its bottom surface A101 b. The annular shelf extends around the perimeter of the void A101 c. As shown, the second polymer layer forming the flexible membrane A102 occupies at least a portion of the annular shelf and thus the annular shelf acts as a surface onto which the flexible membrane A102 may be formed, thereby increasing the strength of the bond between the flexible membrane and the first polymer layer. The top surface of the flexible membrane may either be flush with the annular shelf (as shown in figure 18a), or be within the void A101 c in the first polymer layer, such that the second polymer layer occupies a portion of the void A103c (as shown in Figure 18b).

Whilst the annular shelf A104 of figure 18b could extend around the perimeter of the void and meet the void at its periphery, figure 18c shows an alternative embodiment, wherein an annular ledge is provided extending radially inwardly around the perimeter of the void, wherein the annular ledge forms part of the annular shelf. In that case, the size of the surface onto which the flexible membrane A102 may be formed is further increased, thereby further increasing the strength of the bond between the flexible membrane and the first polymer layer.

In the embodiment of figure 18d, the first polymer layer A101 further comprises a depression A106 recessed into its bottom surface A101 b. If an annular shelf is formed in the bottom surface A101 b of the first polymer layer (as with the embodiment of figure 18d), the depression may be formed in that annular shelf. The depression of the illustrated embodiment is an annular ring A105 which surrounds the void, but the depression may take various other forms and in particular need not be annular. The second polymer layer A102 occupies at least a portion of the depression (e.g. annular ring A105) to form a membrane A102a over the void which can more easily spread forces exerted upon it to the first polymer layer A101. In a preferred embodiment, the annular ring A105 further comprises a bevelled edge A106 on its inner periphery to facilitate manufacture.

In all embodiments shown in figures 18a-d, the second polymer layer A102 forms a membrane A102a across the void A101c in the first polymer layer, and is fused to the first polymer layer to form a seal A102b between the membrane A102a and the first polymer layer A101. The seal is formed during the moulding process as will be described with reference to figures 23 to 26 below. When manufactured according to a method of the invention, the second polymer layer A102 is injection-overmoulded onto the first polymer layer A101 and forms a seal anywhere the second polymer layer A102a contacts the first A101. Figures 19 to 23 show preferred embodiments of valves and bellows for use in the exemplary fluidic cartridge in more detail. In the embodiment of figure 19, the first polymer layer A101 further comprises a fluid passageway A101d having an opening A101e which opens into the void A103c in the first polymer layer so as to enable transmission of a positive or negative fluid pressure into the void for moving the membrane A102a formed over the void. The fluid passageway A101d may be coupled to a fluid interface such that when the cartridge is inserted into a reader, the passageway A101 d may be connected to a source of positive or negative fluid pressure in the reader. In a particular embodiment, the fluid interface is a pneumatic interface for connecting to a source of positive or negative gas pressure.

As shown in figures 20 to 22, the third polymer layer A103 further comprises first and second fluid passageways A201 , A202. The first and second fluid passageways A201 , A202 have openings A201 a, A202a in the top surface A103a of the third polymer layer, such that the flexible membrane can seal against openings A201a, A202a to prevent fluid from flowing between the first and second passageways.

In circumstances where the fluidic control device is operated as a valve (see figures 19, 20 and 22), the membrane A102a of the fluid control device A100, is movable between:

• a closed position, wherein a positive pressure is applied to the void such that the membrane A102a is sealed against the openings of the first and second fluid passageways A201 a, A202a to prevent fluid communication between the first and second passageways A201a, A202a; and

• an open position, wherein a vacuum or gauge pressure is applied to the void such that the membrane A102a is spaced apart from the openings of the first and second fluid passageways A201 a, A202a to permit fluid communication A203 between the first and second passageways A201a, A202a.

An exemplary illustration of the membrane in the closed position is illustrated in figure 20a, whereas an exemplary illustration of the membrane in an open position is shown in figure 20b.

In circumstances where the fluidic control device is operated as a bellows pump (see figure 21) it is advantageous to maximise the movement of the membrane A102a, and there is also no requirement for the membrane to default to a closed position, as with the devices described above. Hence, in an embodiment of a bellows pump, there is provided a cavity A600 between the third polymer layer and the membrane. The first and second fluid passageways A201 , A202 may further have openings A201 a, A202a in the top surface A103a of the third polymer layer wherein the top surface of the third polymer layer A103a and the bottom surface of the flexible membrane A102 define the cavity A600. The membrane A102a of the fluid control device A100 illustrated in figure 21 is thus movable to vary the volume of the cavity and act, in cohort with the upstream and downstream bellows valves described above, as a bellows.

When operable as a valve, it is sometimes advantageous to limit the volume of the valve when the membrane is in its open position. This minimises the dead volume within the valve when the valve is open by restricting the extent to which the flexible membrane can move into the void in its open position. Hence, the fluid control device of figure 22 further comprises an abutment A700, such as a protrusion, cage, lip or cross structure, within the void, wherein the abutment limits movement of the membrane in its open position. The abutment may be positioned between the flexible membrane and the roof of the void. The displacement of the membrane from its closed to its open position is preferably between 0.1 and 0.5 mm, more preferably between 0.2 and 0.4 mm, and there preferably exists a gap of between 0.3 and 0.7 mm, more preferably between 0.4 and 0.6 mm between the top of the abutment A700 and the top surface A101 a of the first polymer layer.

In the embodiments described above, the void A101c defined in the first polymer layer may be uniform in cross-section. The cross-section of the void A101c may be substantially circular, circular or elliptical in plan. Where the fluid control device is operable as a valve and the void cross-section is circular, the diameter of the void A101 c is preferably between 2 and 10 mm, more preferably between 3 and 7 mm and even more preferably between 4 and 6 mm. Most preferably, the void diameter is 5 mm. Where the fluid control device is operable as a bellows pump and the void cross-section is circular, the diameter of the void A101c is between 14 and 24 mm, preferably between 16 and 20 mm and more preferably between 17 and 19 mm. Most preferably, the void diameter is 18 mm.

2.2 Manufacture of a fluidic control device

According to a method of the invention, a fluid control device 100 may be manufactured using an injection-moulding process described below.

Figure 23 is a diagrammatic flow diagram illustrating a method A400 according to the invention for forming a first polymer layer A101 and a second polymer layer A102 of a fluid control device A100. As shown in step A401 , the method A400 includes providing a first tool portion A400a and a second tool portion A400b so as to define a first cavity A401 a. The method subsequently comprises injection-moulding a first polymer into said cavity to form a first polymer layer A401 a having a top surface, a bottom surface and a void extending through the first polymer layer from the top surface to the bottom surface.

As shown in steps A402 and A403, the method subsequently comprises separating the second tool portion A400b from the first tool portion A400a (and the first polymer layer A401 a already formed) and then applying a third tool portion A400c to the first tool portion A400a (and the first polymer layer A401a already formed). The first tool portion A400a, first polymer layer A101 and third tool portion A400c define a second cavity A403a, into which a second polymer A102 is be injection- overmoulded onto at least a portion of the first polymer layer A101. The second polymer may be injected-overmoulded into the cavity A403a before the first polymer layer has cooled to a setting temperature.

As shown in step A404, the method subsequently comprises separating the first and third tool portions A400a, A400c from the first and second polymer layers A101 , A102, thereby forming a component of the fluid control device A100.

The process of injection-overmoulding the second polymer onto at least a portion of the first polymer layer A101 provides a membrane A102a across the void in the first polymer layer A101c which is fused to the first polymer layer A101 c. If the second polymer is injection-overmoulded onto the first polymer layer A101 before that layer has had a chance to cool to a setting temperature (i.e. whilst that layer is still hot and not completely solidified from the injection-moulding process used to form it), the second polymer layer A102 bonds with the first polymer layer A101 , forming an improved seal A102b between the second polymer layer A102 and the first polymer layer A101 when compared with prior art manufacturing techniques.

Figures 24 to 26 are diagrammatic flow diagrams providing methods of making particular embodiments of the invention described above in reference to figures 18a-d, where the first polymer layer and second polymer layer take different geometries. The method is the same for all geometries, but the tool portions used to define the injection moulding cavities vary depending on the geometry of the first and second polymer layers to be achieved. It should be understood that these embodiments are merely by way of example, and that methods for forming the first and the second polymer layers A101 , A102 with different geometries not illustrated in the figures may also be covered by the present invention. Furthermore, the features illustrated or described in connection with one embodiment may be combined with features of any other embodiment. During the step of overmoulding the second polymer layer, the seal A102b between the second polymer layer A102 and the first polymer layer A101 may optionally be formed by the bonding of the second polymer layer A102 to the first polymer layer A101. The bonding of the two layers may involve one or both of the interdiffusion of polymer molecules from the second polymer layer A102 into the first polymer layer A101 and/or the interdiffusion of polymer molecules from the first polymer layer A101 into the second polymer layer A102.

As illustrated in figure 27, the step of injecting the second polymer into the second cavity A403a defined by the first tool portion A400a, first polymer layer A101 and third tool portion A400c comprises injecting the second polymer through a runner A1201 in the third tool portion A400c. The location of the runner A1201 is offset from the void A101 c in the first polymer layer such that the injection point A1202 in the second polymer layer A102 is spaced apart from the membrane A102a. It will be appreciated that the fluidic control device formed in this way will be recognisable by a skilled person, since the location of the runner is usually discernible from the formed component itself. The advantage of having the injection point spaced apart from the membrane is such that any distortion around the injection point does not affect the sealing of the membrane.

The setting temperature of the first polymer layer will depend on the specific polymer used. In one embodiment, the setting temperature of the first polymer may be between 10 °C and 100°C, preferably between 20°C and 80°C, preferably between 30°C and 70°C, preferably between 40°C and 60°C. In another embodiment, the setting temperature of the first polymer may be between 100 °C and 200°C, preferably between 1 10°C and 190°C, preferably between 120°C and 180°C. The skilled person would understand that a polymer may set over a temperature range, instead of at a well-defined single temperature. The temperature range may correspond to the glass transition of the polymer.

An additional step in the methods described above involves forming a third polymer layer A103, comprising a top surface A103a and a bottom surface A103b, and arranging the third polymer layer A103 with the first and second polymer layers A101 , A103 such that the second polymer layer A102 is disposed between the first and third polymer layers A101 , A103, and such that the bottom surface of said first polymer layer A101 b and the top surface of said third polymer layer A103a are in contacting arrangement around at least a portion of the perimeter of the second polymer layer.

The step of forming a third polymer layer A103 may include pre-forming the third polymer layer using a technique known to those skilled in the art, such as, but not limited to, injection moulding. The step of arranging the layers may further comprise joining or bonding the bottom surface of the first polymer layer A101 b to the top surface of the third polymer layer A103a using a technique known to those skilled in the art, such as, but not limited to, mechanical joining methods, for example, clamping, or bonding methods, for example, chemical bonding, heat sealing, diffusion bonding, adhesive bonding, ultrasonic welding or laser welding.

2.3 Peel Strength Test

The strength of the seal formed between the first and second polymer layers A101 , A102 can be tested using standard peel strength tests. In particular, the seal could be tested using the RTP Company peel strength test (RTP TP-55), which is a variation of ASTM D6892/D903. This test can be used to determine the peel strength characteristics of the bond formed between the first and second polymer layers to establish the relative strength between two different seals.

2.4 Use of the fluid control device

The fluid control device A100 described herein can be operable as a valve or bellows pump.

In use, the fluid passageway A101d in the first polymer layer A101 may be coupled to a fluid interface for connecting to a source of positive or negative fluid pressure. The fluid passageway A101 d enables the transmission of a positive or negative fluid pressure to the void for moving the membrane A102a.

When a positive pressure is applied to the void via the fluid interface, the membrane A102a is sealed against the openings of the first and second fluid passageways A201 a, A202a to prevent fluid communication between the first and second passageways A201 a, A202a: the membrane is in the 'closed position'. On application of a negative pressure or vacuum to the void, the membrane A102a is spaced apart from the openings of the first and second fluid passageways A201a, A202a to permit fluid communication A203 between the first and second passageways A201 a, A202: the membrane is in the Open position'. By altering the applied pressure, the membrane can be moved between the closed and open positions and in doing so is operable as a valve or bellows pump, depending on the particular structure and disposition within the exemplary cartridge.

It will be recognised that equivalent first, second and third polymer layer structures may be substituted for the layers illustrated and described herein and also that many different arrangements of fluid passageways could be provided to alter the mechanism of the valve or pump described herein. Other structures may be employed to implement the claimed invention. Numerous variations, modifications and substitutions for the different layers are contemplated within the scope of the claimed invention. 3. Additional isolated inventive aspects

The following is a non-exhaustive list of isolated aspects of the exemplary cartridge described above which may be claimed. These aspects are described with reference to figures 11 to 15. The inclusion of this section does not preclude there being further aspects of the exemplary cartridge described above which may also be claimed.

3.1 Valves for minimising dead volume

An advantageous arrangement for a valve in a fluidic cartridge will now be described, which may form an isolated inventive aspect.

Hence, in one aspect, there is provided a valve for a fluidic cartridge, the valve comprising:

a valve cavity having first and second openings connected to first and second passageways, respectively; and

a flexible membrane movable between a closed position, in which the flexible membrane seals against the first and second openings to prevent fluid flow between the first and second passageways, and an open position, in which the flexible membrane is spaced apart from the first and second openings to permit fluid to flow between the first and second passageways;

wherein the a valve cavity comprises a roof and a floor, the floor comprising said first and second openings; and further comprising:

an abutment between the flexible membrane and the roof of the valve cavity, such that the abutment restricts movement of the membrane in its open position.

Preferably the abutment is provided on the flexible membrane, and comprises one or more of a protrusion, a cage, a lip or a cross structure.

It is sometimes advantageous to limit the extent to which the flexible membrane in a valve described herein is able to travel in its open position. That is, it is desirable to minimise the distance which the valve membrane moves to its open, and thus minimise the distance it must travel to close. By minimising this distance, the dead volume within the valve cavity is reduced, improving the reactivity of the valve.

Hence, as shown in more detail in figure 1 1 , preferred embodiments of a valve 300 further comprise an abutment 302. The abutment of the illustrated example is a cross structure, but in different embodiments may be a protrusion, cage, lip or similar, attached to the upper surface of the flexible membrane 304 so as to contact the roof 306 of the valve cavity and thus limit movement of the membrane in its open position.

It should be appreciated that the channels and openings of the valve are not shown in figure 1 1.

The abutment is particularly advantageous when filing the amplification chambers of the exemplary cartridge, because it reduces the dead-volume in the valve cavity and thus limits the distance between the bottom surface of the flexible membrane and the openings in the valve cavity, thereby permitting a more precise volume of fluid to be metered into the amplification chambers.

3.2 Crack pressure in valves

An advantageous arrangement for a valve in a fluidic cartridge will now be described, which may form an isolated inventive aspect.

Hence, in one aspect, there is provided a valve for a fluidic cartridge, the valve comprising:

a valve cavity having first and second openings connected to first and second passageways, respectively;

a flexible membrane within the valve cavity movable between a closed position, in which the flexible membrane seals against the first and second openings to prevent fluid flow between the first and second passageways, and an open position, in which the flexible membrane is spaced apart from the first and second openings to permit fluid to flow between the first and second passageways; wherein

the valve is configured such that a pressure required in the first passageway to move the flexible membrane from the closed position to the open position is higher than a pressure required in the second passageway to move the flexible membrane from the closed position to the open position.

It will be appreciated that within the valve cavity there is a portion (known as the valve chamber) between the flexible membrane and the floor. There is also a portion within the valve cavity on the other side of the flexible membrane to the valve chamber. This portion will have a volume. The pressure within that volume may be changed by applying a positive or gauge pressure to the volume via an actuation channel, for example. The actuation channel may be connected to a source of positive or gauge pressure via a pneumatic interface, for example. The pressure within the volume is known as the actuation pressure. This operation is described in more detail above. In a preferred arrangement, the first and second openings may be arranged such that fluid in the first passageway acts on the flexible membrane only over a relatively small cross-sectional area whereas fluid in the second passageway acts on the flexible membrane over a larger cross- sectional area, preferably substantially the whole membrane.

The effect of this is that the valve is able to withstand a much greater pressure in the first passageway that in the second passageway.

Preferably the valve cavity has a floor comprising the first and second openings and one or more walls between which the flexible membrane extends; and wherein the second opening is coupled to a recess in the floor between the opening and the flexible membrane, the recess having a larger cross-sectional area than the opening.

Preferably the first opening is located centrally within the floor and the recess extends around the first opening, such that the second opening is located between the first opening and a wall of the valve cavity. In a particularly preferred arrangement, the valve cavity has a circular cross section, and the recess is an annular recess which surrounds the first opening.

Preferably the opening of the second fluid passageway is located adjacent the perimeter of the valve chamber. Preferably the valve chamber has a diameter of between 2 and 10 mm, preferably between 3 and 7 mm and more preferably 4 and 6 mm. More preferably, the second opening is offset by 2 mm from the first opening.

An exemplary valve is shown in figure 12 in its closed position. The valve 310 may be used in place of any of the valves of the exemplary fluidic cartridge shown above. The valve comprises a valve cavity 312 having a flexible membrane 314 overlying a cavity floor 316 in which first 318 and second 320 apertures are provided, leading to first 322 and second 324 fluid passageways, respectively.

The cavity 312 is formed from a void in a first polymer layer (preferably the fluidic layer 1 14 of the exemplary cartridge) together with a second polymer layer (preferably the second fluidic layer 115 of the exemplary cartridge).

The flexible membrane 314 is shown lying across the floor 316 of the cavity such that the valve is shown in its closed position. The valve is movable from this position to an open position (where it is spaced from the floor 316 and the apertures 322, 324 to form a valve chamber), as described herein. The first opening 318 of the valve is centrally located within the perimeter of the void formed in the first polymer layer, and is therefore centrally located in the valve cavity 312. The second opening 324 of the valve is offset from the first opening 322. The second opening is coupled to an annular recess 326 in the floor, and thus the cross-sectional area over which the fluid in the second passageway 324 acts on the flexible membrane 314 is much larger than the cross-sectional area over which the fluid in the first passageway 322 acts on the flexible membrane.

The pressure of a fluid in the first passageway acts on the flexible membrane only over a relatively small cross-sectional area of the flexible membrane. Thus, because the pressure of a fluid in the valve cavity on the other side of the flexible membrane acts over the whole membrane, it may be lower without allowing the membrane to move to its open position.

In contrast, the pressure of a fluid in the second passageway acts on the flexible membrane over a relatively large cross-sectional area of the flexible membrane. Since the respective cross-sectional areas are closer, so too is the pressure in the second passageway which the flexible membrane is able to withstand vis-a-vis the pressure in the valve cavity.

Preferably, the respective cross-sectional areas of the openings of the fluid passageways allows the membrane to resist pressures around 2.5 times the actuation pressure on the first, central, fluid passageway, but only pressures equal to the actuation pressure (i.e. the pressure in the valve cavity) on the opening of the second, offset, fluid passageway.

3.3 Entry port design

An advantageous arrangement for an entry port on a fluidic cartridge will now be described, which may form an isolated inventive aspect.

Hence, in one aspect, there is provided a fluidic cartridge for processing a liquid sample, the cartridge having a sample mixing chamber comprising:

a sample inlet aperture for introducing a liquid sample into the sample mixing chamber; a cage surrounding the inlet aperture and extending into the sample mixing chamber, the cage further comprising one or more protrusions extending radially inwardly to abut against a sample delivery device introduced through the sample inlet.

The body of the cage may be formed from one or more elongate bars, or one or more solid walls, depending from the roof of the sample mixing chamber. If solid walls are provided, there is preferably an aperture in the lower portion of the walls through which a liquid sample introduced by the sample delivery device can pass. Preferably the bars or wall forming the body are tapered to conform to the nib of a conventional sample delivery device introduced through the sample inlet.

Solid walls have the additional advantage that they provide a barrier to prevent fluid introduced into the mixing chamber from escaping out of the inlet aperture, which is particularly useful if the cartridge is turned upside-down during use.

If the cage is formed from solid walls, the protrusion may be a ledge extending inwardly from the walls leaving an aperture. Preferably the protrusion extending from the sides of the inlet aperture is positioned above the floor of the sample mixing chamber; more preferably above a liquid fill level of the sample mixing chamber. This prevents liquid sample from being sucked back into the sample delivery device once introduced into the mixing chamber.

Preferably a vent is provided in the sample mixing chamber to allow air to escape from the chamber during the introduction of the sample. This is particularly useful when the inlet aperture is sealed by the sample delivery device.

Preferably a guide channel is provided within the sample mixing chamber (a portion of which is preferably directly underneath the inlet aperture) to direct the sample introduced by a sample delivery device into a visual indicator region. An exemplary visual indicator region is described above in connection with the exemplary cartridge.

Preferably a change in refractive index of the visual indicator region described herein identifies when a sample has been introduced. The visual indicator region may comprise a narrow fluid passageway, which becomes filled by the fluid sample via capillary action. The filling of the narrow fluid passageway changes the refractive index of the visual indicator region and a colour change identifies when a sample has been introduced.

A preferred embodiment of this aspect will now be described with reference to the exemplary fluidic cartridge. The housing 1 11 (see figure 4) comprises a sample inlet aperture 126 through which a sample may be introduced into the sample mixing chamber 10 of the cartridge 100 using a pipette, for example. As shown in more detail in figure 13a, the sample mixing chamber 10 is formed from the pneumatic layer 114, which has a roof adjacent the housing 1 11 in the region of the inlet aperture, and a corresponding inlet aperture through which a sample may be introduced into the sample mixing chamber 10. The roof of the mixing chamber 10 comprises a cage structure formed by walls 330 surrounding the inlet aperture 126 which extend into the sample mixing chamber 10 from the roof, and a ledge 332 extending radially inwardly from the walls 330. The shape of the cage structure allows a sample delivery device, such as a pipette, to be located in the correct position in the sample mixing chamber 10, and the ledge 332 prevents the pipette contacting the surfaces of the sample mixing chamber 10, thereby reducing the risk of contamination. The walls 330 can be tapered to further increase the engagement with the pipette.

Once the sample delivery device is located through the aperture, the user can dispense the sample. The ledge 332 is positioned above a nominal liquid fill level (not shown) of the sample mixing chamber so as to prevent the user from accidentally sucking the sample back up after dispensing it into the chamber.

A vent 334 into the chamber is provided to allow air to escape in the event that the inlet aperture is sealed by the sample delivery device.

A guide 336 is provided within the sample mixing chamber 10, a portion of which is directly underneath the inlet aperture 126 to direct the sample introduced by a sample delivery device into a visual indicator region 338. An exemplary visual indicator region is described above in connection with the exemplary cartridge.

3.4 Thermal Isolation pockets

An advantageous arrangement for thermal isolation pockets for a nucleic acid amplification chamber on a fluidic cartridge will now be described, which may form an isolated inventive aspect.

In nucleic acid amplification and detection, it is preferable to apply heat evenly throughout the liquid sample. Whilst it is possible to do this without difficultly in a laboratory by placing heat sources equidistantly around the sample, it is much harder to achieve in a cartridge.

Hence, in one aspect, there is provided a fluidic cartridge for performing nucleic acid amplification on a liquid sample, the cartridge comprising at least one sample processing chamber and a thermally insulating region adjacent the chamber to prevent heat loss from the chamber through the thermally insulating region. Preferably the at least one sample processing chamber is one or both of a nucleic acid amplification chamber and a nucleic acid detection chamber (hence forth 'processing chamber'). Preferably the nucleic acid processing chamber is adjacent a surface (preferably a bottom surface) of the cartridge for accepting heat from an external source, the chamber situated between the thermally insulating region and the surface such that heat passing from the external source through the surface and thence the chamber is not lost out of the other side of the chamber owing to the presence of the thermally insulating region. This arrangement is found to make the change in temperature inside the chamber (for instance when turning the heat source on and off) as fast as possible, which is beneficial for performing rapid PCR, for example.

This is particularly advantageous because a single heat source may be placed adjacent the cartridge to supply heat for the amplification process from one side (the heated side), and yet the sample within the cartridge will be heated substantially and the amount of heat lost through the unheated side minimised as far as possible.

Preferably the cartridge is comprised of at least a fluidic layer and a pneumatic layer in contacting arrangement. The nucleic acid processing chamber may be formed in the fluidic layer and the thermally insulating region may be formed in the pneumatic layer. Preferably the fluidic cartridge further comprises a fluidic foil underneath the fluidic layer, the foil forming the aforementioned surface for accepting heat. The use of a thin foil maximises the heat transfer from the external source. The material of the foil may be chosen to optimise the heat transfer. For instance, a metal foil may be used, but it is preferred that a polyethylene terephthalate / polypropylene composite is used due to the advantages in ease of manufacture of the cartridge, together with material strength and acceptable heat transfer properties.

Preferably the thermally insulating region is formed from one or more sealed thermal isolation pockets formed in the pneumatic layer and sealed by a pneumatic foil. The pockets may be filled with gas such as air or may be evacuated during the manufacturing process such that they provide a vacuum.

A preferred embodiment of this aspect will now be described with reference to the exemplary fluidic cartridge. As shown in figure 3, the exemplary cartridge 100 comprises, from top to bottom, a housing 11 1 , a blister sub-assembly 112, a pneumatic foil 113, a pneumatic layer 114, a fluid layer 1 15 and a fluidic foil 1 16.

Referring to figures 6A and 6B, which shows the pneumatic layer, six thermally insulating regions 140a-b, 141a-d are provided. The insulating regions 140a-b are located adjacent two corresponding amplification chambers formed in the fluidic layer 1 15, whilst insulating regions 141 a-d are located adjacent four corresponding detection chambers formed in the fluidic layer 115, when the cartridge is assembled. As shown, the insulating regions 140a-b consist of a plurality of thermal isolation pockets, whereas insulating regions 141a-d each consist of a single pocket.

During nucleic acid amplification and detection, thermocycling of the amplification and detection chambers takes place. The chambers in the fluidic layer may be heated by applying heat to the bottom of the cartridge 100, adjacent the fluidic layer 115. The thermal isolation pockets retain the heat within the cartridge, minimising heat loss from the fluidic layer 1 15 into the pneumatic layer 1 14. The thermal isolation pockets also eliminate the need for heating of the fluidics cartridge from both the top and bottom surfaces e.g. heating both the fluidics layer and the pneumatic layer, simplifying the overall design of the cartridge and reader.

The thermal isolation pocket may comprise one large pocket or multiple smaller pockets. The advantage of using multiple smaller pockets is that the risk of convection currents being set up is reduced, thus providing maximal thermal insulation.

3.5 Capture column

An advantageous arrangement for a filtering device in a fluidic cartridge (preferably a 'capture column') will now be described, which may form an isolated inventive aspect.

Hence, in one aspect, there is provided a fluidic cartridge comprising a channel through which a liquid sample may pass, the channel having a filter for capturing biological components and further comprising:

an upstream portion and a downstream portion; and

a capture portion between the upstream and downstream portions in which the filter is arranged; wherein:

the diameter of the capture portion is greater than the diameter of the upstream and downstream portions.

Preferably the capture portion is a chamber within the channel, the chamber having an inlet surface having an opening coupled to the upstream portion of the channel and an outlet surface having an opening coupled to the downstream portion of the channel.

Preferably the fluidic cartridge comprises at least two polymer layers, wherein the upstream portion and an upstream part of the capture portion of the channel are formed in a first polymer layer and the downstream portion and a downstream part of the capture portion of the channel are formed in a second polymer layer; and wherein the filter is clamped between the first and second polymer layers.

Preferably the inlet surface of the chamber comprises distribution conduits leading radially outwardly from the opening so as to direct a liquid sample passing through the opening in the inlet surface radially outwardly.

Preferably the outlet surface of the chamber comprises distribution conduits leading radially inwardly toward the opening so as to direct a liquid sample which has passed through the filter radially inwardly toward the opening in the outlet surface.

A preferred embodiment of this aspect will now be described with reference to the exemplary fluidic cartridge. In the exemplary cartridge described herein, a capture column 24 is provided along the main channel (see figure 1). As shown in figures 14a and 14b, the capture column 24 has filter 340 which binds DNA from lysed material before releasing it during elution. As shown in figure 14a, capture column 24 comprises an inlet channel 342 leading into a capture chamber 344 at an upstream end 346, and an outlet channel 350 leading from capture chamber 344 at a downstream end 348.

A filter 340 is provided in chamber 344, perpendicular to the direction of flow of fluid through the main channel, such that fluid must pass through filter 340 when passing from the upstream end of the main channel 342 to the downstream end 350 of the main channel.

Referring now to figure 14b, the inlet and outlet walls (only one is shown) of the chamber comprise distribution conduits 352 configured to direct fluid radially outwardly into the chamber 344 as it enters the chamber, and radially inwardly toward the exit aperture after it has passed through the filter 340.

3.6 Waste chamber

An advantageous arrangement for waste chamber in a fluidic cartridge will now be described, which may form an isolated inventive aspect.

Hence, in one aspect, there is provided a fluidic cartridge comprising a channel through which a liquid sample may pass and a waste chamber for receiving fluid from the channel, the waste chamber comprising: a pipe, coupled to the channel, extending from a bottom surface of the waste chamber and having an opening elevated above the bottom surface to pass fluid from the channel into the chamber; and

a vent within the waste chamber configured to vent the waste chamber to atmosphere.

Preferably the vent comprises a second pipe, coupled to a vent channel within the cartridge, extending from the bottom surface of the waste chamber and having an opening elevated above the bottom surface. Preferably the vent passageway comprises at least one Anderson impactor.

Preferably at least one absorbent pad is provided within the waste chamber.

A preferred embodiment of this aspect will now be described with reference to the exemplary fluidic cartridge. In the exemplary cartridge described herein, a waste chamber is provided for collecting and storing waste fluid which is produced during washing etc. Waste chamber 10 is shown in more detail in figures 15a and 15b. Waste chamber 38 comprises a pipe 360, extending substantially vertically from a bottom surface 362 of waste chamber 38. The pipe 38 defines a channel having a first end 364 connected to the bottom surface of the waste chamber 38 and fluidly connected to the main channel 16. A second end 366 of fluid pipe 360 is disposed within waste chamber 38, and has an opening through which fluid can flow into the waste chamber.

Preferably, pipe 360 is substantially vertical, and perpendicular to the bottom surface of the waste chamber 38. The opening at the second end of pipe 360 is located near the top of the waste chamber 38 as shown in figure 15b. By providing the first opening near the top of the waste chamber, the risk of leakage is minimised should the cartridge be turned upside down.

Absorbent pads 368 are also provided in the waste chamber. Preferably, the upper surface of absorbent pads 368 should also be near the top of waste chamber 38, even more preferably, the top of absorbent pads 368 should be substantially level with the opening at the second end 366.

In the exemplary cartridge described herein, a second opening 370 is provided in waste chamber 38 as shown in figure 15b. The second opening 370 is configured to vent main channel 16 via waste chamber 28 to atmospheric pressure. This avoids putting a back pressure along the main channel as the waste channel fills with fluid. Preferably, the second opening 370 is provided at the end of a second pipe 372 protruding from the bottom surface of waste chamber 38. The second opening 370 may be fluidly connected to a vent passageway (not shown) which has an opening outside of the cartridge housing to allow the waste chamber to remain at atmospheric pressure. However, venting the waste chamber outside the cartridge carries a small risk of aerosol contamination. To reduce this, the vent path has impact traps and vents under the cartridge cover.

The skilled person will be capable of modifying the exemplary cartridge to implement the inventive aspects described herein in various ways depending on circumstances. It is intended that the scope of the present invention is defined by the following claims.