The disclosure relates to quality control for a batch of assay devices. Modern assays and tests are often provided in the form of a microfluidic device (or cartridge) for use with a reader. The device allows a sample to be introduced and may carry other liquids in storage arrangements such as blister packs so that they can also be introduced into the device, for example by pressing on the blister pack. The device is then inserted into a reader for processing the sample and obtaining a read out of the assay, for example to determine the concentration of a analyte, such as proteins (e.g., glycated haemoglobin, C- Reactive Protein), enzymes (e.g., AST, ALT), hormones (e.g., TSH), sugars (e.g., glucose), lipids (e.g., cholesterol, triglycerides) and the like, in a sample. The devices are typically provided in batches and some form of quality control is typically required for users to make sure that both the reading system and the batch of devices used to carry out the assay are performing to the required standards. Usually, quality control is performed by the user by taking an assay device, loading it with a control reagent sample instead of an unknown sample and running the assay. This is inconvenient, since control reagents provided in liquid form are expensive and have a very limited shelf-life once opened, resulting in a considerable expense and inconvenience for the user. Moreover, control reagents provided in solid form need to be solubilized or suspended in a liquid carrier (e.g., water or a buffer) before use, which represents an inconvenience for the user.

In overview, a batch of assay devices comprising a quality control device, a quality control device and a method of manufacturing a batch of assay devices comprising a quality control device, as well as a method of using the quality control device to carry out quality control are disclosed.

In a first aspect, a batch of devices is disclosed. Each device comprises a sample inlet port, a liquid inlet port and a liquid handling structure defining a detection chamber and a flow path between the liquid inlet port and the detection chamber. A reagent for interacting with an analyte in a sample introduced into the device through the sample inlet port is disposed in each device to enable detection of the analyte in the detection chamber. At least one of the devices is configured as a quality control device and comprises a deposit of the analyte in the flow path to reconstitute a reconstituted sample of known concentration for detection in the detection chamber when the liquid flows along the flow path in the at least one device and the remaining devices do not comprise the deposit of the analyte. Within a batch of devices, the assay devices do not include the deposit of analyte but are otherwise substantially identical to a quality control device. The assay devices may differ from the quality control device, for example, in that the sample port, which is not needed in a quality control device, is permanently sealed in the quality control device, while it may be sealable once a sample has been applied in the remainder of the batch (although the port may also be initially open and sealable in the quality control device). Further differences may, for example, relate to details of any surface marking or decoration applied to the devices or packaging in which they are packaged, for example to identify the quality control device as such.

Advantageously, by providing a batch of devices with a quality control device that incorporates a sample of the analyte, quality control is made easier for the user, as described in more detail below. The reagent may have specificity for the analyte, for example specifically bind to the analyte. The reagent may be an antibody or enzyme, mix of antibodies and enzymes, etc., and the analyte may be an antigen or set of antigens, protein or carbohydrate molecule to which the antibody(ies) specifically binds or the enzyme(s) is specific for. Other reagents may be used in the assay, for example as discussed below, and may be involved in the assay detection, such as in the case of fluorophores. The reagent specific to the analyte may be immobilised or bound to a physical support such as a surface of the device or carrier particles, such as latex beads, for example, which can also be considered as reagents. In some cases, the analyte may bind to a support like latex beads and the antibodies bind to the analyte-support complex. For example, in the case of latex beads or similar particles, antibody to target binding can be detected by the resulting agglutination of the latex beads with attached analyte and antibodies. Generically, antibodies are examples of probe molecules specifically binding to target molecules such as antigens. The disclosed devices and methods are equally applicable to any type of specific reagent, antibody, probe molecule, stain, fluorophore or otherwise. It will be appreciated that the disclosure is not limited to a single specific reagent but that the assay may involve more than one specific reagent. Reference to "a specific reagent" does not exclude the presence and use of additional specific reagents. The reagent or reagents may be provided in dry form in the flow path or may be provided in a liquid storage arrangements, such as a blister pack, secured adjacent the liquid inlet port. Reagents may comprise particle suspensions, functionalised latex beads or nanoparticles, reaction mixtures, one or more buffers, saline solution, antibodies, enzymes or a combination thereof. Latex beads or nano-particles and a functionalising reagent may be stored separately in the device and the latex beads or nanoparticles may be functionalised during the microfluidic protocol when the device is used.

The analyte may comprise a biomolecule or a mixture of biomolecules and the analyte deposit may further comprise one or more of: one or more stabilisers selected from glucose, sucrose, reducing monosaccharide sugars and reducing disaccharide sugars; a buffer; one or more preservatives (for example, an antimicrobial agent); serum; a serum matrix; and blood. Prior to use of a device by introduction of a fluid, for example, to carry out an assay, the deposit may be a dry deposit substantially free from solvent, for example a liquid sample dried in situ or a deposit of an already dried substance. Alternatively, the deposit may be in a gel state, for example where the analyte comprises trehalose or gelatin. In either case, the deposit may be imbibed in an absorbent material, such as filter paper, or coated on a carrier material, such as polymer substrate. The assay may be to detect a characteristic, for example concentration, of an analyte or to detect the presence of the analyte. For example, where the assay device is for measuring a concentration of particular biomolecule, the deposit comprises a sample of said biomolecule, thereby providing a control run for the assay. Thus, the assay device is provided with an integrated control. This may be of particular advantage when a batch of devices is produced, one of which comprising the integrated analyte deposit, as it provides a control run for the batch of assays. It may also provide for calibration of the apparatus used to measure the result of the assay. The analyte may be deposited such that upon pick up of the analyte by the fluid flowing through the device, sufficient control analyte, preferably of a known concentration is present in the liquid to provide a control for the analyte.

In some embodiments, the or each device comprises a body defining an internal liquid handling structure. The body defines a first aperture in an external surface thereof to provide a liquid inlet port to the liquid handling structure and a second aperture in the external surface to provide a sample inlet port to the liquid handling structure, wherein the sample inlet port is sealed or sealable. The device comprises liquid storage arrangement storing a liquid and secured to the external surface of the body overlapping the first aperture to dispense the liquid through the first aperture when a pressure exceeding a threshold pressure is applied to the liquid storage arrangement. The device further comprises a reagent specific to the analyte to enable detection of the analyte in the assay. The liquid handling structure defines a detection chamber to receive liquid from the liquid inlet port and enabling detection of a signal indicative of a reaction between the analyte and the reagent and a first fluid flow path between the liquid inlet port and the detection chamber. In a quality control device, a deposit comprising the analyte is deposited within the first fluid flow path. When the liquid flows along the flow path it interacts with the deposit to reconstitute a sample of known analyte concentration to interact with the reagent, for example in the detection chamber or as liquid flows from the liquid receiving chamber to the detection chamber.

In some embodiments, the first liquid flow path and a second liquid flow path from the sample inlet port to the detection chamber join in a common flow path common to both the first and second flow paths, and the deposit comprising the analyte is disposed in the common flow path.

In a second aspect, a device configured to carry out quality control for an assay to detect an analyte is disclosed. A device body defines an internal liquid handling structure, a first aperture in an external surface of the body to provide a liquid inlet port to the liquid handling structure and a sample inlet port to the liquid handling structure, wherein the sample inlet port is sealed or sealable. For example, in light of the use as a quality control device, the inlet port may be permanently sealed by not providing a corresponding aperture in the external surface, or an aperture may be provided but (permanently or reversibly) sealed. Alternatively, the sample inlet port may be configured as for an assay (rather than quality control device), in association with a sealed or sealable aperture in the external surface. A liquid storage arrangement storing a liquid is secured to the external surface of the body overlapping the first aperture to dispense the liquid through the first aperture when a pressure exceeding a threshold pressure is applied to the liquid storage arrangement. The device comprises a reagent specific to the analyte to enable detection of the analyte and the liquid handling structure defines a detection chamber to receive liquid from the liquid inlet port and enabling detection of a signal indicative of a reaction between the analyte and the reagent. The liquid handling structure further defines a detection chamber to receive liquid from the liquid inlet port and enable detection of a signal indicative of a reaction between the analyte and the reagent. The liquid handling structure also defines a first liquid flow path from the liquid inlet port to the detection chamber and a second liquid flow path from the sample inlet port to the detection chamber. The first and second liquid flow paths join in a common flow path to the detection chamber and a deposit comprising the analyte is disposed in the common liquid flow path, wherein when the liquid flows along the common flow path it interacts with the deposit to reconstitute a sample of known analyte concentration to interact with the reagent. Advantageously, by depositing the analyte in the common flow path, the similarity of the assay and quality control devices is increased, since the portion of the first flow path not exposed to analyte in the assay device remains so in the quality control device, which may improve quality control. The increased similarity facilitates the aim of the quality control device to control the quality of a batch of substantially identical assay devices.

The device or devices may be configured for rotation about an axis of rotation to drive liquid flows in the device. The deposit may be disposed in an unvented chamber of the liquid handling structure, which may be configured to enable the liquid to be moved back and forth repeatedly between the unvented chamber and a portion of the liquid handling structure upstream of the unvented chamber to pick up the deposit with the liquid and mix the liquid by varying the speed of rotation (thereby changing the balance between a centrifugal force and a pressure in the unvented chamber). The device or devices may be a microfluidic device(s). For the avoidance of doubt, the term "microfluidic" is referred to herein to mean devices having a fluidic element such as a reservoir or a channel with at least one dimension below 1 mm. The device may be configured to handle volumes of liquid on the scale of nanolitres to microlitres or up to 100 -microliters. Some but not necessarily all the structures on such a device may be microfluidic.

In some embodiments, the device or devices comprise a feature which defines the axis of rotation and which is configured to be coupled to a rotational element to drive rotation of the device. For example, the device or devices may be configured as a centrifugal disc, such as a microfluidic disc. The device or devices, disc-shaped or otherwise, may comprise a central hole which is configured to engage with a spindle of a drive system, the spindle being coupled to a motor for driving rotation of the spindle, which in turn drives rotation of the engaged device. It will be understood that the terms "vented" and "unvented" as used herein are used such that a vented chamber is connected to the atmosphere external to the device or a closed air circuit so that pressure can equilibrate as liquid flows in or out of respective ports, for example inlet and outlet ports, of the vented chamber. Conversely, an unvented chamber is neither connected to external air nor to a closed air circuit such that, once liquid fills any inlet and outlet ports of the unvented chamber any difference in respective flow rates in and out of the unvented chamber leads to a change in pressure in the unvented chamber. In other words, in an unvented chamber the only fluid flow paths in or out of the unvented chamber are through one or more liquid ports part of a liquid flow circuit of the device.

It will be understood that the cavities referred to herein may be described as vented or unvented, as the case may be.

Where the term "level" is used in relation to a chamber or other liquid containing structure, it will be understood that this does not necessarily refer to a straight level as would be observed in a chamber filled with liquid under gravity, but that the term includes curved levels which may be curved due to a centrifugal force acting on the liquid or due to surface tension effects. The term "level" refers to a geometric locus, e.g. relative to a centre of rotation.

Any reference to a fill level of a liquid containing structure (e.g. a chamber or conduit) rising will be understood to refer to the liquid level moving radially inwards, towards the axis of rotation. Similarly, any reference to a fill level of a liquid containing structure (e.g. a chamber or conduit) falling will be understood to refer to the liquid level moving radially outwards away from the axis of rotation. It will be understood that reference to a structure 'A' being disposed radially inwards of a structure 'B' should be taken to mean that a distance between structure Ά and the axis of rotation of the device is less than a distance between structure 'B' and the axis of rotation of the device. Equally, it will be understood that reference to a structure Ά being disposed radially outwards of a structure 'B' should be taken to mean that a distance between structure 'A' and the axis of rotation of the device is greater than a distance between structure 'B' and the axis of rotation of the device. It will be understood that reference to a structure extending radially inwards should be taken to mean that the structure extends towards the axis of rotation. Equally, it will be understood that reference to a structure extending radially outwards should be taken to mean that the structure extends away from the axis of rotation. In a third aspect, a method of manufacturing a batch of devices each configured to carry out an assay, wherein each device comprises a sample inlet port, a liquid inlet port and a liquid handling structure defining a detection chamber a flow path between the liquid inlet port and the detection chamber and a reagent for interacting with an analyte in a sample introduced into the device through the sample inlet port to enable detection of the analyte in the detection chamber. The method comprises manufacturing the batch of devices and depositing a sample of the analyte in the flow path of a quality control device of the batch of devices to reconstitute a reconstituted sample of known concentration for detection in the detection chamber when the liquid flows along the flow path in the quality control device.

In some embodiments, the method comprises drying the deposited sample or depositing the sample in dry form. The sample may comprise an amount of analyte and the liquid handling structure is configured to meter an amount of liquid flowing along the flow path to reconstitute the reconstituted sample to a known concentration of analyte. The deposited sample may comprise a biomolecule, for example in a concentration of about 0.01 ng/ml to about 1 g/dl and the volume. The sample may be deposited in liquid form and the volume of the deposited sample may be 1 nanolitre to 100 microlitres. The method may comprise depositing the sample in liquid form and drying the sample in situ.

In some embodiments, the device may be configured as disclosed herein.

In yet a further aspect, a method of carrying out quality control for a batch of devices as disclosed herein or manufactured according to a method disclosed herein is disclosed. The method comprises causing the liquid to reconstitute the deposited sample in the quality control device, performing the assay to obtain a result of the assay and comparing the result to an expected result. In some embodiments, causing the liquid to reconstitute the deposited sample may comprise applying a mechanical pressure to the exterior of the quality control device to introduce the liquid into the quality control device, introducing the device into a reader and rotating the device inside the reader according to a rotation protocol defined for the batch of devices. Obtaining the result of the assay may comprise analysing a signal from the quality control device. The signal may be indicative of light reflected from or transmitted through the detection chamber. In some embodiments, the method may comprise adjusting one or more parameters of the reader based on the comparison between the result and the expected result, for example to calibrate the reader or making other adjustments, for example bias adjustments.

In an additional aspect, there is provided a method of manufacturing a device as described herein for carrying out an assay to detect a characteristic of an analyte, the method comprising: providing a substrate defining a liquid handling structure on a surface of the substrate, wherein the liquid handling structure comprises a liquid receiving chamber and a fluid flow path between the liquid receiving chamber and a detection chamber; depositing a sample of a reagent comprising the analyte having a characteristic on a portion of the surface of the substrate within the fluid flow path; drying the sample of a reagent to form a reagent deposit on the surface of the substrate; and sealing the substrate with a seal to enclose the surface defining a liquid handling structure within which the reagent deposit is dried. The seal used to seal the substrate may be a second substrate or a film. Disclosed embodiments may use this process to manufacture a batch of disks.

Embodiments are now described by way of example to illustrate aspects and principles of the disclosure, with reference to the accompanying drawings, in which:

Figure 1 illustrates a centrifugal microfluidic device;

Figure 2 illustrates a specific flow path;

Figure 3 illustrates a specific implementation of the centrifugal microfluidic devices;

Figure 4 illustrates a device reader; and

Figure 5 illustrates a method of operation of the device reader.

With reference to Figure 1 , a centrifugal microfluidic device 100 comprises a sample inlet port 102, a liquid inlet port 104 and a liquid storage arrangement 106, such as a blister pack, secured to the device 100 in a fully overlapping relationship with the liquid inlet port 104. When the liquid storage arrangement 106 is pressed on, it ruptures in the region of the liquid inlet port 104 and liquid is introduced from the liquid storage arrangement 106 into the device 100. The liquid may be a buffer and/or a reagent solution to be mixed with a sample introduced into the device through sample inlet port 102. The sample inlet port 102 is sealable once the sample has been introduced and the device comprises an internal air circuit to provide pressure equilibration to vented structures in the device as liquid flows. The sample inlet port 102, and in some embodiments a vent port connected to the internal air circuit, may be sealable with an adhesive flap provided separately or secured to the device 100.

The device 100 defines a flow path 108 from the liquid inlet port 104 to a detection chamber 1 12. A reaction between an analyte in the sample and a reagent, specific to the analyte, in the device 100 (for example provided inside the liquid storage arrangement 106 or in dry form at one or more points along the flow path 108, for resuspension in liquid flowing along the flow path) can be detected in the detection chamber 1 12. A flow path 1 10 connects the sample inlet 102 to the flow path 108, to allow the sample to interact with the liquid and flow to the detection chamber 1 12, for detection of a reaction between the reagent and the analyte. Further reagents may be provided in dry form along the flow path 1 10 for processing and/or conditioning of the analyte, for example lysing blood cells in a blood sample. It will be appreciated, thus, that the flow path 108 comprises a portion of a flow path between the sample inlet 102 and the detection chamber 1 12, that is there is a common flow paths for liquids from both ports.

When the reagent(s) is provided at one or more points along the flow path 108, the reagent(s) may be provided as one or more discrete dry reagent deposits along the flow path 108, arranged to provide pick-up of the reagent by the liquid (for example, a buffer). Pick-up of reagent may be achieved by rehydration, dissolution or re-suspension of reagent by the liquid or by digestion of reagent by an enzyme within the liquid. Accordingly, the reagent may be hydrophilic, water soluble and/or enzymatically degradable. Additional reagents may be provided, acting in combination or separately from the reagent, at adjacent or different positions along the flow path 108.

The device is configured so that liquid flows in the device are driven by centrifugal forces when the device is rotated and comprises a cut-out feature 1 14, in the fashion of a CD or DVD, to allow a spindle in a reader for the device 100 to engage the device 100 and rotate it about an axis of rotation, in this case centred on the feature 1 14. The reader (not shown) is configured to rotate the device according to a temporal profile of rotation speed to drive liquid flows in the device in accordance with the specific assay protocol implemented in the device and comprises a reading arrangement comprising a light source and a light sensor to detect light from the light source that is transmitted through or reflected by the detection chamber 1 12. Occurrence of a reaction between the reagent and the analyte in the device alters the light received from the detection chamber, so that the light sensor signal can be used infer the presence or a characteristic (such as concentration) of the analyte in a sample in the device 100. It will be understood that the flow paths 108 and 1 10 are illustrated in Figure 1 in a highly schematic fashion and will in practice comprise various liquid handling elements, for example for processing the sample, such as separating the sample into phases, selectively flowing selected phases, mixing the sample with the liquid and with chemicals and other substances provided in dry form, metering a volume of the sample and/or liquid, producing aliquots, etc. Equally, there may be multiple such flow paths and multiple detection chambers 1 12 to provide the same or different respective assays, that is detection of a reaction between respective pairs of a reagent specific to the analyte and the analyte. One example of a reagent analyte pair is a pair of a biomolecule and an antibody specific to the biomolecule, where the antibody may for example be immobilized in the detection chamber or may be attached to particles such as latex beads or nanoparticles that are or get suspended in the liquid. Typically, the flow path 108 will at least comprise a metering chamber to meter a well-defined volume of liquid for interaction with the sample downstream, although in some embodiments, the liquid storage arrangement is configured to provide a precisely defined volume of liquid.

Batches of the device 100 are manufactured and are characterized and tested to produce calibration data for a reader. Each device 100 is packaged in an individual packaging in some embodiments, in some advantageous embodiments in sealed vacuum pouches. These batches are distributed to customers for use with a reader in batches that may comprise all devices 100 in a manufacturing batch or subsets thereof. Each batch distributed comprises one or more quality control devices that are now described in detail. Each quality control device is identical to the other devices 100 in the batch, save for, of course, manufacturing tolerances and other inconsequential differences such as different surface decoration or packaging labeling. A further difference may be that, as will become clear, there is no need to provide for the sample port 102 to be sealable. Therefore, in some embodiments, the sample port 102 is sealed, for example with an adhesive flap attached at manufacture or by providing only the internal structures corresponding to the sample port 102 with a corresponding cut-out providing access to the sample inlet port 102 being omitted from an external layer of the device 100 at the time of manufacture.

A yet further difference is that a deposit containing an amount of analyte (or a substance reacting with the reagent in substantially the same way as the analyte) is deposited in the flow path 108 at the time of manufacture, as indicated schematically by arrow 1 16 in Figure 1 . In some embodiments, the analyte is deposited in the common flow path common to liquid from both inlet ports. The amount of analyte is chosen so that a sample of known concentration is reconstituted as a metered volume (metered in a metering chamber or in the liquid storage arrangement 106) of liquid flows in the flow path 108, without any sample being introduced into the device from outside. This provides, in effect, a device 100 with an integrated quality control and/or calibration sample, obviating the need for customers to separately procure and/or store and apply samples of quality control solutions. Additionally, providing the quality control sample in dried form in a device 100 sealed in a vacuum pouch improves the shelf-life of the quality control sample. The analyte may be a known biomolecule or mixture of biomolecules. It will be appreciated that the exact nature of the analyte, and hence the reagent, will depend on the particular assay to be carried out. Examples of biomolecules that may be used as analtyes in the present invention include antigens, biomarkers, proteins (e.g., glycated haemoglobin, C- Reactive Protein, myoglobin etc), enzymes (e.g., aspartate transaminase (AST), alanine aminotransferase (ALT), etc.), hormones (e.g., thyroid-stimulating hormone (TSH), etc.), carbohydrates (e.g., sugars, such as glucose), polynucleotides, lipids (e.g., cholesterol, triglycerides), cells, fragments of cells and the like. The biomolecule may be isolated from human tissue. The analyte deposit may be provided on a serum, blood or plasma based matrix or a buffer.

The deposit may further comprise additional excipients to provide for increased pick up or improved flow of the analyte within in the device. The device may further comprise: a) one or more stabilisers selected from glucose, sucrose, reducing monosaccharide sugars and reducing disaccharide sugars; b) a buffer; c) one or more preservatives (for example, an antimicrobial agent) and d) serum or blood derivative. The deposit may further comprise additional proteins or enzymes.

A skilled person is familiar with many different samples that could be used to provide the analyte deposit in devices of the present invention. It will be appreciated that the choice of analyte sample will depend on the particular assay for which the device is intended. Examples of analytes that can be used for quality control purposes include: Seronorm™ HbA1 c Liquid L-1 and L-2, liquid human-based hemolysate samples produced from blood collected from non-diabetic voluntary blood donors; Seronorm™ CRP Liquid L-1 , L-2 and L-3, liquid human-based control serums produced from blood collected from voluntary blood donors; Liquichek™ Diabetes Control, a liquid, human whole blood based control designed to monitor the precision of hemoglobin test procedures associated with diabetes monitoring; Lyphochek® Diabetes Control, a human whole blood based product intended to monitor the precision of hemoglobin tests procedures used in diabetes monitoring, including Hemoglobins A1 , A1 C, F and Total Glycated Hemoglobin. Further analyte samples are readily available to a skilled person.

The deposit is arranged to provide pick-up of the analyte by the liquid, wherein pick-up of analyte is achieved by rehydration, dissolution or re-suspension of analyte by the liquid or by digestion of analyte by an enzyme within the liquid. Accordingly, the analyte may be hydrophilic, water soluble and/or enzymatically degradable. With reference to Figure 2, a specific, although still simplified, flow path 108 comprises a vented chamber 202, and an unvented chamber 204. The sample and liquid inlet ports 102, 104 are connected to the vented chamber 202 by respective flow paths 1 10 and 206 that are illustrated schematically in Figure 2, which omits some details such as, for example, a metering structure in the flow path 206 to meter a volume of liquid coming from the liquid inlet port 104. The vented chamber 202 is connected to the air circuit (not shown) by a vent conduit 208, to the unvented chamber 204 by a flow path 210 and to the detection chamber 1 12 by a flow control arrangement 212 and flow path 214. The flow control arrangement 212 is configured to retain liquid in the vented chamber 202 until a time when it is desired to flow downstream, for example by means of controlling the profile of rotations speeds of the device. In some specific embodiments, the flow control arrangement 212 may be a capillary siphon valve, with which a person skilled in the art is very familiar. Other flow control arrangements, for example a surface tension valve, sacrificial valve, metering (non- capillary siphon) and any other flow control arrangement is of course equally possible. As before, flow paths 210 and 214 are presented in a simplified fashion for the sake of clarity of presentation but may each comprise respective liquid handling structures such as various chambers and conduits. A dried deposit 216 of analyte is provided in the unvented chamber for those devices 100 that are configured as quality control devices. In operation of a (non-quality-control) device 100, a sample is introduced via sample port 102, the sample port 102 is sealed and the liquid storage arrangement 106 is ruptured to introduce the liquid into the device through the liquid inlet port 104. The sample may, for example, be a lysed or a whole blood sample. The device is then inserted into the reader for rotation in accordance with a speed profile to cause processing of the sample and liquid and a signal is then read out from the detection chamber 1 12. A similar process is followed for a quality control or calibration run, although without the introduction of a sample, as the quality control sample is reconstituted inside the device 100 as liquid flows through the device 100. Specifically, in the example of an embodiment as described above with reference to Figure 2, in a non-quality-control device, after any intervening processing steps liquid from both inlets 102, 104 is combined in the vented chamber 202. To promote mixing of the liquid from the two ports, the rotation profile comprises a portion of oscillating velocity timed to coincide with both liquids being present in the vented chamber 202, which cause the combined liquid to flow back and forth between the vented and unvented chambers 202, 204 due to the interaction of the time varying centrifugal force and pressure in the unvented chamber 204 as liquid is forced into it by an increasing centrifugal force and forced back out as a decreasing centrifugal force does not balance the pressure in the unvented chamber 204 leading to an expansion of the air compressed by previous liquid ingress and corresponding expulsion of liquid from the chamber 204. This is repeated for a set number of cycles and the flow control arrangement 212 is then caused to allow liquid to flow out of the vented chamber 202 to the detection chamber 1 12 on continued rotation. For example, in the case of a capillary valve, the device 100 is slowed or stopped to prime the siphon by capillary force.

In the case of a quality control device 100, the device is substantially the same as explained above and substantially the same rotation protocol is used. Hence the liquid flows are substantially the same, with the exception, of course, that no liquid flows from the sample port 102. Instead, only the liquid from the liquid inlet port 104 flows into the vented chamber 202 and back and forth between the vented and unvented chambers 202, 204. As the liquid flows back and forth between the two chambers, it picks up a deposit of analyte 216 in the unvented chamber 204 and eventually flows as a reconstituted sample downstream to the detection chamber 1 12 once the flow control arrangement 1 12 has been caused to allow liquid to flow downstream.

A specific embodiment of the device 100 is now described with reference to Figure 3. A centrifugal microfluidic device 300 is configured for an assay to detect the HbA1 c (glycated haemoglobin) concentration in a blood sample. It will be appreciated that a device of a same or similar structure can implement a variety of assays of blood components and markers.

The device 300 comprises structural units A-E indicated by dashed outlines in Figure 3 implementing five corresponding functional blocks: (A) lysing a blood sample from sample inlet 102 and metering an aliquot of lysed blood; (B) metering two aliquots of buffer liquid comprising a suspension of latex beads from the liquid storage arrangement 106;(C) diluting the aliquot from block (A) with one of the aliquots of block (B); (D) diluting the dilution from block (C) with the other one of the aliquots of block (B); and (E) resuspending a dried antibody specific to HbA1 c to promote an agglutination reaction that is a function of antibody HbA1 c binding, so that a measure HbA1 c concentration can be detected based on a measurement of the turbidity of the liquid from block (E), measured in the detection chambers 312a, b. When configured as an assay device (i.e. a non-quality control device without an analyte deposit) to perform the HbA1 c blood test, the device 300 contains three main reagents: a liquid comprising a suspension of unfunctionalised latex beads provided in the liquid storage arrangement 106 (for example, a blister pack); a dry deposit of a hemolysing agent 314 in block (A) and a dry deposit 318 of a reagent specific to the analyte, specifically a mixture of antibodies, in block (E). To conduct the regular test, a user applies pressure to the liquid storage arrangement 106 to dispense the liquid into the liquid receiving chamber 316 of the device 300. A blood sample of about 5 microlitres to be investigated with the assay is introduced into the device 300 through the inlet port 102 and the device sealed with an adhesive flap. The blood sample may be lysed when it comes into contact with hemolysing agent on re-suspension of the deposit of the hemolysing agent 314. In some embodiments, the user may choose to insert a lysed blood sample irrespective of the presence of a lysing agent in the device or, in some embodiments, the lysing agent may be omitted.

The device is introduced into the reader and a rotational protocol is executed to carry out the fluidic operations required to conduct the HbA1 c assay. Specifically, the following steps are carried out:

Step #1 : Metering an aliquot of the lysed blood sample on structural block [A] and metering two separate aliquots of the liquid from the liquid storage arrangement in structural block [B].

Step #2: Performing a first dilution (e.g. 1 :50) of the sample aliquot with one aliquot of the liquid in structural block [C], actively promoting mixing by moving the liquids back and forth between the vented chamber 202 and the unvented chamber 204 using at least one acceleration and deceleration cycle.

Step #3: Metering a smaller aliquot of the diluted sample and further diluting it with the second aliquot of the liquid to reach the sample dilution required for the assay (e.g. 1 :2500). This step is executed in structural block [D].

Step #4: Re-suspending the dry deposit 318 of the reagent specific to the analyte with the final dilution of the sample to promote the latex agglutination reaction. This step is executed in structural block [E].

Step #5: Routing the reaction mixture to detection chambers 312a and 312b.

The assay reaction is then detected, for example followed in real-time using suitable detection means, as is well known in the art.

When configured as an integrated quality control device, the device 300 contains the dry deposit of an analyte 216 downstream from the sample inlet port 102, for example in the unvented chamber 204 in block C. In such case, the deposit of the hemolysing reagent 314 may not be required if, for example, the analyte is provided in the deposit readily available (i.e. not requiring any lysing step) and would need to be included in the flow path from the liquid storage arrangement 106 rather than in block A as in the assay device otherwise The device is otherwise substantially identical as described above.

A user applies pressure to the liquid storage arrangement 106 to dispense the liquid into liquid receiving chamber 316 of the device 300. The device is then introduced into the reader and the same rotational protocol is executed. The only differences in operation are: in Step #1 no sample metering is required (although the corresponding rotation step may be included for simplicity or consistency) and, in Step #2, the dry analyte deposit 216 is re- suspended in the first aliquot of liquid yielding a known dilution similar to that obtained when the device is used to run a sample of the same concentration, using the acceleration and decceleration described above to promote mixing and resuspension. In some embodiments resuspension may instead occur in the second dilution in step #3. It will thus be appreciated that the position of the analyte deposit 216 may be varied. For example, the deposit 216 may be disposed in block D in a chamber corresponding to unvented chamber 204 in block C. Thus, the rotational protocol may be identical or substantially the same for the assay and quality control runs of the respective devices.

An exemplary embodiment of a reader 400 is now described with reference to Figure 4. The reader 400 comprises a drive 402 under control of a controller 404 for controlling a rotation profile of a device 100 in the drive and having a spindle 406 for engaging the feature 1 14 as the device is loaded into the reader, for example in the manner of CD or DVD disk loading tray. A light source 408 is configured to shine light onto or through the detection chamber 1 12 of the device 100 when loaded into the reader and a sensor 410 is configured to receive light from the detection chamber 1 12 as described above. A processor 412 is connected to the sensor 410 to process a signal indicative of the received light to carry out the assays or perform quality control or calibration.

A method of quality control or calibration implemented by the processor 412 and controller 404 (or the processor 412 controlling the other components of the reader 400) is now described with reference to Figure 5. At a first step 502, a device 100 is loaded. At step 504, the drive is controlled to implement a rotation protocol to implement the assay with the device and at step 506 a signal is read and processed to obtain an assay result, for example a concentration of analyte. At step 508 the reader checks whether the device is a quality control device, for example by detecting a machine-readable indicium on the device 100 or by way of a user input, for example entered prior to inserting the device and stored. If it is not, the reader reports the result of the assay via a user interface (not shown) at step 510. If it is, the reader, at step 512, compares the obtained assay result with an expected result to perform quality control or calibration at step 514. The expected result may be stored in the reader, for example in firm ware, may be entered by a user when providing an input indicating a quality control device, or may read by the reader from the or another machine- readable indicium. Quality control may comprise determining if the obtained result is within an acceptable margin of the expected result and calibration may comprise adjusting a parameter of the reader, for example calibration data used by the reader to obtain the assay result, so that the obtained result matches the expected result.

In some embodiments, whether configured in a disc-shape or otherwise, the device is manufactured by forming the liquid handling structures (channels, conduits, etc.) in a substrate, for example by injection moulding or stamping the substrate. The substrate is then sealed by bonding a polymer film to the surface in which the liquid handling structures are defined, with appropriate cut-outs for fluidic access to the liquid handling device. In other embodiments, the device may be formed by bonding together two substrates, which may both define respective liquid handling structures, for example, in cooperation, or by a sandwich of a bonding film between two substrates.

The deposit of analyte may be applied to the device by first applying drops of solution or suspension containing the reagent(s) to the relevant substrate, in the region which, once the substrate is bonded with its counterpart (either the polymer film or another substrate), will form the appropriate structure, for example the unvented chamber 204. The drops of solution or suspension are then allowed to dry, thus leaving behind the dry reagent(s) on the substrate. Alternatively, a solution containing the one or more reagents may be applied to a body of absorbent material, which is then allowed to dry, leaving behind dry reagent(s) on the material. The material can then be disposed on the substrate, in the region which will form the appropriate structure, prior to or after the substrate has been bonded with its counterpart.

The above description has been made in terms of specific embodiments for the purpose of illustration and not limitation. Many modifications and combinations of, and alternatives to, the features described above will be apparent to a person skilled in the art and are intended to fall within the scope of the invention, which is defined by the claims that follow.

For example, while conduits have been described above with reference to drawings depicting channel shaped conduits, it will be understood that the term "conduit" covers any arrangement providing a flow path conveying or conducting liquid from one part of the device to another. Accordingly, a conduit with a bend or crest, for example as described above for the flow control device 212 can, for example, be implemented as a bent channel as depicted schematically in the drawings, or more generally as any structure that can contain liquid, has an inlet, and an outlet and is shaped or configured so that liquid flowing from the inlet to the outlet first flows radially outward (or, respectively, inward) to an inflection point and then flows radially inward (or, respectively, outward). The conduits (and other flow paths) described herein in various embodiments are thus defined by their function and a shape or configuration necessary to achieve that function, rather than being limited to any specific shape or configuration beyond that which is necessary to achieve the respective described functions. Likewise, while chambers have been described above with reference to drawings depicting chambers of a certain form factor, it will be appreciated that the disclosure is not so limited and that the described chambers may take any suitable shape or configuration, for example have varying depth, be significantly elongate to resemble a channel, for example a serpentine or meandering channel, be formed by a network of channels or cavities, contain pillars, comprise interconnected volumes, etc.

Embodiments have been described with reference to microfluidic devices, specifically centrifugal microfluidic devices, but it will be apparent that the concept of integrating a quality control sample in an assay device for later reconstitution is not limited to any particular device or flow mechanism but is, for example, equally applicable to embodiments with other flow mechanisms, for example pressure, gravity, electrophoresis, electro osmosis, chromatographic and so on driven flow. It will be appreciated that the disclosure extends to all such embodiments and is also not limited to any specific way of manufacturing the devices or any specific form factor. Likewise, the disclosure is not limited to the specific assays, analytes and reagents described here but extends to all assays that can benefit from an arrangement that enables reconstitution of an integrated quality control sample, be that for the purpose of quality control or calibration.