An assay device and method for performing an assay

Abstract

Disclosed is an assay device and methods for its use. The assay device comprises an analysis chamber containing one or more beads configured to move freely in the analysis chamber. The relative sizes of the bead(s) and the analysis chamber are defined by either of: a ratio of a cross section of the analysis chamber to the bead(s) of between about 3 and 25; or a ratio of a volume of the analysis chamber to a total volume of the bead(s) of between about 5 and 100. The assay device may be positioned on a rotatable disc. The method of use comprises exerting rotational forces on a fluid in contact with the bead in the analysis chamber by modulating an angular acceleration of the analysis chamber and measuring a characteristic of the fluid in the analysis chamber.

Description

[0001] AN ASSAY DEVICE AND METHOD FOR PERFORMING AN ASSAY

[0002] FIELD OF THE INVENTION

[0003]

[0001] This invention relates to an assay device and a method for performing an assay, in particular a microfluidic assay device for use in a centrifugal assay assembly, and methods related thereto.

[0004] BACKGROUND

[0005]

[0002] Rapid mixing and precise timing are critical for biomedical assays that rely on measurement of reaction kinetics. These considerations come to the fore particularly for point-of-care (POC) systems. Microfluidic techniques offer the possibility to bring the timing under control, by accurately knowing when the reaction begins and when the measurement is taken. One issue is achieving rapid and reliable mixing, of analyte fluids with diluents and reagents, in order to define the start time and ensure homogeneity throughout the reaction volume. Where reactions take place at a surface, rapid mass transport to the reaction surface is also required, in order to ensure that the system is surface reaction-rate controlled and uniform over the reaction surface area. Many methods have been proposed and extensively reviewed. Amongst the microfluidic methods, centrifugal microfluidics offers the substantial advantage of simple and precise control of the fluid flow, for timing accurately the beginning and end of the incubation steps, and of an extensive toolbox of structures that can be employed on the centrifugal disc for implementation of a range of necessary unit operations that can conveniently be strung together to give the required process flow. However, the small channel dimensions in microfluidic devices mean that flow is generally laminar. Mixing is then by diffusion only and so can be slow.

[0006]

[0003] There is a desire for an easily manufactured system, assembly and / or device that achieves rapid, uniform mixing and mass transfer for fast and accurate POC based on a small sample volume of an enzyme or substrate concentration, or of an antigen or antibody.

[0007]

[0004] It is an object of at least preferred embodiments of the present invention to address one or more of the above-mentioned disadvantages and / or to at least provide the public with a useful alternative.

[0008]

[0005] In this specification where reference has been made to patent specifications, other external documents, or other sources of information, this is generally to provide a context for discussing features of the invention. Unless specifically stated otherwise, reference to such external documents or sources of information is not to be construed as an admission that such documents or such sources of information, in any jurisdiction, are prior art or form part of the common general knowledge in the art.

[0009] SUMMARY OF THE INVENTION

[0010]

[0006] In one aspect, there is provided an assay device, comprising: an analysis chamber configured to receive fluid; the analysis chamber containing a bead configured to move freely in the analysis chamber. In some embodiments, the assay device may be a microfluidic device.

[0011]

[0007] In another aspect, there is provided an assay device configured for use with a centrifugal system exerting centrifugal force on the assay device, comprising: an analysis chamber configured to receive fluid; the analysis chamber containing one or more beads configured to move freely in the analysis chamber; wherein: a ratio of a cross-sectional area of the analysis chamber to a total cross sectional area of the one or more beads is between about 3 and 25, or between 3 and 10, wherein the total cross sectional area of the analysis chamber is determined by the two largest dimensions of the analysis chamber; or a ratio of a volume of the analysis chamber to a total volume of the one or more beads is between about 5 and 100, or between 5 and 50, or between 5 and 25.

[0012]

[0008] In some embodiments, the bead may have a density that is slightly greater than a density of the fluid. For example, the bead may have a density that is between about 2% and about 10% more than a density of the fluid. In alternative embodiments, the bead may have a density that is slightly lower than a density of the fluid. For example, the bead may have a density that is between about 2% and about 10% lower than a density of the fluid. In some embodiments, the bead has a density that is within about 10% of a density of the fluid.

[0013]

[0009] In some embodiments, the bead may have a diameter of between 100 and 2500 microns, and the analysis chamber may have a major internal dimension of between 1 and 10 mm, or between 1 and 8 mm, or between 1 and 6 mm. In some embodiments, the bead may have a diameter of between about 1 mm and about 2 mm.

[0014]

[0010] The bead may be configured to rotate and translate in the analysis chamber when the analysis chamber is subjected to a change in rotational acceleration. The bead may be configured to move through a middle portion of the analysis chamber when the analysis chamber is subjected to a change in rotational acceleration.

[0015] [Oil] In some embodiments, the bead may comprise a surface-bound capture reagent configured to interact with a target in the fluid. The surface-bound capture reagent may be uniformly distributed on the surface of the bead. The bead may have a predetermined level of surface-bound capture reagent on its surface.

[0016]

[0012] In some embodiments, a ratio of a major internal dimension of the analysis chamber to a diameter of one or more of the beads is between about 2 and 5, between about 2 and 4, between about 2 and 3, or between about 2 and 2.5.

[0017]

[0013] In some embodiments, a ratio of a cross-sectional area of the analysis chamber to a total cross-sectional area of the one or more beads is between about 3 and 25, between about 3 and 20, between about 3 and 15, or between about 3 and 10, wherein the cross- sectional area of the analysis chamber is determined by the two largest dimensions of the analysis chamber. For example, when the assay device is on a rotatable disc, the cross- sectional area is parallel with the rotatable disc.

[0018]

[0014] In some embodiments, a ratio of a volume of the analysis chamber to a total volume of the one or more beads is between about 5 and 100, or between 5 and 50, or between 5 and 25.

[0019]

[0015] In some embodiments, the analysis chamber may comprise an elongate shape. The elongate shape may comprise a major internal dimension aligned with a direction of centrifugal force in use. For example, when the assay device is on a rotatable disc, the major internal dimension may be oriented with the direction of centrifugal force or (the opposing direction) centripetal force

[0020]

[0016] In some embodiments, the analysis chamber comprises side walls which taper inwardly towards an inner end of the analysis chamber proximate the axis of rotation from an opposing outer end of the analysis chamber.

[0021]

[0017] In some embodiments, the cross-sectional area of the analysis chamber has an aspect ratio of between about 1 and 4.

[0022]

[0018] In some embodiments, the analysis chamber comprises a trapezoidal type of shape, a triangular-type shape or an hourglass-type shape.

[0023]

[0019] In some embodiments, a side wall of the analysis chamber has one or more curved corners, wherein the radius of curvature of the one or more curved corners is at least 1 mm. In some embodiments, a minimum radius of curvature in the analysis chamber side wall(s) is at least 1 mm.

[0020] The analysis chamber may comprise an inlet port for receiving the fluid. The analysis chamber may comprise a window configured to allow optical analysis of the fluid in the analysis chamber.

[0024]

[0021] The assay device may comprise an inlet chamber for receiving the fluid. The assay device may comprise a microfluidic channel providing fluid communication between the inlet chamber and the analysis chamber. In some embodiments, substantially all of the internal surfaces of the analysis chamber, microfluidic channel and inlet chamber, may comprise a hydrophobic coating. In some embodiments, substantially all of the internal surfaces of the assay device comprise a hydrophobic coating.

[0025]

[0022] The assay device may comprise a valve at or between the inlet chamber and the inlet port of the analysis chamber for controlling the flow of the fluid into the analysis chamber. The valve may comprise a capillary stop. The capillary stop may be positioned in the microfluidic channel.

[0026]

[0023] The inlet chamber, microfluidic channel and analysis chamber may lie along a single plane.

[0027]

[0024] The analysis chamber may contain a single bead. Alternatively, the analysis chamber may contain six or fewer beads, for example two or three beads.

[0028]

[0025] In another aspect, there is provided a centrifugal assay assembly comprising one or more assay device(s) as described herein provided on a rotatable disc. The analysis chamber of each of the assay devices may be located at the same distance from the axis of rotation of the disc.

[0029]

[0026] In some embodiments, the rotatable disc may define, in the analysis chamber, a top wall, a bottom wall, one or more side walls, an inner end proximate an axis of rotation, an outer end opposite the inner end, one or more inlet ports positioned at the inner end for receiving fluid from an inlet chamber, and one or more waste ports at the outer end for fluid egress to a waste chamber.

[0030]

[0027] In some embodiments, substantially all of the internal surfaces of the rotatable disc comprise a hydrophobic coating.

[0031]

[0028] Each analysis chamber may be defined by a top wall, a bottom wall, one or more side walls, an inner end proximate to an axis of rotation and an outer end opposite the inner end. One or more beads is contained within the analysis chamber. Each analysis chamber may further comprise one or more inlet ports for receiving fluid, and one or more waste ports for fluid egress. The beads may be permanently contained in the analysis chamber, such that they cannot fit through the inlet ports or the waste ports.

[0029] In another aspect, there is provided a microfluidic assay device comprising an inlet chamber and an analysis chamber in fluid communication via a channel, wherein substantially all of the internal surfaces of the inlet chamber, the analysis chamber, and the channel comprise a hydrophobic coating.

[0032]

[0030] In some embodiments, substantially all of the internal surfaces of the assay device comprise a hydrophobic coating.

[0033]

[0031] In another aspect, there is provided a method for performing an assay on a fluid comprising a target, the method comprising providing an assay device as described herein, wherein at least one of the one or more beads comprise a surface-bound capture reagent configured to bind with the target, the method comprising the steps of: introducing the fluid into the analysis chamber; exerting rotational forces on the fluid and the bead by modulating an angular acceleration of the analysis chamber; and measuring a characteristic of the fluid in the analysis chamber.

[0034]

[0032] In some embodiments, the rotational forces may be oscillating rotational forces, the oscillating rotational forces comprising an oscillating period of 1 to 3 times per second.

[0035]

[0033] In some embodiments, modulating the angular acceleration of the analysis chamber may comprise a product of T. number. (dQ / dt) that is 2000 rpm.mm or greater, 2500 rpm.mm or greater, 3000 rpm.mm or greater, 3500 rpm.mm or greater, 4000 rpm.mm or greater, or 4500 rpm.mm or greater, wherein T is the oscillating period in s, rchamber is the radius of the analysis chamber in mm, and dQ / dt is the angular acceleration of the analysis chamber in rpm.s-1.

[0036]

[0034] For non-circular analysis chambers, the variable rchamber may be determined as (r ajor + rminor) / 2, where r ajor is half the length of the major internal dimension and r inor is half the length of the minor internal dimension perpendicular to the major dimension.

[0037]

[0035] Alternatively, rchamber may be determined as two separate variables, Tmajor and rminor. Modulating the angular acceleration of the analysis chamber in this embodiment may comprise two products - T. rminor. (dQ / dt) and T. rmajor. (dQ / dt) - wherein each product is 2000 rpm.mm or greater, 2500 rpm.mm or greater, 3000 rpm.mm or greater, 3500 rpm.mm or greater, 4000 rpm.mm or greater, or 4500 rpm.mm or greater, wherein T is the oscillating period in S, Tchamber iS the radius of the analysis chamber in mm, and dQ / dt is the angular acceleration of the analysis chamber in rpm.s-1.

[0038]

[0036] The method may comprise a quantitative determination of the target in the fluid.

[0037] Measuring the characteristic may be performed at one or more known times after the introduction of the fluid. The known time may be a predetermined time after the introduction of the fluid.

[0039]

[0038] The method may comprise measuring a rate of change of the measured characteristic.

[0040]

[0039] The method may comprise calculating or inferring a reaction rate from the rate of change of the measured characteristic. The characteristic may be measured at a constant angular velocity or a constant angular acceleration. The method may comprise measuring the characteristic at an area of the analysis chamber that is unimpeded by the bead. The measured characteristic may comprise one or more of: light colour, light transmission, light reflectance, light emission, light fluorescence, light scattering and / or light absorption. Measuring the characteristic may comprise taking an optical measurement of the sample, or recording an image of the fluid or of the bead. The optical measurement may comprise a measurement of light absorption, light transmission or fluorescence. The optical measurement may comprise measuring a colorimetric signal.

[0041]

[0040] Modulating the angular acceleration may comprise: inducing translational and rotational movement of the bead; inducing abrupt motion of the bead; and / or modulating the angular velocity of the assay device. Modulating the angular acceleration of the analysis chamber may comprise periodically or intermittently changing a direction of the rotational velocity of the assay device.

[0042]

[0041] In another aspect, there is provided a method of mixing two or more fluids in a microfluidic chamber, the method comprising contacting a first fluid and a second fluid in a microfluidic chamber comprising a bead having a density substantially equivalent to the first or second fluids, and modulating the angular acceleration of the microfluidic chamber.

[0043]

[0042] In another aspect, there is provided a method of mixing two or more fluids in a microfluidic chamber, the method comprising: contacting a first fluid and a second fluid in a microfluidic chamber comprising one or more beads configured to move freely in the analysis chamber, and modulating the angular acceleration of the microfluidic chamber; wherein: a ratio of a cross sectional area of the analysis chamber to a total cross sectional area of the one or more beads is between about 3 and 25, or between 3 and 10, wherein the total cross sectional area of the analysis chamber is determined by the two largest dimensions of the analysis chamber; or a ratio of a volume of the analysis chamber to a total volume of the one or more beads is between about 5 and 100, or between 5 and 50, or between 5 and 25.

[0044]

[0043] This invention may also be said broadly to consist in the parts, elements and features referred to or indicated in the specification of the application, individually or collectively, and any or all combinations of any two or more said parts, elements or features. Where specific integers are mentioned herein which have known equivalents in the art to which this invention relates, such known equivalents are deemed to be incorporated herein as if individually described.

[0045]

[0044] The term 'comprising' as used in this specification and claims means 'consisting at least in part of'. When interpreting statements in this specification and claims that include the term 'comprising', other features besides those prefaced by this term can also be present. Related terms such as 'comprise' and 'comprised' are to be interpreted in a similar manner.

[0046]

[0045] It is intended that reference to a range of numbers disclosed herein (for example, 1 to 10) also incorporates reference to all rational numbers within that range and any range of rational numbers within that range (for example, 1 to 6, 1.5 to 5.5 and 3.1 to 10). Therefore, all sub-ranges of all ranges expressly disclosed herein are hereby expressly disclosed.

[0047]

[0046] As used herein the term '(s)' following a noun means the plural and / or singular form of that noun. As used herein the term 'and / or' means 'and' or 'or', or where the context allows, both.

[0048] BRIEF DESCRIPTION OF THE DRAWINGS

[0049]

[0047] The present invention will now be described by way of example only and with reference to the accompanying drawings in which:

[0050]

[0048] Figure 1 is a plan view of an assay device in accordance with an embodiment of the present disclosure.

[0051]

[0049] Figure 2 is a section view of the device of Figure 1, taken through line A-A.

[0052]

[0050] Figures 3A to 3C are plan views of an assay device in accordance with an embodiment of the present disclosure.

[0053]

[0051] Figure 4 is a plan view of an assay device in accordance with an embodiment of the present disclosure.

[0054]

[0052] Figures 5A to 5D are plan views of an assay device in accordance with an embodiment of the present disclosure.

[0053] Figure 6 is a plan view of an analysis chamber in accordance with an embodiment of the present disclosure

[0055]

[0054] Figures 7A to 7C are plan views of an assay device in accordance with an embodiment of the present disclosure.

[0056]

[0055] Figure 8 is a plan view of a disc with a plurality of assay devices arranged peripherally around the disc, for use in a centrifugal system, in accordance with an embodiment of the present disclosure.

[0057]

[0056] Figure 9 is a perspective view of a system in accordance with an embodiment of the present disclosure.

[0058]

[0057] Figures 10A to 10G illustrate a method of operation of the device of Figure 3, where Figure 10A shows a step of introducing fluid into the inlet chamber; Figure 10B shows a step of rotating the device to deliver the fluid to the entrance of channel; Figure IOC shows the progress of fluid down the channel towards the analysis chamber; Figure 10D shows fluid fully transferred to the analysis chamber and in contact with the bead for mixing; Figure 10E shows the progress of fluid from the analysis chamber towards a waste chamber; Figure 10F shows the analysis chamber being emptied; and Figure 10G shows the analysis chamber being fully emptied and the waste chamber containing the fluid.

[0059]

[0058] Figure 11 is a chart of rotational velocity and rotational acceleration of the disc shown in Figure 8 over time, according to an embodiment of the method of the present invention. The solid line relates to the disc's rotational velocity (Q / rpm) and the dashed line relates to the disc's angular acceleration (rpm I s).

[0060]

[0059] Figure 12 is a chart of rotational velocity of the disc shown in Figure 8 over time, according to an embodiment of the method of the present invention.

[0061]

[0060] Figure 13 is a chart showing passing mixing trials for assay devices comprising various rchamber / rbead values and Trchamber(d disc / dt) values.

[0062]

[0061] Figure 14 is a chart of the rotational velocity of the disc during the method described in Example 3. Inset is a zoom-in of a mixing cycle shown in the chart.

[0063]

[0062] Figure 15 is a chart of optical transmission scanning across the analysis chamber during the method described in Example 3, showing development of solution uniformity with increasing number of cycles of disc angular velocity.

[0064] DETAILED DESCRIPTION OF A PREFERRED EMBODIMENT

[0065]

[0063] While the present invention may be embodied in many different forms, for the purpose of promoting an understanding of the principles of the present invention, reference will now be made to the figures, which show exemplary embodiments of the invention.

[0066]

[0064] Figures 1-7 show several exemplary embodiments of an assay device comprising an analysis chamber for receiving fluid, the analysis chamber containing one or more beads configured to move freely within the analysis chamber.

[0067]

[0065] With reference to Figures 1-4, the device 1, 101 is configured for use in a centrifuge device or other system configured to apply rotational forces to the device. Figure 8 shows one example in which the device 1, 101 is arranged on a disc 201 that is rotatable to exert rotational forces on the device 1, 101.

[0068]

[0066] A fluid received by the device may comprise a target to be analysed. The fluids may comprise a target, which may comprise biological fluids (such as a whole blood sample), suspensions of biological material, processed biological material, fractions or dilutions thereof. In an example, the fluid may comprise a whole blood sample, plasma or serum. The fluid may further comprise one or more of assay reagents, washing fluids and rinsing fluids. The fluids may be aqueous fluids or aqueous suspensions.

[0069]

[0067] The small channel dimensions in microfluidic devices mean that flow in microfluidic scales is generally laminar. Mixing is then by diffusion only, and so can be slow. Stretching, splitting and / or folding of the flow improves mixing at microfluidic scales. This may be achieved by the present disclosure which comprises a method of mixing two or more fluids in a microfluidic chamber, or of achieving compositional uniformity in a fluid where a substance is being generated or consumed by reaction at a surface. The method may comprise contacting a first fluid and a second fluid, or a fluid and a reactive surface in a microfluidic chamber comprising a bead having a density substantially equivalent to the first or second fluids, and modulating the angular acceleration of the microfluidic chamber. The reactive surface may be the surface of the bead or all or part of the surface of the chamber.

[0070]

[0068] With reference to Figures 1 to 4, the device 1, 101 may comprise one or more inlet chambers 7, 107, to receive the fluid from a respective inlet port 5. The inlet chamber 7, 107 is configured to hold a volume of fluid therein prior to addition to the analysis chamber 3, 103. The inlet chamber 7, 107 may be sized to hold a predetermined volume of fluid. For example, the capacity of the inlet chamber 7, 107 may correspond to a volume of fluid required or desired in the analysis chamber for the assay.

[0071]

[0069] The device 1, 101 may comprise one or more inlet ports 5 to receive the fluid and facilitate loading of the fluid into the device. In one embodiment, the inlet port 5 is located on a wall of the inlet chamber 7, 107. Some embodiments may include a plurality of inlet ports 5. Where there are a plurality of inlet chambers, one or more of the plurality may comprise an inlet port.

[0072]

[0070] The inlet port 5 and inlet chamber 7, 107 are in fluid communication with the analysis chamber 3, 103. A channel 9, 109 may provide the fluid communication between the inlet chamber 7, 107 and the analysis chamber 3, 103, such that fluid may be delivered to the analysis chamber 3, 103. Channel 9, 109 is a microfluidic channel that promotes laminar flow therethrough. Preferably, the channel 9, 109 is a linear channel. The channel 9, 109 may have any suitable cross-section, for example it may have a round cross-section such that it is substantially cylindrical. Alternatively, the cross-section may be rectangular with or without rounded corners.

[0073]

[0071] The device 1, 101 may include one or more valves 12, 112 in fluid communication with the inlet chamber and the analysis chamber. Valve 12, 112 may be located between inlet port 5 and analysis chamber 3, 103. The, or each, valve 12, 112 is configured to control flow between the inlet chamber 7, 107 and the analysis chamber 3, 103. The valve 12, 112 may have two or more states, for example, a valve may have two states being a flow state and a non-flow state.

[0074]

[0072] The channel 9, 109, or a portion thereof, may comprise or define the microfluidic valve. For example, an inner surface of channel 9, 109 may comprise a hydrophobic material or coating selected to decrease the wettability at the valve. The valve 12, 112 preferably comprises a passive microfluidic valve. One example of a passive microfluidic valve is a capillary stop. Valve 12, 112 may be located at a terminal end of the channel 9, 109 or located along the channel 9, 109. For example, valve 12, 112 may be provided at or adjacent a first end of the channel 9, 109, a second end of the channel 9, 109, a midportion of the channel, or substantially the whole length of the channel 9, 109 may form the valve 12, 112.

[0075]

[0073] In the present embodiments the channel 9, 109 or a portion thereof is sized and shaped to create a capillary stop when the device 1, 101 is stationary and such that, at a given rotational speed, resulting rotational forces acting on the device 1, 101 and fluid in the inlet 7, 107 will create sufficient pressure to overcome a resistive force of the capillary stop and drive the fluid through the channel 9, 109 towards the analysis chamber 3, 103. The dimensions of the channel 9, 109 are selected depending on the properties of the fluid, the anticipated environmental conditions, and the speed of rotation at which it is desired for the capillary valve to switch between the flow state and the non-flow state. These parameters could be readily calculated by a person skilled in the art.

[0074] Analysis chamber 3, 103 inlet chamber 7, 107 and channel 9, 109 may be defined by a substantially flat bottom wall, a substantially flat top wall, and one or more side walls. The chamber may comprise an inner end proximate an axis of rotation and an outer end opposite the inner end. Each analysis chamber may further comprise one or more inlet ports for receiving fluid, and one or more waste ports for fluid egress. The beads may be permanently contained in the analysis chamber, such that they cannot fit through the inlet ports or the waste ports.

[0076]

[0075] Analysis chamber 3, 103 comprises a mixing area and is configured to house one or more beads 15. The analysis chamber comprises one or more entry inlet 4, 104 in fluid communication with the one or more inlets 5 and configured to receive fluid from one or more inlet chambers 7, 107 via channel(s) 9, 109.

[0077]

[0076] Analysis chamber 3, 103 has a shape that, in conjunction with one or more beads, facilitates movement of fluid in the analysis chamber 3, 103 about the bead(s) to induce rapid and uniform mixing of fluids and / or contact of fluids with the surface of a bead 15 while minimising or eliminating any 'dead' regions of stagnant fluid within the analysis chamber 3, 103. In the devices shown in Figures 1-4, the analysis chamber 3, 103 comprises a circular cross-sectional shape. However, the analysis chamber 3, 103 may have a regular or irregular cross-sectional shape. For example, the analysis chamber cross-section may be generally circular or elliptical, or another suitable shape. In some embodiments, the analysis chamber 3, 103 may comprise one or more wavy or undulating side walls. The analysis chamber 3, 103 cross-section may have one or more axes of symmetry or may be an asymmetrical shape.

[0078]

[0077] References herein to cross sectional shapes or two-dimensional shapes of the analysis chamber refer to the chamber's dimensions observed in plan view, which may be the two largest dimensions of the analysis chamber. When positioned on a rotatable disc, the shapes of the analysis chamber described herein relate to the two dimensions aligned with the plane of the disc.

[0079]

[0078] The analysis chamber 3, 103 may comprise one or more principal internal dimensions measured in the plane of rotation of the device. The principal dimensions are indicative of the overall size of the analysis chamber 3, 103. For example, where the analysis chamber 3, 103 has a cylindrical shape, the principal dimension comprises the diameter of the analysis chamber 3, 103.

[0080]

[0079] In an embodiment where the shape of the analysis chamber 3, 103 is non-circular, such as elliptical or irregular, the principal dimensions may comprise the major internal dimension (for example, along a major axis) and a minor internal dimension (for example, along a minor axis). The minor internal dimension may be perpendicular to the major internal dimension.

[0081]

[0080] The major dimension and the minor dimension may be defined as average dimensions.

[0082]

[0081] An internal dimension of the analysis chamber may be between 1 and 10 mm. Where the analysis chamber is circular, this converts to a chamber radius of between about 0.5 and 5 mm.

[0083]

[0082] With reference to Figure 2, the depth D of the analysis chamber is perpendicular with the plane of the rotatable disc, and may be defined by straight side walls in the depth dimension. The side walls may be defined by sides of a middle sheet 16. The side walls may be substantially perpendicular to the top and bottom walls of the analysis chamber, where the top and bottom walls are defined by lower and upper sides of a top layer and a bottom layer, respectively.

[0084]

[0083] The depth D of the analysis chamber 3, 103 is selected to facilitate movement of fluid in the chamber about the bead(s) in a direction that is out of the plane of rotation of the device. Preferably, the analysis chamber 3, 103 has a minimum depth greater than 1.1 times the diameter of the bead for allowing movement of the bead within the analysis chamber 3, 103. The analysis chamber 3, 103 may have a maximum depth less than about 2 times the diameter of the bead, such that the mixing effects of the bead are maximised. The chamber depth may be defined by the distance between bottom sheet 11 and top sheet 21. In some examples, analysis chamber 3, 103 may have an average depth of between 1 and 3 mm. Analysis chamber 3, 103, may have a constant depth within the chamber.

[0085]

[0084] The analysis chamber may have a circular or non-circular shape. Non-circular shapes of the analysis chamber may include trapezoidal type shapes, triangular type shapes or hourglass type shapes.

[0086]

[0085] Analysis chambers having non-circular shapes may be configured for use such that the major internal dimension is oriented with the direction of centrifugal force or (the opposing direction) centripetal force, in use, and a minor internal dimension oriented orthogonally with the major internal dimension and the depth.

[0087]

[0086] Such analysis chambers comprise an inner end proximate the centre of rotation (in use) and an outer end distal from the centre of rotation. The outer end may be at or proximate to an outer perimeter of a disc.

[0087] One or more channels 9, 109, opens to the analysis chamber at the inner end, and conduit 129 opens to the analysis chamber at the outer end.

[0088]

[0088] Preferably the internal walls of the analysis chamber 3, 103 form a smooth internal surface that is free from sharp corners, side chambers, or small concavities. These may cause fluid to become trapped and stagnant.

[0089]

[0089] The corners of the analysis chamber (as defined by the major and minor dimensions described above) may be rounded. Without wishing to be bound by theory, it is believed that rounding of the corners improves the motion of the bead within the analysis chamber and therefore improves the mixing of the bead and fluid. If the side walls are too sharply curved, the beads may be pressed up against the wall in use. A larger radius of curvature of the wall of the chamber at its outer radius with respect to the centre of rotation means the bead can move further in response to the fluid motion. Therefore, where the analysis chamber comprises corners (e.g., where the analysis chamber comprises a trapezoidal type shape or a triangular type shape), the corners may be substantially rounded. For example, corners of the shape of the analysis chamber may comprise a radius of curvature of at least 1 mm. For example, analysis chamber 603 comprises corners at an inner end 605 and outer end 606 have a radius of curvature of about 1.5 mm.

[0090]

[0090] Corners of the analysis chamber defined by side walls with the top wall or bottom wall (i.e., corners in the analysis chamber comprising a depth dimension) may be rounded or substantially right angled.

[0091]

[0091] Where the analysis chamber is non-circular, the major internal dimension may be between about 1 and 10 mm. The minor internal dimension may also be between about 1 and 10 mm.

[0092]

[0092] Circular analysis chambers such as analysis chambers 3, 103, have a substantially constant radius and therefore an aspect ratio of about 1.

[0093]

[0093] For non-circular analysis chambers, the major internal dimension and the minor internal dimension may be each selected such that the analysis chamber has a desired aspect ratio. The aspect ratio may be the ratio of the average length of the major dimension to the average length of the minor dimension.

[0094]

[0094] The shape of the analysis chamber may be elongate. The analysis chamber may be configured so that its aspect ratio is less than about 4, and at least about 1. In some situations, it may be preferable for the analysis chamber to have an aspect ratio less than 3 and at least about 1. This is because a very narrow analysis chamber has the potential to limit the fluid motion because of the effect of the hydrodynamic boundary layer at the walls of the chamber.

[0095]

[0095] The aspect ratio of the analysis chamber influences the efficiency of mixing. Without wishing to be bound by theory, as the Euler force is proportional to radial distance from the centre of rotation, the radial distance across the fluid determines the gradient of Euler force which in turn drives rotational movement of the fluid and the bead. However, if the chamber is constricted along the circumferential direction, then the mass of fluid that is being accelerated is limited and the momentum change is thus smaller. A very narrow chamber further will limit the fluid motion because of the effect of the hydrodynamic boundary layer at the walls of the chamber.

[0096]

[0096] Figures 5A to 5D show a device 600 comprising an elongate analysis chamber 603 containing between zero and three beads 615, 615a, 615b, 615c. The shape of the analysis chamber may be substantially symmetric along a major dimension. The side walls of the analysis chamber 603 comprise an inner end 605 which may be proximate an axis of rotation (when analysis chamber 603 is positioned on a rotatable disc) and an outer end 606 opposite the inner end 605. Side walls may taper inwardly towards the inner end 605 from the outer end 606. Thus, the width of the analysis chamber at the outer end 606 is wider than the width of the analysis chamber at the inner end 605. Analysis chamber 603 may thus comprise a substantially "avocado" or "pear" shape.

[0097]

[0097] Elements of device 600 other than the analysis chamber 603 may be substantially the same as those described herein for device 1.

[0098]

[0098] Analysis chamber 603 may be arranged with direction R oriented with the direction of centrifugal force (the direction of the arrow), in use. Analysis chamber 603 comprises one or more rounded corners transitioning from inner end and outer end to the side walls. The radius of curvature of the one or more corners at inner end 605, Rmner, and the radius of curvature of the one or more corners of the outer end 606, Router, may be each at least 1 mm. For example, in analysis chamber 603 shown in Figure 6, the radius of curvature at the inner end 605 and outer end 606, Rmner and Router, is 1.5 mm. Analysis chamber 603 may further comprise and substantially straight or gently curving lateral side walls located between the inner end 605 and outer end 606.

[0099]

[0099] Analysis chamber 603 shown in Figure 6 comprises an average major dimension of about 8 mm and an average minor dimension of about 3.5 mm. Chamber depth is about 2.1 mm. Therefore, analysis chamber 603 has an aspect ratio of about 2.3. Cross sectional area is about 30 mm2and volume is about 60 mm3. Whilst analysis chamber 603 is suitable for beads having a range of diameters, an exemplary range of bead diameters for analysis chamber 603 is between 1 and 2 mm.

[0100]

[0100] In another example, Figures 7A to 7C show a device 700 comprising an analysis chamber 703 containing between zero and two beads 715, 715a, 715b. The shape of the analysis chamber 703 may be substantially symmetric along a major dimension. Analysis chamber 703 comprises a substantially peanut or hourglass shape. Analysis chamber 703 comprises an inner section 705, an outer section 706, and a middle section 707 between the inner and outer section, wherein the width of the chamber is narrowed at one or more points of the middle section. Analysis chamber 703 shown in Figures 5A-5C may have an aspect ratio of less than about 4, or less than about 3. The radius of curvature of the corner at inner end 705, Rinner, and the radius of curvature of the corner of the outer end 706, Router, may be each at least 1 mm.

[0101]

[0101] In use with bead 15, modulation of rotational forces causes both the fluid in the analysis chamber and the bead 15 to swirl around the analysis chamber, and contact the whole of the side walls of the analysis chamber.

[0102]

[0102] In an example, the device 101 shown in Figure 4 and disc 201 shown in Figure 8, comprise a plurality of inlet chambers 107 and corresponding channels 109 each in fluid communication with analysis chamber 103 via respective inlet entries 104. It will be appreciated that the devices 101 and disc 201 may be configured with any analysis chambers described herein, including analysis chambers 600 and 700.

[0103]

[0103] Each inlet chamber 107 may be configured to receive and store a different fluid. Fluid communication between each inlet chamber 107 and the analysis chamber 3, 103 may be independently controlled by respective valves 112. Each of the inlet chambers 107 may have the same capacity or they may have different capacities. Where they are different, their different capacities may correspond to the required fluid ratios for the assay. Although Figures 4 and 8 show two inlet chambers, it will be apparent that the device 101 may comprise three, four or more inlet chambers.

[0104]

[0104] The one or more beads 15 are free to move within at least a portion of the analysis chamber 3, 103. The one or more beads are contained within the analysis chamber and their movement is constrained by the analysis chamber side walls.

[0105]

[0105] In some embodiments, the analysis chamber 3, 103 contains a single bead 15, as illustrated in Figures 3A, 4, 5B, and 7B. In an alternative embodiment, the analysis chamber 3, 103 may contain a small number of beads, for example less than six beads, for example two or three beads. For example, Figures 3B, 3C, 5C, 5D, and 7C show an embodiment of device 1 comprising two or three beads.

[0106]

[0106] Each of the one or more beads may be constructed of the same material or alternatively may be constructed of different materials. The beads may have the same size dimensions, or one or more may have different dimensions.

[0107]

[0107] The size and density of the bead(s) 15 is selected to induce rapid and uniform mixing of fluids and / or improve a dynamic contact of the fluid with the surface of a bead in the analysis chamber 3, 103.

[0108]

[0108] The relative size of the bead and analysis chamber is selected to achieve efficient mixing. If the diameter or size of the bead in relation to the analysis chamber is too large, the bead may tend to roll around the lateral internal walls of the analysis chamber in response to reversals in angular acceleration. Beads that are too small are less effective at inducing rapid and uniform mixing.

[0109]

[0109] The relative size of the analysis chamber and the one or more beads may be selected based on one or more of: relative radii (or diameters), relative cross sectional areas, and relative volumes.

[0110] [HO] In an example, the diameter of bead 15 is preferably equal to or less than 50% of a principal internal dimension of the analysis chamber. That is, the ratio of the principal internal dimension of the analysis chamber to the diameter of the bead is preferably equal to or greater than 2. This allows the fluid to flow and rotate around the bead during mixing. This bead size also allows for a sufficient region of fluid to be unimpeded by the bead to facilitate optical measurement of the fluid, for example by permitting a light beam to pass unimpeded through a portion of the chamber for measurement (as described in more detail below). In some embodiments, for example, where the analysis chamber 3, 103 has a circular cross section, the analysis chamber 3, 103 comprises a principal dimension of between about 1 mm and about 6 mm and each bead 15 comprises a diameter between about 100 pm and about 2500 pm. In exemplary embodiments the, or each, bead 15 comprises a diameter between about 1 mm and about 2 mm.

[0111] [Ill] In an example, one or more beads 15 may comprise a diameter that is between about 20% and about 50% of the principal internal dimension of the analysis chamber. That is, the ratio of the principal internal dimension of the analysis chamber to the diameter of one or more beads may be between about 2 and 5. For example, one or more beads may have a diameter between about 40% and about 50% of the principal internal dimension of the analysis chamber or a diameter of 33% or less of the principal internal dimension of the analysis chamber. That is, the ratio of the principal internal dimension of the analysis chamber to the diameter of one or more beads (i.e., equivalent to rChamber / rbead) may be between about 2 and 2.5, or greater than 3.1.

[0112]

[0112] For non-circular analysis chambers, the variable Tchamber may be determined as (rmajor + rminor) / 2, where rmajor is half the length of the major internal dimension and rminor is half the length of the minor internal dimension perpendicular to the major dimension. Alternatively, rchamber may be determined as two separate variables, Tmajor and Tminor.

[0113]

[0113] In an example, the ratio of the cross sectional area of the chamber (Achamber) to the cross sectional area of the one or more beads (Abead i + bead 2 + bead n) - i.e., AChamber / (Abead 1 + bead 2 + bead n) is between about 3 and 100, or between about 3 and 50, or between about 3 and 25.

[0114]

[0114] In an example, the ratio of the volume of the chamber (Vchamber) to the total volume Of the One or more bead(s) (Vbead 1 + bead 2 + bead n)>i.e., Vchamber / (Vbead 1 + bead 2 + bead n)—iS between 1.5 and 200, or between 5 and 150, or between 5 and 100, or between 5 and 50.

[0115]

[0115] The relative density of the bead and the fluid for use in the analysis chamber may be selected to achieve efficient mixing. If the bead density is significantly higher or lower than the density of the fluid, the bead tends to roll around the lateral internal walls of the chamber in response to reversals in angular acceleration, whereas if the bead density is too low, the beads will float and can stick to the meniscus at a periphery of the analysis chamber.

[0116]

[0116] The bead or beads 15 may have a substantially neutral buoyancy in the fluid to be analysed. For example, the bead or beads 15 may have a density that is slightly greater than a density of the fluid to be analysed in the analysis chamber 3, 103. For example, the bead 15 may have a density that is between about 2% and about 10% more than the density of the fluid. Alternatively, the bead or beads 15 may have a density that is slightly lower than a density of the fluid to be analysed in the analysis chamber 3, 103. For example, the bead 15 may have a density that is between about 2% and about 10% lower than the density of the fluid. The bead or beads may therefore have a density that is within about 10% of the density of the fluid.

[0117]

[0117] The bead or beads 15 may comprise any suitable material. Some example materials include polystyrene, polycarbonate, glass or metal (for, example magnetic beads). The bead or beads may comprise a single material, or alternatively may comprise a combination of materials. For example, the bead or beads may comprise two or more layers or zones of different materials. The beads may be solid or alternatively the beads may have one or more hollow portions.

[0118]

[0118] The bead or beads 15 may include at least one capture reagent, which preferably establishes a plurality of target binding sites on the surface of the bead or beads 15. The capture reagent is configured or selected to interact with a target in the fluid. The bead preferably comprises a known or predetermined amount of surface bound capture reagent, and / or the method of operation of the assay comprises a calibration of the beads to allow the determination of the assay results. The capture reagent may comprise one or more of an enzyme, enzyme substrate, antibody or antigen covering at least a portion of the surface of the bead(s). The surface-bound capture reagent is immobilised and uniformly distributed on the surface of the bead. The capture reagent is selected to interact with a target in the fluid received by the analysis chamber 3, 103. Surface-functionalised beads prepared by well-known methods are suitable, and an example of a particular method of preparation is described below in Example 1. The bead 15 preferably comprises a uniform distribution of capture reagent on its surface.

[0119]

[0119] The device 1, 101 comprises a pressure release outlet 13, 113 in fluid communication with analysis chamber 3, 103. This pressure release outlet 13, 113 allows air to vent from the analysis chamber 3, 103 and improve the flow of fluid into the analysis chamber. The pressure release outlet 13, 113 may be connected to the analysis chamber 3, 103 via a channel 19, 119. In Figures 1-4, the channel 19, 119 connects directly to the analysis chamber 3, 103. Preferably, channel 19, 119 is connected to the analysis chamber 3, 103 or channel 9, 109 such that fluids are not driven into the channel 19, 119 or towards the outlet 13, 113 by rotational forces exerted on the device 1, 101 in use.

[0120]

[0120] The device 1, 101 may comprise a waste chamber 121 in fluid communication with the analysis chamber 3, 103 via channel 129 for receiving fluids from the analysis chamber 3, 103, during or at the end of an assay. Waste chamber 121 and channel 129 are preferably connected to the analysis chamber 3, 103 such that fluid in the analysis chamber 3, 103 may be driven to the waste chamber 121 by rotational forces exerted on the device 1 in use.

[0121]

[0121] The device may comprise one or more valves 127 in fluid communication with the analysis chamber 3, 103 and the waste chamber 121 configured to control flow therebetween. Valve 127 may be located between analysis chamber 3, 103 and waste chamber 121. The valve 127 may have two or more states, for example, a flow state and a non-flow state.

[0122] The waste chamber 121 may facilitate washing or rinsing of the interior of the device, for example to enable re-use. The waste chamber 121 may also include a pressure release outlet 123 which can be in fluid communication with the waste chamber via a conduit 125.

[0122]

[0123] The device 1, 101 may be constructed using any suitable construction process. Figure 2 shows a cross section of device 1 and shows a construction comprising stacked layers of three or more sheet components. Alternative construction methods, such as injection moulding, hot embossing, three-dimensional printing, moulding, laser cutting or lithographic techniques would also be suitable and readily apparent to those skilled in the art.

[0123]

[0124] Figure 2 shows the device 1 comprising three layers - bottom sheet 11, top sheet 21, and middle sheet 17, with adjacent sheets bonded together with an adhesive layer 14, 16. The sheets 11, 21, 17 may be bonded with other methods known in the art. The middle sheet 17 is cut or etched to define the lateral dimensions of the components of the device, including the inlet chamber 7, 107, channel 9, 109, and analysis chamber 3, 103. Forming the majority of the components in a single layer improves the efficient construction of the device. Top sheet 21 comprises inlet port 5. Vents such as pressure release outlet 13 may be formed in the top sheet 21 or bottom sheet 11.

[0124]

[0125] The sheets may comprise a thermoplastic, for example an acrylic material like poly(methylmethacrylate), a polycarbonate, a polystyrene or a cyclic polyolefin. In this embodiment, each layer may comprise a thickness of about 2 mm, but in alternative embodiments each layer may be thinner or thicker, or the three layers may have different thicknesses.

[0125]

[0126] The internal surfaces of the device are preferably coated with a hydrophobic material, such as a hydrophobic fluoropolymer. The hydrophobic material may coat substantially all of the internal fluid-contacting surfaces of at least the analysis chamber 3, inlet chamber 7 and channel 9.

[0126]

[0127] At least one wall defining the analysis chamber 3, 103 may include a window to allow optical analysis of the contents of the chamber. In some embodiments, at least a portion of the top wall and / or bottom wall of the analysis chamber 3, 103 may be transparent to an optical measuring instrument. For example, the top sheet 21 and / or bottom sheet 11 may be visually transparent or comprise a visually transparent portion such that colour changes in the fluid in the analysis chamber 3, 103 may be measured during the assay.

[0128] The device 1, 101 may be arranged on an assembly configured for use with a centrifuge system configured to exert a rotational force on the fluid in use. The assembly may be removably engageable with the system.

[0127]

[0129] Figure 8 shows a top view of a centrifugal assembly 201 comprising a plurality of devices 101 arranged on a rotatable disc 203. This arrangement advantageously enables processing of a plurality of samples simultaneously, giving a precise measurement result with short total assay time. Centrifugal assembly 201 may additionally, or alternatively, comprise one or more devices 600 or 700, as shown in Figures 5 and 7.

[0128]

[0130] The construction of the rotatable disc 203 may comprise a sandwich construction, comprising a bottom layer, a top layer, and one or more middle layers between the bottom layer and the top layer. The device 101 may be formed within one or more of the middle layers, wherein the middle layer comprises cut out spaces defining the components of the device 101, such as the inlet chamber 107, microfluidic channel 109, and analysis chamber 103. Accordingly, such components may lie along a single plane defined by an upper surface of the bottom layer of the disc 203.

[0129]

[0131] The devices 101 may be removably fixed to the rotatable disc 203. This may enable removal from and replacement of device 101 on the disc 203. Alternatively, each device 101 may be provided on a modular component such that they interlock or otherwise connect with each other to form a disc or other arrangement for rotation.

[0130]

[0132] The disc 203 may be controlled to vary the rotational direction of the disc 203 between clockwise and anti-clockwise directions (shown with the circular arrows on Figure 8).

[0131]

[0133] Each device 101 is radially spaced from a rotation axis 205 (shown in Figure 8 coming out of the page) of the disc 203 such that the inlet chamber(s) 107 and the analysis chamber 3, 103 circle the rotation axis 205 during use. In this embodiment, the devices 101 are provided at or near a periphery of disc 203.

[0132]

[0134] The analysis chamber 3, 103 is configured to be positioned closer to a periphery of the disc than the inlet chamber(s) 107 such that the analysis chamber 3, 103 undergoes higher absolute velocities and rotational forces than the inlet chamber(s) 107 for a given rotational speed of the disc 203. The inlet chamber(s) 107 are positioned closer to the rotation axis 205 than the analysis chamber 3, 103.

[0133]

[0135] Generally, the disc 203 can be any size and the devices may be positioned any distance from the rotation axis. The further the devices 101 are from the rotation axis, the less angular speed is required to generate a rotational force (e.g., to move / flow / transfer / mix fluids / materials).

[0134]

[0136] One or more devices 101 may be positioned on a disc radially outward from the rotation axis at a distance x of between about 30 and 50 mm from the rotation axis. In the arrangement shown in Figure 8, the distance x from the rotation axis to the centre of analysis chamber is about 40 mm. In embodiments comprising the device 1 (shown in Figures 1-3) arranged on a rotatable disc, they may be arranged such that the inlet port 5, inlet chamber 7, 107, channel 9, 109, and analysis chamber 3, 103 are aligned colinearly radially with respect to the rotation axis 205. With reference to Figure 1, device 1 may be arranged with direction R oriented with the direction of centrifugal force (the direction of the arrow), in use.

[0135]

[0137] The centrifugal assembly 201 is rotationally driven by any suitable known driving means configured to control motion of the disc 203. The driving means is controllable to rotate the assembly 201 at a selected rotational speed and to change the rotational speed according to a selected steady or varying acceleration. The driving means may be configured to change the direction of rotation with control of acceleration in an oscillatory manner. The driving means can be configured to follow a protocol of changing rotation speed, rotational velocity, and / or rotational direction to control the motion of the fluids within the disc including mixing, resuspension and dissolution of solids, and timed transfer of fluids between the inlet chambers and analysis chambers.

[0136]

[0138] The assembly 201 is configured for use in a diagnostic system 501. The system 501 may be designed for analysing fluids in a device 1 on an assembly 201. The system 501 may comprise a centrifuge or system configured to exert a rotational force on the fluid in use.

[0137]

[0139] Figure 9 shows a system 501 configured to receive the assembly 201 and one or more measurement instruments 503 for measuring a characteristic of the fluid and / or bead. Assembly 201 may be removable from the system 501, such that assembly 201 may be removed or replaced. System 501 may comprise a driving means such as one or more motors 505 configured to control motion of the assembly 201, one or more controllers 509 for controlling the one or more motors 505 and / or measurement instruments 503, supports 507 for one or more of the aforementioned components, a power supply, and a base 511 for one or more of the aforementioned components.

[0138]

[0140] The measurement instrument 503 may comprise an optical measurement instrument such as a colorimeter, a photographic or videographic camera, or an instrument that can measure light transmission, reflectance, emission, fluorescence, and / or absorption.

[0139]

[0141] The device 1, 101, assembly 201 and system 501 described herein is useful for performing a centrifugal assay on one or more fluids.

[0140]

[0142] The method of performing the assay comprises introducing fluid containing a target compound into the analysis chamber 3, 103, exerting rotational forces on the fluid and the bead 15 by modulating an angular acceleration of the analysis chamber, and measuring a characteristic of the fluid in the analysis chamber.

[0141]

[0143] As will be described in more detail below, the method may comprise the following steps:

[0142]

[0144] Figure 10A shows a step of introducing fluid into the inlet chamber via inlet port.

[0143]

[0145] Figure 10B shows a step of rotating the device to exert a rotational force on the fluid and deliver the fluid to the entrance of channel. Flow of the fluid through the channel is controlled by the actuation of the valve.

[0144]

[0146] Figure 10C shows the progress of fluid down the channel towards the analysis chamber as the valve is opened, or other resistive force in the channel is overcome, and rotational force drives the fluid.

[0145]

[0147] Figure 10D shows fluid fully transferred to the analysis chamber and in contact with the bead. One or more mixing and measuring steps are performed.

[0146]

[0148] Figure 10E shows the progress of fluid from the analysis chamber and through a waste channel towards a waste chamber, as a waste valve is opened, or other resistive force in the channel is overcome, and rotational force drives the fluid.

[0147]

[0149] Figure 10F shows the analysis chamber being emptied of fluid.

[0148]

[0150] Figure 10G shows the analysis chamber being fully emptied and the waste chamber containing the fluid.

[0149]

[0151] An embodiment of the method is generally described below with reference to device 1 shown in Figures 1-3 and with reference to Figures 11-15 which chart rotational motion (velocity and, in Figure 9, acceleration) of the device on a rotating disc during several stages of the method. It will be readily apparent that the method may be applied to other embodiments of the device described herein.

[0152] With reference to Figures 10A and 10B, the method may comprise loading fluid into an inlet chamber 7, 107 via inlet port 5. The inlet chamber 7, 107 is configured to store the fluid prior to initiation of the assay reaction.

[0150]

[0153] With reference to Figures IOC, 10D, 11 and 12, the method may comprise a fluid introduction stage 301, in which fluid is introduced to the analysis chamber 3, 103 from the inlet chamber 7, 107 via channel 9, 109 by one or more external forces. Fluid is preferably introduced into the analysis chamber 3, 103 by a rotational force caused by a rotation of the device 1, such as described with reference to assembly 201. Alternative or additional external forces for introducing the fluid into the analysis chamber 3, 103 may include mechanical, pneumatic, or hydraulic forces.

[0151]

[0154] In some embodiments of the method, two or more assay fluids may be introduced to analysis chamber 3, 103. Each of the two or more assay fluids may be introduced from the same or separate inlets, or from the same or separate channels.

[0152]

[0155] At least one fluid may be stored in inlet chamber 7, 107 prior to initiation of the assay. As noted above, fluid communication between inlet chamber 7, 107 and the analysis chamber 3, 103 may be controlled by a valve 12 or by resistive forces in the intervening channel 9, 109. Actuation of the valve 12 or overcoming the resistive forces with sufficient pressure on the fluid may initiate the flow of the fluid into the analysis chamber 3, 103.

[0153]

[0156] For assays that measure, or are dependent on, reaction kinetics in the analysis chamber 3, 103, fluid is preferably introduced to the analysis chamber rapidly as a single dose. Figure 11 shows the introduction of the fluid at period 301 in the device accelerates to a maximum velocity to drive the fluid to the analysis chamber 3, 103. Rapid introduction of fluid to the analysis chamber facilitates the setting of an accurate reaction initiation time. Preferably, the time taken to completely introduce the fluid is less than one second.

[0154]

[0157] Following an acceleration to a maximum velocity, the rotational velocity may reduce, as indicated in period 301. In embodiments where two or more fluids are separately introduced to the analysis chamber 3, 103, these may be introduced simultaneously or sequentially.

[0155]

[0158] The presence of air or air bubbles in the analysis chamber 3, 103 may be minimised or avoided. The volume of the fluid to be introduced to the analysis chamber 3, 103 may be selected to reduce or avoid the presence of an air gap in the analysis chamber 3, 103. For example, the volume of the fluid to be introduced may be substantially equivalent to the volume of the analysis chamber 3, 103, accounting for the presence of the one or more bead(s) and any other components housed in the analysis chamber 3, 103. Alternatively, the volume of fluid to be introduced to the analysis chamber may be deliberately less than the volume of the analysis chamber.

[0156]

[0159] The time of actuation of the valve may be recorded and may be used to calculate or infer the time of the assay reaction initiation. Alternatively, the time of initiation of angular acceleration, or a short period thereafter, of the device may be recorded and may be used to calculate or infer the time of reaction initiation, as shown in period 301 of Figure 11. In embodiments where two or more fluids are separately introduced, calculations or inferences of initiation time may be based on the time of introduction of the fluid comprising the target that reacts with the capture reagent on the bead 15.

[0157]

[0160] The bead 15 is configured to move freely within at least a portion of the analysis chamber 3, 103. The analysis chamber 3, 103 may house the bead 15 prior to addition of fluid. Alternatively, the bead 15 may be introduced separately to the analysis chamber 3, 103 following introduction of the fluid. In this embodiment, the time of reaction initiation may be set by the time the bead 15 is introduced to the analysis chamber 3, 103.

[0158]

[0161] In an embodiment, the device 1, 101 is mounted on a rotatable disc, such as that described above with reference to Figure 8. Rotation of the disc exerts rotational forces to the device and, by extension, to the fluid and bead 15 in the analysis chamber 3, 103.

[0159]

[0162] The disc may be controlled to modulate an angular acceleration of the device 1 during the assay. Modulation of angular acceleration may be achieved by varying the rotational speed of the disc, for example between 0 and 5000 rpm, at a selected constant or varying rate.

[0160]

[0163] The disc may be controlled to vary the rotational direction of the disc between clockwise and anti-clockwise directions.

[0161]

[0164] The modulation of angular acceleration may induce a circulation of the fluid throughout the analysis chamber.

[0162]

[0165] The bead 15 is configured to move freely within at least a portion of the analysis chamber 3, 103 and interact with the circulating fluid. Thus, modulation of angular acceleration may induce translational and rotational movement of the bead 15 within the analysis chamber 3, 103.

[0163]

[0166] The device 1, 101 is subjected to one or more mixing cycles, each mixing cycle comprising one or more changes in angular acceleration on the device. In one embodiment, a modulation of angular acceleration comprises one or more changes in rotational direction. Preferably, the modulation of angular acceleration comprises one or more abrupt changes in rotational direction.

[0164]

[0167] The assembly may be configured to intermittently or periodically vary rotational direction between clockwise and anti-clockwise directions. In an alternative embodiment, modulation of angular acceleration may be achieved without a change in rotational direction. That is, angular acceleration may be varied without a reversal in the rotational direction of the device. For example, the rotational velocity may be increased and decreased in a single rotational direction.

[0165]

[0168] Figure 11 shows a mixing cycle 303 comprising plurality of changes in rotational acceleration and direction, in which angular velocity during mixing cycle 303 reaches positive and negative maxima. Each change in rotational direction is accompanied by a modulation of angular acceleration. Figures 11, 12 and 14 show mixing cycles in which the rotational velocity of the disc moves through zero. The disc undergoes positive acceleration to a maximum positive rotational velocity, and then negative acceleration, through a rotational velocity of zero, to a maximum negative rotational velocity.

[0166]

[0169] In an embodiment, the duration of mixing cycle 303 is between one and 60 seconds. More preferably, the duration of a mixing cycle 303 is between one and 10 seconds.

[0167]

[0170] In an embodiment, the rotational velocity during mixing cycle 303 is between 100 and 5000 rpm, more preferably between 200 and 1000 rpm.

[0168]

[0171] In an embodiment, the rotational acceleration is modulated between one and 120 times in a mixing cycle 303. More preferably, rotational acceleration is modulated between one and 20 times in a mixing cycle 303. In an example, rotational acceleration is modulated at least once every 1 second.

[0169]

[0172] Modulation of the angular acceleration also causes a movement of the bead 15 within the analysis chamber. Angular acceleration drives a circulatory motion of the fluid within the analysis chamber. Modulation of the angular acceleration causes a variation in the circulatory motion. For example, reversal of the sign of the angular acceleration (such as by one or more abrupt changes in rotational direction) causes a reversal and a folding of the fluid flow. Inducing a variable circulatory motion in two or more fluids that are in contact with each other but each in laminar flow may improve the mixing of the fluids. Inducing a variable circulatory motion in a fluid in contact with a surface where a reaction is occurring may promote uniformity of composition of the fluid with respect to substances dissolved in it that are reacting at the surface.

[0173] Mixing may be further improved where angular acceleration is modulated in a time frame that is less than that of the hydrodynamic relaxation time of the fluid but also long enough that the length scale for damping of flow oscillations is significant with respect to the length scales of the device (chamber radius and depth, and bead radius). For fluids having a kinematic viscosity of or approximately that of water («1 mm2s'125°C) the mixing stage comprises a frequency of changes in rotational direction of approximately 1 to 3 times per second.

[0170]

[0174] Effective mixing may be achieved where the mixing of the disc has a product of rChamberT(d disc / dt) of 2000 rpm.mm or greater, 2500 rpm.mm or greater, 3000 rpm.mm or greater, 3500 rpm.mm or greater, 4000 rpm.mm or greater, or 4500 rpm.mm or greater.

[0171]

[0175] During mixing cycle 303, changes in angular acceleration cause the bead to move about the analysis chamber 3, 103. The device is configured to allow the bead to translate within at least a portion of the analysis chamber 3, 103. Preferably, bead 15 translates away from the lateral internal walls of the chamber during the mixing cycle, and more preferably translates through a middle portion of the analysis chamber 3, 103.

[0172]

[0176] Movement of bead 15 during mixing cycle 303 causes reconfiguration of eddies in the fluid, which improves mixing of the fluid. The bead 15 rotates and translates within the analysis chamber 3, 103 when subjected to a change in rotational acceleration. As noted above, the bead surface comprises a capture reagent that reacts with a target in the fluid. As the reaction between the surface-bound capture reagent and target occurs at the surface of the bead, it is desirable to improve the mixing of the fluid and the corresponding exchange of fluid-borne products and reactants at the fluid-surface interface of the bead. It is also desirable to improve uniformity of reaction rate across the surface of the bead. One or both of these desirable outcomes may be achieved by modulating the angular acceleration of the device such that the bead translates and rotates within the fluid.

[0173]

[0177] The circulation of the fluid within the analysis chamber 3, 103 induces a fluid velocity gradient across the surface of bead 15, which causes a rotation in the bead 15. Consequently, the bead experiences a lift force that moves it away from the lateral internal walls of the analysis chamber. Rotation of the bead 15 in the fluid improves the uniformity of mass transport over the surface of the bead during the mixing cycle 303. Accordingly, in preferred embodiments the bead not only translates around the chamber wall in response both to the oscillating forces from the disc motion and to the fluid motion, but also spins in response to the gradient of fluid velocity.

[0178] The method comprises one or more measurement stages 305. Preferably, the measurement stage 305 comprises the measuring of a characteristic of the fluid and / or bead in the analysis chamber 3, 103. Measurement stage 305 may comprise obtaining one or more measurements of the characteristic of the fluid and / or bead by one or more measuring instruments that are external from the analysis chamber 3, 103 and device 1. For example, it may comprise obtaining an optical measurement and / or a recording of an image of the fluid and / or bead. Optical measurements may include one or more measurements of light colour, transmission, reflectance, emission, fluorescence, scattering and absorption. Recording an image may include photography or videography. The analysis chamber 3, 103 may comprise a window that is sufficiently transparent to one or more wavelength(s) of light used to obtain the measurement.

[0174]

[0179] The characteristic to be measured is preferably a characteristic of the fluids in the chamber. Preferably, the bead 15 does not impede a measurement of the fluid during measurement stage 305. This may be achieved by controlling the rotation of the device during measurement stage 305. In a preferred embodiment, the rotational velocity of the device is constant during measurement stage 305. This may reduce the likelihood of the bead 15 translating within the analysis chamber 3, 103 during the measurement stage 305 and potentially impeding the measurement of the fluid.

[0175]

[0180] The rotational velocity of the device may also be constant during measurement stage 305, for example, as shown in Figures 11 and 12. For example, the rotational velocity may be constant at between 0 and 5000 rpm, more preferably between 0 and 1000 rpm. Alternatively, or in addition, the measurement may be obtained from a measurement region of the analysis chamber s, 103 in which the likelihood of the bead 15 being located in that portion is relatively low.

[0176]

[0181] The method may comprise plurality of measurement stages 305. In preferred embodiments, there is a mixing stage 303 between two measurement stages. Figure 12 shows a method comprising fluid introduction stage 301, and a plurality of alternating mixing stages 303a, 303b, 303c, 303d and measuring stages 305a, 305b, 305c, 305d. Each of the plurality of mixing stages 303a-d may have durations that are the same or different to each other. Each of the measuring stages 305a-d may have durations that are the same or different to each other.

[0177]

[0182] Preferably, the measurement of the characteristic comprises a measurement or a determination of the change in the measured characteristic. Accordingly, the characteristic is preferably repeatedly measured over time and the method therefore comprises two or more measurements of the characteristic over time. For example, the method may comprise taking two or more measurements during the assay. The two or more measurements may be periodic or intermittent. The time at which each measurement is taken is preferably recorded to facilitate the calculation of a change in the measured characteristic over time. The time of each measurement is taken preferably corresponds to an elapsed time after the introduction of the fluid or from the time of initiation of the assay reaction.

[0178]

[0183] A measurement of the characteristic of the fluid at the time of introduction or reaction initiation may be simply assumed based on the known properties of the fluid being measured. Hence, a physical measurement may not be required at or before introducing the fluid to the analysis chamber to generate a measurement of the characteristic at reaction initiation.

[0179]

[0184] The analysis chamber 3, 103 may be emptied of fluid by driving the fluid out under one or more external forces. The fluid may be driven through an opening in the analysis chamber 3, 103 to a waste chamber, such as waste chamber 121 shown in Figure 4, via a conduit such as conduit 129. Fluid communication between analysis chamber 3, 103 and waste chamber 121 may be controlled by a valve 127 or by resistive forces in the conduit 129. Actuation of the valve 127 or overcoming the resistive forces with sufficient pressure on the fluid may initiate the flow out of the fluid into the analysis chamber 3, 103.

[0180]

[0185] A quantitative determination of the assay target may be calculated or inferred from one or more measured characteristics.

[0181]

[0186] Where a measurement is taken once the reagent-target reaction has reached equilibrium, a quantitative determination of the target may be calculated or inferred from a measured characteristic at equilibrium.

[0182]

[0187] Measurement of the rate of change may facilitate a quantitative determination of the target prior to the reaction between target and reagent reaching equilibrium. A rate of change of the measured characteristic may be calculated or inferred from one or more measured characteristics. Quantitative determination of the target may be calculated or inferred from the calculated or inferred rate of change of the measured characteristic.

[0183]

[0188] The amount of target bound to a functionalised bead surface may be determined based on a calculation or inference of the enzyme-loading dependent rate constant for colour development, which may be derived from the optical measurement. Methods include taking the measurement at a known time following the reaction initiation (i.e., the time of the target's first contact with the bead). Alternatively, the dynamic range of the assay is extended by using an empirical fit (e.g., polynomial) to derive an initial slope of a signal-time curve. In another method, a range of measurements are fit to a model for the reaction kinetics to derive a rate constant for the reaction.

[0184] Examples

[0185] Example 1 - Assay device and centrifuge construction

[0186]

[0189] A disc comprising nine microfluidic assay devices, each assay device having the construction shown in Figure 1, were constructed from three stacked discs of poly(methylmethacrylate). Chambers and channels were laser cut through the central layer and through-valves (vents) were laser cut through the top layer. The diameter of the analysis chamber was 5mm. The depth of the analysis chamber in each device was 2.3 mm.

[0187]

[0190] The bottom layer supported the central layer and provided the lower wall to the chambers and channels. Top, central and bottom layers were joined with adhesive. All surfaces were coated with a hydrophobic fluoropolymer (Cytopel 500, Cytonix, LLC, MD, USA) before assembly. The disc comprised nine devices 1, 101 arranged circumferentially evenly around the centre of the disc. The mixing chamber was cylindrical with radius 3 to 6 mm. The radial position of the centre of the reaction chamber was 45.6 mm from the centre of the disc. Each analysis chamber was loaded with a polished polystyrene bead (1.58±0.0125 mm in diameter, supplied by Redhill Precision (Prague, CZ)).

[0188]

[0191] The disc was mounted on a centrifuge instrument comprising a controller, motor drive. The motor was a brushless DC motor (Anahiem Automation BLWS232D) where speed was computer controlled and locked to the signal from a shaft encoder (4096 counts I revolution). For imaging, a strobe light was synchronised with the motor drive. The motor and controller response time to a rotation rate step change software command was ~0.1 s. An optical pulse from an index hole and pulse counting from the shaft encoder were used to control and synchronise the disc rotation programme and data acquisition.

[0189]

[0192] An optical analyser was provided in the form of an intensity-stabilised red (650 nm) laser diode converging to a beam diameter of «0.04 mm at the reaction chamber, and diverging to cover ~50% of the surface of a detection photodiode mounted above the chamber.

[0190] Example 2 - Bead size analysis

[0191]

[0193] Beads of different diameter were used to explore the effect on mixing. Bead surface was coated with horse-radish peroxidase (HRP), by adsorption of an HRP-labelled antibody as described in Example 3. The fluid injected into the chamber comprised a solution containing 4-4' tetramethylbenzidine (TMB) and hydrogen peroxide. Reaction of TMB catalysed by HRP on the bead surface generates a blue colour which becomes dispersed by mixing through the solution. Effects of disc rotational acceleration, chamber radius and bead radius on a mixing timescale of five mixing cycles with cycle time 0.34 to 0.5 s were evaluated by determining the ratio of standard deviation to mean of the optical transmission across the chamber as a measure of compositional uniformity. Bead radius, chamber radius, rotational acceleration, oscillation period each influenced compositional uniformity.

[0192]

[0194] Figure 13 shows a dependence of mixing on the ratio of chamber radius to bead radius, Chamber / rbead, and product rChamber(d disc / dt), with oscillation period of angular acceleration, T. Chambers analysed were circular in cross section with diameters 3.1, 4 and 5 mm each. Chamber cross sectional area corresponds to 7.5, 13 and 20 mm2, respectively, and chamber volumes correspond to 17, 29 and 45 mm3, respectively. Beads analysed were spherical with diameters 1, 1.58 and 2 mm each. Bead cross sections correspond to 0.79, 2.0 and 3.1 mm2, respectively and bead volumes correspond to 0.52, 2.1, and 4.2 mm3, respectively.

[0193]

[0195] Analysis of the radius, cross section and volume ratios of the analysis chamber and one bead are shown in Table 1.

[0194] Chamber : Chamber : Bead Chamber Bead radius Bead radius cross section Chamber : Bead radius (mm) (mm) ratio ratio volume ratio

[0195] 2.5 0.5 5.0 25 85

[0196] 2.5 0.79 3.2 10 22

[0197] 2.5 1.0 2.5 6.3 11

[0198] 2.0 0.50 4.0 16 55

[0199] 2.0 0.79 2.5 6.4 14

[0200] 2.0 1.0 2.0 4.0 6.8

[0201] 1.55 0.5 3.1 9.6 33

[0202] 1.55 0.79 2.0 3.9 8.3

[0203] 1.55 1.0 1.6 2.4 4.1

[0204] Table 1

[0196] A mixing 'pass' was determined as the standard deviation / mean of optical transmission on the laser path across the chamber less than an arbitrary constant threshold after five cycles. For the range of oscillation period and rotational acceleration considered, the product rChamberT(d disc / dt) is an approximate measure of the change of momentum of the fluid when the rotational acceleration changes sign. Figure 13 shows that the larger this parameter, the more effective the mixing. For example, effective mixing may be achieved where the mixing of the disc has a product of rChamberT(d disc / dt) of 2000 rpm.mm or greater, 2500 rpm.mm or greater, 3000 rpm.mm or greater, 3500 rpm.mm or greater, 4000 rpm.mm or greater, or 4500 rpm.mm or greater.

[0205]

[0197] Fig. 11 also indicates that there is an optimum range for mixing with rChamber / rbead between 2 and 2.5 and an optimum range where mixing is improved when rChamber / rbead > 3.1.

[0206] Example 3 - Method of operation

[0207]

[0198] The method of operation was demonstrated with the system described in Example 1 to measure surface-bound HRP. The rotational velocity of the device during the method is shown in Figure 14. The inset chart in Figure 14 shows a zoomed in section of the chart and identifies two points at which measurements were taken (M).

[0208]

[0199] Beads were sensitised using passive adsorption of Goat anti-Human IgG Fc horse radish peroxidase (HRP: Thermofisher #A18817, RRID AB_2535594 ). HRP loading on beads was altered by change of concentration of the HRP solution. Antibody diluted from 1 mg / mL stock solution was shaken in the dark with 16 beads in a 4 mL glass vial for 1 hr. The beads were washed three times then stored in buffer at 4°C. Immediately before use, beads were removed from the buffer and dabbed dry with a tissue before insertion into the disc reaction chambers, after which the disc was closed with the top layer. The coefficient of variation in the amount of enzyme adsorbed was separately assessed, by conventional measurement on a sample of 8 beads for each loading (128 in total) each in a sample well, and measurement of the colour intensity using a spectrophotometer following quenching the reaction with strong acid, as 4.5%.

[0209]

[0200] The inlet chamber was loaded, via an inlet port, with fluid comprising an enzyme substrate, tetramethylbenzidine (TMB) with H2O2 (Surmodics TMBW1000-01, TMB concentration approx. 2 mM, H2O2 concentration approx. 30 mM, pH 3.4) with a volume selected to exactly fill the reaction chamber. The fluid was held in the inlet chamber by pinning the contact line at the entry to the channel, by virtue of the capillary force exerted by the hydrophobic surfaces of the channel.

[0201] The measurement was initiated at t = tO by initiation of disc rotation to pass the constriction and inject the reagent into the reaction chamber. Increasing angular velocity to approx. 3200 rpm forced the fluid into the channel against the effect of surface tension and injected the fluid into the analysis chamber. Video imaging showed fluid contacting the bead at t-tO = 0.7 ±0.1 s and that the chamber was full within approximately 1 s following a software command to increase angular velocity.

[0210]

[0202] Mixing into the solution of reaction product from the surface of the bead was assessed by measurement of the variation of optical transmission as the reaction chamber moved past the laser beam, following injection of fluid into the analysis chamber containing a bead with surface-adsorbed HRP.

[0211]

[0203] Mixing by oscillation of rotational acceleration and rotation direction was initiated at t-tO = 2.6 s. With increasing number of cycles during the mixing phase, the solution composition, measured by the optical transmission, became more uniform. A mixing cycle comprised the following parameters: (d _disc) / dt = ±2500 rpm / s; disc,max=±550 rpm; and disc oscillation period, T = 0.88 s. The product of rChamberT(d disc / dt) is accordingly 5500 rpm. mm.

[0212]

[0204] For measurement, the disc rotation rate was held at 550 rpm ( disc,measurement= 550 rpm), which moved the bead out of the laser path, and the measurement made during the period of constant rotation rate. The first measurement was made at t- 10=10.5 s (range 10.45 - 10.6s) and thereafter at a fixed time interval of At = 9.7 s (range 9.6 - 9.9) up to a total reaction time of 300 s. The full range of measurement was 0 to 3000 counts; the noise due to variations of the laser intensity and photodiode noise was ~6 counts (range). Total variation at full scale including variations in optical scattering from the disc material was ~ 40 counts (range). The signal was linear in light intensity over the full range. The photodiode signal was sampled at 230 kHz; 5 samples were taken and averaged for each trigger pulse from the shaft encoder. For measurement of reaction kinetics, the average light intensity across the centre of the reaction chamber for a single rotation of the disc was determined for each chamber (14 measurement points).

[0213]

[0205] After each measurement, mixing was paused for 1 min with the disc rotating at constant speed, in which the bead held at the radial position in the analysis chamber most distant from the disc centre. During this time, the solution would be static (no Euler force) and the blue reaction product would be being dispersed only by diffusion from the bead surface. A mixing cycle was then re-imposed following which the variation of optical transmission was measured again.

[0206] One cycle of acceleration-deceleration was then performed. Optical absorption was measured again. This cycle of pause, measure, mix and measure, was repeated with increasing number of mixing oscillations. Uniformity of solution composition was assessed by measuring the spatial variation of signal across the reaction chamber.

[0214]

[0207] Figure 15 shows an optical absorption scanning across the measurement region of an analysis chamber during a measurement stage. It shows development of solution uniformity with increasing number of cycles of disc angular velocity. Relative optical transmission in photodiode counts against position from the initial intersection of the laser beam with the edge of the analysis chamber. The curves are labelled with the number of oscillation cycles after each pause; 5mm diameter reaction chamber, 1.58 mm diameter bead.

[0215]

[0208] Preferred embodiments of the invention have been described by way of example only and modifications may be made thereto without departing from the scope of the invention.

Claims

CLAIMS1. An assay device configured for use with a centrifugal system exerting centrifugal force on the assay device, comprising: a) an analysis chamber configured to receive fluid; b) the analysis chamber containing one or more beads configured to move freely in the analysis chamber; wherein: a ratio of a cross sectional area of the analysis chamber to a total cross sectional area of the one or more beads is between about 3 and 25, or between 3 and 10, wherein the total cross sectional area of the analysis chamber is determined by the two largest dimensions of the analysis chamber; or a ratio of a volume of the analysis chamber to a total volume of the one or more beads is between about 5 and 100, or between 5 and 50, or between 5 and 25.

2. The assay device as claimed in claim 1, wherein the bead has a density that is within about 10% of a density of the fluid.

3. The assay device as claimed in claim 1 or claim 2, wherein the analysis chamber contains between one and six beads.

4. The assay device as claimed in any one of the preceding claims, wherein the analysis chamber contains a single bead.

5. The assay device as claimed in any one of claims 1 to 3, wherein the analysis chamber contains two or three beads.

6. The assay device as claimed in any one of the preceding claims, wherein the analysis chamber has a major internal dimension of between 1 and 10 mm.

7. The assay device as claimed in any one of the preceding claims , wherein the diameter of the one or more beads is between about 1 mm and about 2 mm.

8. The assay device as claimed in any one of the preceding claims, wherein the analysis chamber comprises an elongate cross-sectional area.

9. The assay device as claimed in claim 8, positioned on a rotatable disc having a central axis of rotation, wherein a major internal dimension of the analysis chamber is aligned with a direction of centrifugal force, in use.

10. The assay device as claimed in claim 9, wherein side walls of the analysis chamber taper inwardly towards an inner end of the analysis chamber proximate the axis of rotation from an opposing outer end of the analysis chamber.

11. The assay device as claimed in any one of claims 8 to 10, wherein an aspect ratio of the average length of the major dimension and the average length of a minor dimension perpendicular to the major dimension is between about 1 and 4.

12. The assay device as claimed in any one of claims 8 to 11, wherein a minimum radius of curvature in the analysis chamber side wall(s) is at least 1 mm.

13. The assay device as claimed in any one of the preceding claims, wherein the analysis chamber comprises a window configured to allow optical analysis of the fluid in the analysis chamber.

14. The assay device as claimed in any one of the preceding claims, wherein the assay device is a microfluidic device.

15. The assay device as claimed in any one of the preceding claims, further comprising a fluid inlet chamber and a microfluidic channel providing fluid communication between the inlet chamber and the analysis chamber, wherein substantially all of the internal surfaces of the analysis chamber, microfluidic channel and inlet chamber comprise a hydrophobic coating.

16. A centrifugal assay assembly comprising a plurality of assay device(s) as claimed in any one of claims 1 to 15 provided on a rotatable disc having a central axis of rotation, wherein the analysis chamber of each of the plurality of assay devices is located at the same distance from the axis of rotation of the disc.

17. A method for performing an assay on a fluid comprising a target, the method comprising: providing an assay device of any one of claims 1 to 15 or the centrifugal assay assembly of claim 16, wherein at least one of the one or more beads comprise a surface-bound capture reagent configured to bind with the target, the method comprising the steps of:- introducing the fluid into the analysis chamber;- exerting rotational forces on the fluid and the bead by modulating an angular acceleration of the analysis chamber; and- measuring a characteristic of the fluid in the analysis chamber.

18. The method of claim 17, wherein the rotational forces are oscillating rotational forces, the oscillating rotational forces comprising an oscillating period of 1 to 3 times per second.

19. The method of claim 17, wherein modulating the angular acceleration of the analysis chamber comprises a product of T. Chamber. (dQ / dt) that is 2000 rpm.mm or greater, 2500 rpm.mm or greater, 3000 rpm.mm or greater, 3500 rpm.mm or greater, 4000 rpm.mm or greater, or 4500 rpm.mm or greater, wherein T is the oscillating period in S, Tchamber iS the radius of a major internal dimension of the analysis chamber in mm, and dQ / dt is the angular acceleration of the analysis chamber in rpm.s'1, wherein Tchamber iS determined as (rmajor + rminor) / 2, where rmajor is half the length of the major internal dimension and Tminor iS half the length of the minor internal dimension perpendicular to the major dimension, or wherein Tchamber iS either rmajor or rminor.

20. A method of mixing two or more fluids in a microfluidic chamber, the method comprising: contacting a first fluid and a second fluid in a microfluidic chamber comprising one or more beads configured to move freely in the analysis chamber, and modulating the angular acceleration of the microfluidic chamber wherein: a ratio of a cross-sectional area of the analysis chamber to a total cross-sectional area of the one or more beads is between about 3 and 25, or between 3 and 10, wherein the total cross-sectional area of the analysis chamber is determined by the two largest dimensions of the analysis chamber; or a ratio of a volume of the analysis chamber to a total volume of the one or more beads is between about 5 and 100, or between 5 and 50, or between 5 and 25.

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