The present invention relates to mixing of liquids within a microfluidic device, in particular although not exclusively in a mixing chamber.

Microfluidic devices, that is devices having fluid handling structures with a smallest dimension of less than one millimeter, for example of the order of tens or hundreds of micrometer, are used in biochemical assays, DNA sequencing, sample preparation and analysis, cell separation and detection, as well as food, beverage and environmental monitoring. Fluid flow in microfluidic devices is highly laminar due to generally low

Reynolds numbers. Consequently, conventional turbulent mixing between two liquids typically does not occur and mixing rates in microfluidic devices are typically limited by diffusion rates and hence inherently slow. To address this issue, various techniques for more efficient mixing have been developed, in particular active methods in which mixing is promoted by agitation of the liquids to be mixed. These methods require external forces to be applied to the microfluidic device. Microfluidic devices with active mixing arrangements typically require complicated structures and hence complex fabrication processes. To date, the integration of such active mixers in a microfluidic device has proved both challenging and expensive.

In a first aspect of the disclosure a method of mixing liquids in a microfluidic device comprises applying a time varying fluid pressure, for example a liquid or gas pressure, to an external wall of the microfluidic device to deform the external wall, thereby agitating liquids to be mixed inside the microfluidic device. Advantageously, deforming an external wall by application of fluid pressure enables mixing in microfluidic devices that are of simple construction and hence cost-efficient.

In some embodiments, the method comprising deforming the external wall in the region of a mixing chamber inside the microfluidic device, thereby agitating liquids to be mixed inside the mixing chamber. The microfluidic device may comprise a substrate defining the mixing chamber. The substrate defines a first surface of the mixing chamber and the external wall, which may be substantially flat, defines a second surface of the mixing chamber on an inner side of the external wall. The substrate may also define a plurality of supply channels to supply respective liquids to be mixed to the mixing chamber

In a second aspect of the disclosure, a microfluidic device comprises a substrate defining a mixing chamber to contain a plurality of liquids and a plurality of supply channels to supply respective liquids to be mixed to the mixing chamber. The substrate defines a first surface of the mixing chamber and an external wall of the microfluidic device defines a second surface of the mixing chamber on an inner side of the external wall. The external wall is configured to have a deflection of at least 0.1 mm when a pressure differential of 10kPa is applied between the mixing chamber and an outer side of the external wall in the region of the second surface. In some embodiments, a pressure in the range of 5 to 10kPa, for example 5kPa, 6kPa, 7kPa, 8kPa, 9kPa or 10kPa is required to achieve this minimum deflection. Advantageously, this arrangement provides a simple and cost- effective active mixing solution, particularly suitable for a method as described above.

In a third aspect of the disclosure, a microfluidic system comprises a microfluidic device in combination with a pressure application device. The microfluidic device comprises a substrate defining a mixing chamber to contain a plurality of liquids. The substrate may also comprise a plurality of supply channels to supply respective liquids to be mixed to the mixing chamber. The substrate defines a first surface of the mixing chamber and an external wall of the microfluidic device defines a second surface of the mixing chamber on an inner side of the external wall. The pressure application device comprises a pressure application zone to seal to the external wall around the region of the second surface and a pressure generator in sealed connection with the pressure application zone to apply a fluid pressure to the external wall when the external wall is sealed to the pressure application zone. The system further comprises a controller to control the pressure generator to vary the output, thereby varying the fluid pressure as a function of time to deform the external wall in the region of the second surface and hence to agitate liquids to be mixed in the mixing chamber.

Advantageously, by controlling the output of the pump to achieve active mixing, active mixing is facilitated with a simple and cost-efficient microfluidic device. Removing some of the complexity to the pressure application device allows the microfluidic device to be provided as a simple and cost-efficient disposable part.

The pressure generator may be a pump that can be controlled to apply a time-varying fluid pressure, for example to diaphragm pump. The pump may be connected in a closed loop arrangement. In some embodiments, the pressure generator may be a piston and cylinder arrangement connected to the pressure application zone, with movement of the piston inside the cylinder varying the fluid pressure. Various detailed embodiments of the above aspects and embodiments are described herein. In some, the supply channels, where present, connect to the mixing chamber through supply ports in the first surface. In some the external wall may be flat. For example, the external wall may comprise a non-elastomeric plastic membrane bonded to the substrate such that an inner surface of the membrane provides the second surface. Specifically, in some embodiments, the membrane has a thickness of 0.1 to 0.4 mm, for example 0.1 mm, 0.2mm, 0.3mm or 0.4mm and a Young's modulus of at least 2GPa, for example 2 to 4GPa, preferably 2GPa, 3Gpa or 4Gpa. In the first and third aspects, the external wall may also be configured to have a maximum deflection of at least 0.1 mm when a pressure differential of 10kPa is applied between the mixing chamber and an outer side of the external wall in the region of the second surface. In some embodiments, a pressure in the range of 5 to 10kPa, for example 5kPa, 6kPa, 7kPa, 8kPa, 9kPa or 10kPa is required to achieve this minimum deflection. Generally there is a relation between the thickness, Young's modulus, size of the mixing chamber and the pressure required to achieve a given displacement, for example 0.1 mm. The largest transverse dimension and shape of the chamber may vary from embodiment to embodiment but in some embodiments, the largest transverse dimension may be between 4 to 10mm. In some embodiment the chamber may have a circular perimeter and the largest transverse dimension would be the diameter.

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

Figure 1 illustrates a microfluidic device;

Figure 2 illustrates a cross-section A--A of the microfluidic device in Figure 1 ; Figure 3 illustrates a cross-section B--B of the microfluidic device in Figure 1 ;

Figure 4 illustrates a microfluidic system; and

Figure 5 illustrates a cross-sectional view of the microfluidic device of Figure 1 , engaged with the system in Figure 4, in a cross-section corresponding to that of Figure 3. With reference to Figure 1 , Figure 2 and Figure 3, a microfluidic device 10 comprises two inlet ports 12 and an outlet port 14. The inlet ports 12 are connected to a mixing chamber 16 by channels 18 and the mixing chamber 16 is connected to the outlet port 14 by a channel 20. The inlet ports 12, outlet ports 14, mixing chamber 16, and channels 18, 20 are formed in a substrate 22, for example by injection moulding of a suitable plastics material, for example. In some embodiments, the substrate defining the microfluidic structure is formed by cut outs in one or more substrate layers that are bonded to each other. In some embodiments, the substrate layers are single or double adhesive layers, for example coated with a suitable adhesive on one or both sides, to bond the substrate layers together. The substrate or substrate layers may be made from PMMA

(polymethylmethacrylates), PC (polycarbonates), COC (Cyclic Olefin Copolymer), etc. In some embodiments, the configuration is such that the ports 12, 14 are defined by, through holes in the substrate 22 and the channels and chambers are defined cooperatively by the substrate 22, together with a membrane 24 bonded to the substrate 22 to define one of the surfaces of the chambers and channels. The membrane 24, may be of the same material as the substrate 22, or a different material, for example polyethylene terephthalate, polyesters (e.g. MYLAR®), polycarbonates, polypropylenes,

polytetrafluoroethylenes, polyimides (e.g. KAPTON®), polymethylmethacrylates, etc. or any other non-elastomeric polymer material. The membrane 24 has in some

embodiments, a thickness in the range of 100 to 400μη"ΐ, for example. In a specific embodiment, the substrate comprises polymethylmethacrylate material and the membrane comprises polymethylmethacrylate material. In some embodiments, the membrane material has a Young's modulus of 2 to 4GPa, for example. As a result, in some embodiments, the device is configured such that the membrane 24 deflects by at least 0.1 mm in response to a fluid pressure of 5kPa being applied to the membrane in the region of the mixing chamber 16.

In summary, in the described embodiments, the microfluidic device 10 has a number of ports 12, 14 formed in one face of it, with the membrane 24 providing the other face of the microfluidic device. The channels 18, 20 and chamber 16 are co-operatively defined between the substrate 22 and the membrane 24. Liquid flows in the microfluidic device 10 may be controlled by any suitable means well known to a person skilled in the art, for example by application of external fluid pressures with a suitable pumping arrangement or by electrophoresis for suitable liquids. With reference to Figure 4 and Figure 5, a microfluidic system comprises a microfluidic device 10 as described above and a coupling device 26. The coupling device 26 comprises a pressure application zone 28 for applying a pressure to the external wall 24 when the microfluidic device 10 is coupled to the coupling device 26. The coupling device 26 comprises, in some embodiments, locating features (not shown) to locate the microfluidic device 10 relative to the coupling device 26, such that the pressure application zone 28 is aligned with the region of the mixing chamber 16 in the microfluidic device 10 and may comprise securing features (not shown) for securing the microfluidic device 10 to the coupling device 26. The pressure application zone 28 is configured as a cavity encompassing the region of the mixing chamber 16 of the microfluidic device 10 and comprises a sealing arrangement 30, for example an O-ring, to seal against the membrane 24 around the region of the mixing chamber 16. An internal conduit 32 inside the coupling device 26 is in fluid communication with the pressure application zone 28 and a pressure connection port (not shown). A pressure hose 34 (or other pressure

connection) couples the pressure connection port to a pressure generator 36, which is coupled to and controlled by a controller 38 to apply a time varying pressure to the membrane 24 when the microfluidic device 10 is coupled to the coupling device 26.

Naturally, the hose 34 may be replaced by a rigid (or any other) conduit and some or all of the components may be integrated in a unitary device.

The pressure generator 36, in some embodiments, comprises a pump, for example a diaphragm pump. The pump may be connected in a closed-loop arrangement with both the intake and outlet of the pump connected the pressure hose 34. Alternatively, one of the intake and outlet of the pump may be connected to the pressure hose 34, with the other one of the intake and outlet of the pump blocked or connected to a dead volume. In some embodiments, the pressure generator 36 comprises a cylinder in fluid connection with the pressure connector via the pressure hose 34. A piston is movable inside the cylinder to create a positive or negative pressure. For example, in some embodiments, the cylinder is coupled to an electric motor controlled by the controller 38 to move the piston and thereby vary the pressure. The pressure may be transmitted from the pressure generator 36 to the device 10 by any suitable medium, for example a gas such as air or a liquid such as water.

In operation, respective liquids to be mixed, for example a sample and a detection reagent in buffer, are supplied to the inlet ports 12, 14. The ports 12, 14 are connected to the relevant driving arrangement (for example, pressure or voltage sources) and the microfluidic device 10 is coupled to the coupling device 26. Liquids are caused to flow into the mixing chamber 16 by application of an appropriate driving force as is well-known to a person skilled in the art. The membrane 24 in the region of the mixing chamber 16 is caused to deform by application of a time varying fluid pressure by the pressure generator 36 under control of the controller 38. In this way, the liquids in the mixing chamber 16 are agitated, causing them to mix. Once the liquids have mixed to a sufficient degree, for example by applying a time varying pressure for a predetermined time, mixed liquid may be retrieved, in some embodiments, at the outlet port 14. In other embodiments, the mixing chamber 16 is configured as a detection chamber, for example, to allow photometric turbicity or absorption measurements, or fluorescence measurements or other immunological assays and a corresponding measurement may be taken from the mixing chamber 16. In yet other embodiments, the microfluidic device is provided with one or more specific detection chambers downstream of the mixing chamber 16 and liquid is caused to flow into the one or more detection chamber for subsequent detection once mixing has occurred. In dependence of the embodiment, the time varying applied fluid pressure may vary between positive and negative pressures (relative to atmospheric pressure), may be only a time varying positive pressure or only a time varying negative pressure. In the latter case, the negative pressure in connection with the sealing arrangement 30 will act to hold the microfluidic device 10 against the coupling device 26, so that the securing features of the coupling device 26 can be dispensed with in some embodiments. The above description of specific embodiments is made by way of example to illustrate aspects of the disclosure, and it will be apparent to a person skilled in the art that many alterations, modifications and juxtapositions of the features of the described embodiments are possible without departing from the disclosure, nor from the scope of the appended claims. For example, while microfluidic devices with supply channels for supplying liquid to a mixing chamber and an outlet channel from the mixing chamber have been described above, it will be appreciated that the disclosure is equally applicable to microfluidic devices having only a mixing chamber, a mixing chamber without supply channels (or with only one or more than two supply channels) or a mixing chamber without an outlet channel.

Likewise, the disclosure extends to microfluidic devices having additional upstream and downstream microfluidic structures, for example, sample component separation structures, diluting, reacting and/or detecting structures. While the microfluidic devices described above have two layers (a substrate defining microfluidic features and a membrane acting as a cover), the disclosure is equally applicable to microfluidic devices with a construction comprising more than two layers, for example a membrane as a cover layer and two or more substrate layers defining microfluidic features of different depths.

In the disclosure above, a membrane secured as a cover layer to a substrate defining microfluidic features provides a deformable wall of the mixing chamber so that liquids can be agitated in the mixing chamber using a time varying fluid pressure. It will, however, be appreciated that the principles of the disclosure are equally applicable to embodiments in which the deformable wall of the mixing chamber is provided by an integral portion of the substrate defining microfluidic features, for example on an aspect of the substrate opposite a cover layer closing the microfluidic device or opposite other substrate layers defining microfluidic features.

It will be understood that, while the above disclosure has been made with reference to drawings depicting a mixing chamber with a circular cross-section, other shapes of mixing chambers are equally envisaged, and are within the scope of this disclosure. For example, the mixing chamber may have an elongate shape, possibly with a high aspect ratio, resembling a channel, may have convex and concave portions and may be defined by other more complicated shapes, for example a shape of a serpentine or meandering channel.