Flow regulators for the control of liquid fronts in microfluidic structures

Abstract

A microfluidic device with controlled fluid flow, comprising at least one pathway having a fluid-containing chamber 1 comprising first and second walls and one or more side walls which together define

Description

Technical Field The present invention relates to flow regulation in microfluidic devices. In particular it relates to flow regulation using one or more passive valve structures for improved uniformity and flow when filling chambers or defined areas in microfluidic devices. Background Microfluidic devices have gained significant prominence as essential tools in the field of molecular diagnostics, particularly in the development of point-of-care (POC) devices. These POC devices, often configured to include cassettes or cartridges, are revolutionising the healthcare industry by enabling rapid and reliable molecular analyses, such as polymerase chain reaction (PCR) and microarray-based assays, to be performed at or near the patient, minimising turnaround times and improving patient care. Microfluidic devices are particularly useful in biomedical applications due to their small volume of samples and reagents, high throughput, and potential for automation. However, despite their numerous advantages, microfluidic devices used in POC molecular diagnostics continue to face technical limitations due to challenges faced with controlling liquid flow as well as the formation and entrapment of bubbles within the fluidic pathways. Challenges with flow control occur when fluid or fluid slugs move through different areas, potentially with different geometries and / or surface characteristics, in a fluidic or microfluidic device. For example, in WO2019 / 077323 there is a fluid pathway that enters a microarray chamber which has a bubble diversion pathway which directs bubbles away from the viewing area. However, it can be challenging to move fluid through such a chamber without air entrapment. Typically, this is dealt with by significantly reducing flow rate through areas and by including additives such as low foaming non-ionic surfactants e.g., Pluronic® and ethanol in the sample liquid and / or hydrophilic array print on the surface of the chamber. This can result in slow use times due to the time required for areas such as chambers to fill, and additional cost and potential downstream issues with including additives and surface additions. The presence of bubbles within fluidic pathways can also cause problems such as disruption of the flow of fluids and can hinder the performance of downstream analyses such as imaging. This is particularly notable in devices where sections are heated e.g., during PCR reactions, then passed for imaging e.g. on a microarray, as the cyclical heating and cooling of liquid in the fluidic pathways can result in increased bubble formation with bubble entrainment occurring at the liquid-air interface of the microarray chamber. Numerous methods and techniques have been proposed to avoid and inhibit bubble generation in PCR processes in microfluidic systems. For example, bubble traps with micro-porous membranes have been described in US20150209783 and upstream bubble traps that retain bubbles are described in EP17926551. Various methods of venting to the external atmosphere have been described, however, an open system with external venting is not always appropriate, particularly in medical devices where potentially pathogenic material is being tested. It would be advantageous to address either the issues of flow control and / or bubble entrainment in microfluidic devices, especially those designed for POC molecular diagnostics. This patent application aims to obviate or mitigate one or more of the issues associated with flow control and / or bubble formation within microfluidic devices, particularly those utilized in POC molecular diagnostic cassettes and cartridges featuring PCR sections and microarrays. Throughout this document the term "microchannel" or microchamber" refers to a channel or chamber with a hydraulic diameter, in at least one dimension, below 1 mm. The term "chamber" in this document refers to any chamber in a microfluidic device, such as sample chambers and detection chambers. The term chamber can also refer to a portion of microfluidic channel where a particular activity occurs or with particular characteristics. The term "fluid communication' refers to a functional connection between two or more areas or chambers that allows fluids to pass between said areas or chambers. The term "liquid front" refers to the leading edge or boundary of a liquid as it advances or spreads into a different area or medium. Summary of the Invention According to the present invention there is provided a microfluidic device with improved fluidic flow regulation, comprising a device body with at least one microfluidic pathway, said pathway comprising a microfluidic chamber which is at least partially fillable with fluid in use, said microfluidic chamber comprising: a first wall, a second wall and one or more side walls which together define a cavity, said first wall comprising: a first base surface; a second base surface adjacent to, and recessed with respect to, the first base surface, said second base surface defining at least one outer channel, said outer channel comprising at least one flow regulation structure and that constricts the flow of liquid by reducing the cross-sectional area of the chamber at that point. Optionally the flow regulation structure is in the form of a raised section which extends out into the chamber from the second base surface of the first wall. Optionally the flow regulation structure is in the form of a raised section which extends out into the chamber from the second wall. Optionally, the top of the raised section (the end of the raised section that extends furthest into the cavity) is below the first base surface (i.e. it does not extend as far as the first base surface) and above the second base surface (i.e. it does extend as out from and further than the second base surface). Preferably, the flow regulation structure is in the form of a raised section which extends out into the chamber from the side wall, reducing the cross-sectional area of the chamber at that point. Optionally, at the point where the raised section extends upwards the side wall also extends inwards, further reducing the cross-sectional area of the chamber at that point. The raised section is in the form of a wall or bump with a front face extending into the cavity substantially perpendicularly to the second base surface, a top section (e.g., the top of the raised section), and a rear face extending back to the second base surface. The phrase "recessed with respect to" is used to describe the spatial relationship between the first and second surfaces. When the second base surface is "recessed with respect to" the first base surface, it means that the first base surface is positioned spatially further inward into the cavity or e.g. spatially lower (when the device is in use) than the second base surface. Advantageously the second base surface provides a moat, or one or more outer channels, which surround the first base surface. The first base surface is essentially a plinth comprising the first base surface and one or more peripheral plinth walls which extend to the second surface. By controlling or regulating the flow of liquid into and through the moat this allows the fluid flow over the plinth, and thus the first surface, to also be managed resulting in more uniform filling of the chamber with reduced, minimal or no bubble entrainment. Advantageously, liquid travels in the outer channel, an upper wall of which is defined by the second base surface, until it reaches the flow regulation structure. This acts to constrict the liquid flow and creates enough 'back-pressure' to force the liquid 'front' across the first base surface (higher resistance) to a point where the liquid front catches up with the liquid held at the flow regulation structure. This acts like a trigger-valve to release the liquid held at the flow regulation structure such that the liquid then passes across, of flows through, the flow regulation structure into the next part of the outer channel formed by the second base surface. Preferably the flow regulation structure is a passive valve. Active valves require an external energy source such as electrostatic, electromagnetic, pneumatic, hydraulic or photothermal. These energy sources can control fluid flow through the deformation of a boundary, or by changing the state of a boundary, for example, by melting wax or a hydrogel. In contrast, passive valves do not require additional driving equipment and their operating state is determined by the fluid under control. Passive valves do not require external systems to control actuators to provide power (although can be present in systems where fluid is being moved under power). Preferably the flow regulation structure is a capillary force valve. Preferably, the chamber has a fluid inlet and a fluid outlet. Preferably, the first base surface is surrounded, on at least one side, by the second base surface. Advantageously, the second base surface forms a moat or outer channel surrounding the first base surface, with said moat or outer channel comprising one or more flow regulator structures. Preferably, the first base surface is surrounded, on both sides, by the moat or outer channel which comprises a plurality of flow regulator structures. In this case, a reference to both sides refers to the second base surface acting as a split channel such that there is effectively a channel that splits into two outer channel sections when it reaches the upstream edge of the first base surface, the two sections surround the first base surface then ultimately rejoin downstream of the first base surface. Each of two sections is considered a 'side'. Optionally the outer channel comprises a plurality of flow regulator structures. In a particular embodiment there are three flow regulators on each side of the first base surface. In a particular embodiment there are at least three flow regulators on each side of the first base surface. In a particular embodiment there are seven or eight flow regulators on each side of the first base surface. Preferably at least some of the plurality of flow regulator structures are arranged in series, such that fluid flowing into and through the microfluidic chamber sequentially flows via each of the sequentially arranged flow regulator structures. Optionally the first wall and side walls of the microfluidic chamber are formed as a groove in a first substrate and a second substrate is overlaid thus enclosing and the microchannel and forming the lower wall. Optionally the first substrate is substantially rigid. Preferably the first substrate is substantially planar. Optionally the second substrate is a film. Preferably the first substrate and second substrate are bonded together. Preferably the first substrate and second substrate are laser welded together. Optionally, the first substrate and second substrate are bonded with an adhesive. In an embodiment of particular interest, the chamber comprises a microarray. Preferably the microarray is formed on the first surface area. Optionally, the microfluidic chamber is at least partially formed in a plug which is insertable into the first or second substrate, said plug adapted to form at least part of the microfluidic chamber. Optionally the geometry of the first and / or second surface area is provided on the surface of the plug that forms part of the microfluidic chamber. Preferably the first microfluidic chamber comprises outer side walls which are curved. It is preferred to have curved walls as opposed to angles or corners as this ensure the fluid flow streamlines and avoids flowing into the corners, termed stagnation zones in fluid mechanics. Optionally the microfluidic device is designed to allow fluid flow by capillary flow. Optionally the microfluidic device is designed to have fluid flow under positive pressure e.g., pushing of the fluid. Optionally the microfluidic device is designed to have fluid flow under negative pressure e.g., pulling or sucking of the fluid. A method of filling a microfluidic chamber in the microfluidic device defined above, comprising flowing fluid into the microfluidic chamber until it reaches the flow regulation structure which acts to constrict the liquid flow and create enough 'back-pressure' to force the liquid 'front' across the first base surface to a point where the liquid front flowing across the first base surface contacts and releases the liquid held at the flow regulation structure such that the liquid then passes across the flow regulation structure into the next part of the outer channel formed by the second base surface. Various further features and aspects of the invention are defined in the claims. Where any reference is made to 'upper' or 'lower' walls or surfaces in a chamber or channel, it is clear that this can be the other way given that as this is referring to microfluidic flow, gravity does not really have a dominant effect, and therefore orientation is not important. Unless defined otherwise, all technical and scientific terms used herein have the meaning commonly understood by a person skilled in the art to which this invention belongs. Brief Description of the Drawings Embodiments of the present invention will now be described by way of example only with reference to the accompanying drawings where like parts are provided with corresponding reference numerals and in which: 5 Figure la is a plan view of a section of a microarray chamber in accordance with the present invention; Figure lb is an expanded view of a portion of Figure la; 10 Figure 2 is a plan view of a section of a microarray chamber in accordance with another embodiment of the present invention; Figure 3 shows fluid flow in the microarray chamber of Figure 2; and 15 Figure 4 is a table summarising the microarray experimental work. Detailed Description In an exemplary embodiment of the invention, there is provided a microfluidics cassette with a continuous flow-through micro-channel. The micro-channel is formed on the inside of the microfluidic cassette, in the desired length and shape so as to allow the passage of a sample, preferably a biological sample, in liquid format along a fluid flow path. The channel is formed in the lower surface of a first substrate, in this embodiment the first substrate is polycarbonate. The first substrate is overlaid with a second substrate that may itself have grooves formed in its upper surface that can be aligned with the channels of the first substrate. By bonding the substrates together, a substantially closed channel is provided (inlets and outlets can be included as required). Again, as this is referring to microfluidic flow, gravity does not really have a dominant effect, and therefore orientation of upper and lower can be the other way round. Any appropriate means of bonding can be used; however laser welding is particularly preferred. Where necessary, the first and second substrates can be aligned prior to bonding. The length and cross-sectional shape of the channel can be any appropriate shape to allow for the desired transport and processing of a sample. For example, the micro-channel 2 can have a cross sectional area of about 0.01 pm2 to 100 mm2. An area or a portion of, or chamber in, the micro-channel is dedicated to performing PCR such that nucleic acids of interest are amplified. This portion may have annealing, extension and denaturation areas. Then, downstream from the PCR portion of the cassette, there is a portion of the channel that forms a microarray chamber that provides for capture of the amplified material. The microarray chamber also allows for the viewing or imaging of the captured material through a viewing surface. For example, a camera can be aligned with the microarray chamber. A plan view of a section of a microarray chamber 1 in accordance with an aspect of the invention is depicted in Figure la. The chamber 1 has a first base surface 2 positioned relatively centrally in the chamber 1, providing a plinth-like structure with a first base surface 2a and a plinth wall 2b extending perpendicularly from the periphery thereof, and a second base surface 3 which is recessed with respect to, and fully surrounds, the first base surface 2 to define a moat or outer channel (effectively a main channel that splits into a first and second split outer channel then rejoins downstream of the first base surface). The first base surface 2a is the microarray surface to which a plurality of biological probes are attached. The second base surface 3, which delimits a wall of the moat has a number of raised bumps 4 or walls (in Figure la there are eight raised bumps 4 on each side of the first base surface 2 which is in the form of a microarray). The chamber also has an inlet 5 at the upstream end of the chamber 1, and an outlet 6 at the downstream end of the chamber 1. In this embodiment the first base surface has a teardrop shape with the narrower portion of the teardrop 9 being at the downstream end of the chamber. This helps to avoid liquid from 1st filled split channel closing off airflow out of 2nd still unfilled split channel in case of asymmetric filling. A more detailed view of a section of the chamber 2 is shown in Figure lb. This shows in more detail the raised bumps 4 in the moat formed on the second base surface 3 (the section of the moat and the raised bump together form a delay valve). It can also be seen that the outer side wall 8 of the chamber is shaped such that it has protruding sections 7a which protrude inwards into the channel formed by the second surface 3 further reducing the cross-sectional area of the outer channel formed by the second surface 3 at that point. Similarly, the external circumference of the first base surface 2 also has protruding sections 7b which are shaped to protrude outwards into the channel formed by the second base surface 3 further reducing the cross-sectional area of the channel formed by the second surface 3 at that point. In this example, the first surface 2 is recessed 170 pm from the upper wall (not shown) which is a window. The total height of the chamber from the lowest point of second base surface (i.e., where there is no bump 4) to the opposite wall is 600 pm. The outer channel or moat at points where there is no bump extends 1.1mm between the outer wall 8 and the plinth wall 2b. The bumps 4 are in the form of walls which extend 200 pm substantially perpendicularly to and from the second base surface 2 (100 pm and 150 pm walls have also been shown to work in this set-up). The top surface of the bump 4 extends 550pm between the outer wall 8 and the plinth wall 2b - due to the protruding sections 7a and 7b - and 250pm in width between its front (upstream) and back (downstream) faces. Another embodiment of a microarray chamber 1' is shown in Figure 2, again as a plan view through a section of the chamber without the upper wall. Again, the chamber 1 has a first base surface 2' positioned relatively centrally in the chamber 1, providing a plinth-like structure with a plinth wall 2b' extending perpendicularly from the periphery of the first base surface 2', and a second base surface 3' which is recessed with respect to, and fully surrounds, the first base surface 2' to provide a moat or outer channel (which effectively splits into two outer channel sections). The first base surface 2' is again the microarray surface to which a plurality of biological probes are attached. The second base surface 3' or moat has a number of raised bumps 4 or walls. In this embodiment there are three raised bumps 4' on each side of the first base surface 2' which is in the form of a microarray. The raised bumps 4', together with the portion of the channel or moat directly upstream of the bump 4' form a delay valve. Reducing the number of bumps 4' (or delay valves) in a portion of channel provides more space for bubble handling if bubbles are present. The chamber 1' also has an inlet 5' at the upstream end of the chamber 1', and an outlet 6' at the downstream end of the chamber 1'. Similar dimensions can be used to those described above for Figure la and lb. In this embodiment the first base surface has an extension 9' at the downstream end of the chamber. This helps to avoid liquid from 1st filled split channel closing off airflow out of 2nd still unfilled split channel in case of asymmetric filling. The basic design principles for the invention are that capillary force valves are used to create a pressure barrier for the interface during initial filling of an outer channel (formed by the recessed second base surface) of a chamber which has a plinth-like structure comprising a first base surface therein. The capillary force valves cause a pressure increase that pushes the liquid towards onto the centre of the chamber and over the plinth-like structure. The outer channel is made as wide as possible to accommodate a greater volume of bubbles in the channels, while maintaining functionality during initial filling. There is a trade-off between number of valves for sufficient control of the initial interface, while optimising volume to accommodate bubbles. Manufacturing constraints also dictate the number and size of valves. Separation between the two channels avoids liquid from 1st filled channel closing off airflow out of 2nd still unfilled channel in case of asymmetric filling. Although the embodiments here describe bumps 4 and 4' and protruding sections 7a, 7a', 7b and 7b' which together provide zones with reduced cross-sectional area. One skilled in the art would understand that either the bumps or the protruding sections could be used. It is however preferred to have at least the protruding sections 7a, 7a', 7b. Figure 3 shows how liquid will flow through the microarray chamber 1' of Figure 2 (although it would be understood that the same flow characteristic would occur in other chambers designed in accordance with the invention e.g., the chamber 1 shown in figure la). When a liquid sample enters through inlet 5' it initially flows onto the upstream section of the second base surface 3'. This initial upstream section widens out in an expanding cone-like shape and has relatively low resistance due to its wider surface area compared to the inlet 5'. When the liquid front meets the front face of the plinth wall 2b' (which in this embodiment extends substantially vertically upward relative to the second surface 3') it splits into two streams, shown as (A) in Figure 3, following the moat-like channel formed by the second surface 3', the outer side wall 8 and the plinth wall 2b. Each stream progresses until it reaches the front wall of a raised bump 4 which temporarily halts the flow as it constricts the liquid flow. This creates enough 'back-pressure' to force the liquid front to flow across the first base surface 2. The liquid front flows across the first base surface until it contacts and releases the liquid held at the raised bumps 4a' (shown as (B) in Figure 3) such that the liquid then passes over the flow regulation structure into the next part of the outer channel formed by the second base surface. It will then follow the same pattern as it will progress until it reaches the front wall of the next raised bump 4b', which again temporarily halts the flow as it constricts the liquid flow. This again creates enough 'back-pressure' to force the liquid front to flow further across the first base surface 2'. The liquid front flows across the first base surface until it contacts and releases the liquid held at the raised bumps 4b' (shown as (C) in Figure 3). Experimental Work In order to test the benefit of filling a chamber using the present inventions, tests were carried out using a version with and without the presence of delay valves. The version without the delay valves did not have any areas of reduced cross-sectional areas in the split channels (the split channels generally having a height and width of 600 pm). The version with the valves was as described above with respect to Figures la and lb. The results of the testing are shown in Figure 4 and it can be seen that there are clear benefits. Explanatory notes regarding the results are as follows: Filling with Water: Array chamber fills with tap water and MQ. water both- reproducible - This is the worst case with regards to surface tension. Yes: Chamber fills with the liquid samples (solvent)- reproducible No: Chamber does not fill properly, fluid has unpredictable behaviour Unreliable: Chamber get filled sometimes only; data not reproducible Filling Time: Time required to fill the chamber fully. Air / Water Bubbles tolerated: Chamber fills when bubbles are contained in water slug, and bubbles are not pushed to recessed array area. Filling with Elution Buffer + 0.1% Pluronic: Sample contains Pluronic to facilitate the filling, filling is not reproducible every time. Filling with Elution: Buffer to elute the DNA from the filter followed by sample PCR before entering in the chamber, chamber gets filled with elute buffer- reproducible. Filling with Elution Buffer with 70ul air slug: Elution buffer combined with 70ul of continuous air slug and elution buffer with bubbles, chamber get fills with elution buffer and no bubble on recessed circle, bubbles and air slug pass through the side trench- reproducible. Washing with PBST: PBS Buffer with non-ionic surfactant tween 20 to wash the array after Hybridisation; chamber gets filled with PBST-reproducible. Max PBST Bubble volume tolerated: How much bubble volume in the PBST slug can be tolerated. All of the features disclosed in this specification (including any accompanying claims, abstract and drawings), and / or all of the steps of any method or process so disclosed, may be combined in any combination, except combinations where at least some of such features and / or steps are mutually exclusive. Each feature disclosed in this specification (including any accompanying claims, abstract and drawings) may be replaced by alternative features serving the same, equivalent or similar purpose, unless expressly stated otherwise. Thus, unless expressly stated otherwise, each feature disclosed is one example only of a generic series of equivalent or similar features. The invention is not restricted to the details of the foregoing embodiment(s). The invention extends to any novel one, or any novel combination, of the features disclosed in this specification (including any accompanying claims, abstract and drawings), or to any novel one, or any novel combination, of the steps of any method or process so disclosed. In particular flow regulation structures may be in the form of bumps or protrusions as described in preferred embodiments, but could also for example be moulded as part of the second substrate and then positioned into the moat, on bonding of the substrate to create a similar valve structure. With respect to the use of substantially any plural and / or singular terms herein, those having skill in the art can translate from the plural to the singular and / or from the singular to the plural as is appropriate to the context and / or application. The various singular / plural permutations may be expressly set forth herein for sake of clarity. It will be understood by those within the art that, in general, terms used herein, and especially in the appended claims are generally intended as "open" terms (e.g., the term "including" or "comprising" should be interpreted as "including but not limited to," the term "having" should be interpreted as "having at least," the term "includes" should be interpreted as "includes but is not limited to," etc.). It will be further understood by those within the art that if a specific number of an introduced claim recitation is intended, such an intent will be explicitly recited in the claim, and in the absence of such recitation no such intent is present. For example, as an aid to understanding, the following appended claims may contain usage of the introductory phrases "at least one" and "one or more" to introduce claim recitations. However, the use of such phrases should not be construed to imply that the introduction of a claim recitation by the indefinite articles "a" or "an" limits any particular claim containing such introduced claim recitation to embodiments containing only one such recitation, even when the same claim includes the introductory phrases "one or more" or "at least one" and indefinite articles such 5 as "a" or "an" (e.g., "a" and / or "an" should be interpreted to mean "at least one" or "one or more"); the same holds true for the use of definite articles used to introduce claim recitations. In addition, even if a specific number of an introduced claim recitation is explicitly recited, those skilled in the art will recognize that such recitation should be interpreted to mean at least the recited number (e.g., the bare recitation of "two recitations," without other 10 modifiers, means at least two recitations, or two or more recitations). It will be appreciatedthat various embodiments of the present disclosure have been described herein for purposes of illustration, and that various modifications may be made without departing from the scope of the present disclosure. Accordingly, the various embodiments 15 disclosed herein are not intended to be limiting, with the true scope being indicated by the following claims.

Claims

1. A microfluidic device with improved fluidic flow regulation, comprising a device body with at least one microfluidic pathway, said pathway comprising a microfluidic chamber which is at least partially fillable with fluid in use, said microfluidic chamber comprising:a first wall, a second wall and one or more side walls which together define a cavity, said first wall comprising:a first base surface;a second base surface adjacent to, and recessed with respect to, the first base surface, said second base surface defining at least one outer channel, said outer channel comprising at least one flow regulation structure and that constricts the flow of liquid by reducing the cross-sectional area of the chamber at that point2. A microfluidic device as in Claim 1 wherein the flow regulation structure is in the form of a raised section which extends out from the second surface of the first wall or from the second wall.

3. A microfluidic device as in Claim 2 wherein the end of the raised section that extends furthest into the cavity does not extend as far as the first base surface and does extend as out from and further than the second base surface4. A microfluidic device as in Claims 2 or 3 wherein the flow regulation structure is in the form of a raised section which extends out into the chamber from the side wall, reducing the cross-sectional area of the chamber at that point.

5. A microfluidic device as in any of the previous claims wherein the flow regulation structure is a passive valve.

6. A microfluidic device as in any of the previous claims wherein the flow regulation structure is a capillary force valve.

7. A microfluidic device as in any of the previous claims wherein the chamber has a fluid inlet and a fluid outlet.

8. A microfluidic device as in any of the previous claims wherein the first base surface is surrounded, on at least one side, by the second base surface, the second base surface comprising at least one flow regulator structure.

9. A microfluidic device as in any of the previous claims wherein the first base surface is surrounded, on both sides, by the second base surface which comprises a plurality of flow regulator structures.

10. A microfluidic device as in any of the previous claims wherein the second base surface comprises a plurality of flow regulator structures.

11. A microfluidic device as in claim 10 wherein at least some of the plurality of flow regulator structures are arranged in series, such that fluid flowing into and through the microfluidic chamber sequentially flows via each of the sequentially arranged flow regulator structures.

12. A microfluidic device as in any of the previous claims wherein the first wall and side walls of the microfluidic chamber are formed as a groove in a first substrate and a second substrate is overlaid thus enclosing and the microchannel and forming the second wall.

13. A microfluidic device as in claim 12 wherein the first substrate is substantially rigid.

14. A microfluidic device as in any of claims 12 or 13 wherein the first substrate is substantially planar.

15. A microfluidic device as any of claims 12 to 14 wherein the second substrate is a film.

16. A microfluidic device as in any of claims 12 to 15 wherein the first substrate and second substrate are bonded together.

17. A microfluidic device as in claims 12 to 16 wherein the first substrate and second substrate are laser welded together.

18. A microfluidic device as in any of the previous claims wherein the chamber comprises a microarray.

19. A microfluidic device as in claim 18 wherein the microarray is formed on the first surface area.

20. A microfluidic device as in any of the previous claims wherein the microfluidic chamber is at least partially formed in a plug which is insertable into the first or second substrate, said plug adapted to form at least part of the microfluidic chamber.

21. A microfluidic device as in any of the previous claims wherein the microfluidic chamber comprises outer side walls which are curved.

22. A microfluidic device as in any of the previous claims wherein the microfluidic device is designed to allow fluid flow by capillary flow.

23. A microfluidic device as in any of the previous claims wherein the microfluidic device is designed to have fluid flow under positive pressure e.g., pushing of the fluid.

24. A microfluidic device as in any of the previous claims wherein the microfluidic device is designed to have fluid flow under negative pressure e.g., pushing of the fluid.

25. A method of filling a microfluidic chamber in the microfluidic device defined in any of claims 1 to 24, comprising flowing fluid into the microfluidic chamber until it reaches the flow regulation structure which acts to constrict the liquid flow and create enough 'back-pressure' to force the liquid 'front' across the first base surface to a point wherethe liquid front flowing over the first base surface contacts and releases the liquid held at the flow regulation structure such that the liquid then passes over the flow regulation structure into the next part of the outer channel formed by the second base surface.

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