Patent Publication Number: US-2021178396-A1

Title: Microfluidic device and a method of loading fluid therein

Description:
RELATED APPLICATIONS 
     This application is continuation application of U.S. application Ser. No. 15/759,685 filed on Mar. 13, 2018, which is a national phase of International Patent Application Serial No. PCT/JP2016/004199 filed on Sep. 14, 2016 which claims priority to GB Application No. 1516430.4 filed on Sep. 16, 2015, the entire disclosures of which are hereby incorporated by reference. 
    
    
     TECHNICAL FIELD 
     The present invention relates to a microfluidic device, and to a method for loading fluid into such a device. More particularly, the invention relates to an Active Matrix Electro-wetting on Dielectric (AM-EWOD) microfluidic device. Electro-wetting-On-Dielectric (EWOD) is a known technique for manipulating droplets of fluid on an array. Active Matrix EWOD (AM-EWOD) refers to implementation of EWOD in an active matrix array incorporating transistors, for example by using thin film transistors (TFTs). 
     BACKGROUND ART 
     Microfluidics is a rapidly expanding field concerned with the manipulation and precise control of fluids on a small scale, often dealing with sub-microlitre volumes. There is growing interest in its application to chemical or biochemical assay and synthesis, both in research and production, and applied to healthcare diagnostics (“lab-on-a-chip”). In the latter case, the small nature of such devices allows rapid testing at point of need using much smaller clinical sample volumes than for traditional lab-based testing. 
     A microfluidic device can be identified by the fact that it has one or more channels (or more generally gaps) with at least one dimension less than 1 millimeter (mm). Common fluids used in microfluidic devices include whole blood samples, bacterial cell suspensions, protein or antibody solutions and various buffers. Microfluidic devices can be used to obtain a variety of interesting measurements including molecular diffusion coefficients, fluid viscosity, pH, chemical binding coefficients and enzyme reaction kinetics. Other applications for microfluidic devices include capillary electrophoresis, isoelectric focusing, immunoassays, enzymatic assays, flow cytometry, sample injection of proteins for analysis via mass spectrometry, PCR amplification, DNA analysis, cell manipulation, cell separation, cell patterning and chemical gradient formation. Many of these applications have utility for clinical diagnostics. 
     Many techniques are known for the manipulation of fluids on the sub-millimetre scale, characterised principally by laminar flow and dominance of surface forces over bulk forces. Most fall into the category of continuous flow systems, often employing cumbersome external pipework and pumps. Systems employing discrete droplets instead have the advantage of greater flexibility of function. 
     Electro-wetting on dielectric (EWOD) is a well-known technique for manipulating discrete droplets of fluid by application of an electric field. It is thus a candidate technology for microfluidics for lab-on-a-chip technology. An introduction to the basic principles of the technology can be found in “Digital microfluidics: is a true lab-on-a-chip possible?” (R. B. Fair, Microfluid Nanofluid (2007) 3:245-281). This review notes that methods for introducing fluids into the EWOD device are not discussed at length in the literature. It should be noted that this technology employs the use of hydrophobic internal surfaces. In general, therefore, it is energetically unfavourable for aqueous fluids to fill into such a device from outside by capillary action alone. Further, this may still be true when a voltage is applied and the device is in an actuated state. Capillary filling of non-polar fluids (e.g. oil) may be energetically favourable due to the lower surface tension at the liquid-solid interface. 
     A few examples exist of small microfluidic devices where fluid input mechanisms are described. U.S. Pat. No. 5,096,669 (Lauks et al.; published Mar. 17, 1992) shows such a device comprising an entrance hole and inlet channel for sample input coupled with an air bladder which pumps fluid around the device when actuated. It is does not describe how to input discrete droplets of fluid into the system nor does it describe a method of measuring or controlling the inputted volume of such droplets. Such control of input volume (known as “metering”) is important in avoiding overloading the device with excess fluid and helps in the accuracy of assays carried out where known volumes or volume ratios are required. 
     US20100282608 (Srinivasan et al.; published Nov. 11, 2010) describes an EWOD device comprising an upper section of two portions with an aperture through which fluids may enter. It does not describe how fluids may be forced into the device nor does it describe a method of measuring or controlling the inputted volume of such fluids. Related application US20100282609 (Pollack et al.; published Nov. 11, 2010) does describe a piston mechanism for inputting the fluid, but again does not describe a method of measuring or controlling the inputted volume of such fluid. 
     US20100282609 describes the use of a piston to force fluid onto reservoirs contained in a device already loaded with oil. US20130161193 describes a method to drive fluid onto a device filled with oil by using, for example, a bistable actuator. 
     SUMMARY OF INVENTION 
     A first aspect of the invention provides a method of loading a microfluidic device with an assay fluid, the method comprising: introducing, into a chamber in the microfluidic device, the chamber having one or more inlet ports, a metered volume of a filler fluid such that the chamber is partially filled with the filler fluid, said device being configured to preferentially maintain the metered volume of the filler fluid in a part of the chamber; and introducing a volume of the assay fluid into the part of the chamber via one of the one or more inlet ports and thereby causing a volume of a venting fluid to vent from the chamber. 
     A second aspect of the invention provides a method of loading a microfluidic device with an assay fluid, the method comprising: substantially completely filling a chamber with a filler fluid or with a fluid mixture containing a filler fluid as one component, the chamber having one or more inlet ports and an outlet port for extracting the filler fluid; inserting a volume of the assay fluid into one of the one or more inlet ports; and extracting sufficient of the filler fluid through the outlet port to enable at least some of the volume of the assay fluid to enter the chamber from the one of the one or more inlet ports. 
     A third aspect of the invention provides a microfluidic device, comprising: a chamber having one or more inlet ports; said device being configured to, when the chamber contains a metered volume of a filler fluid that partially fills the chamber, preferentially maintain the metered volume of the filler fluid in a part of the chamber; and the device being configured to allow displacement of some of the filler fluid from the part of the chamber when a volume of an assay fluid introduced into one of the one or more inlet ports enters the part of the chamber, thereby causing a volume of a venting fluid to vent from the chamber. 
     A fourth aspect of the invention provides a microfluidic device, comprising: a chamber having one or more inlet ports and an outlet port for extracting a filler fluid; whereby in use the chamber is substantially completely filled with the filler fluid, and a volume of an assay fluid introduced into one of the one or more inlet ports is enabled to enter the chamber as sufficient of the filler fluid is extracted through the outlet. 
    
    
     
       BRIEF DESCRIPTION OF DRAWINGS 
         FIG. 1  is a schematic diagram depicting a conventional AM-EWOD device in cross-section. 
         FIG. 2 a    is a schematic diagram depicting a plan view of a microfluidic device in accordance with a first and exemplary embodiment of the invention. 
         FIG. 2 b    is schematic diagrams depicting plan view of a microfluidic device in accordance with a second embodiment of the invention. 
         FIG. 2 c    is schematic diagrams depicting cross-sectional view of a microfluidic device in accordance with a second embodiment of the invention. 
         FIG. 3 a    is schematic diagram depicting a method of loading a microfluidic device in accordance with the first embodiment of the invention. 
         FIG. 3 b    is schematic diagram depicting a method of loading a microfluidic device in accordance with the first embodiment of the invention. 
         FIG. 3 c    is schematic diagram depicting a method of loading a microfluidic device in accordance with the first embodiment of the invention. 
         FIG. 3 d    is schematic diagram depicting a method of loading a microfluidic device in accordance with the first embodiment of the invention. 
         FIG. 4 a    is schematic diagram depicting a method of loading a microfluidic device in accordance with the second embodiment of the invention. 
         FIG. 4 b    is schematic diagram depicting a method of loading a microfluidic device in accordance with the second embodiment of the invention. 
         FIG. 4 c    is schematic diagram depicting a method of loading a microfluidic device in accordance with the second embodiment of the invention. 
         FIG. 4 d    is schematic diagram depicting a method of loading a microfluidic device in accordance with the second embodiment of the invention. 
         FIG. 5 a    is schematic diagram depicting a method of loading a microfluidic device in accordance with a third embodiment of the invention. 
         FIG. 5 b    is schematic diagram depicting a method of loading a microfluidic device in accordance with a third embodiment of the invention. 
         FIG. 5 c    is schematic diagram depicting a method of loading a microfluidic device in accordance with a third embodiment of the invention. 
         FIG. 5 d    is schematic diagram depicting a method of loading a microfluidic device in accordance with a third embodiment of the invention. 
         FIG. 6 a    is schematic diagram depicting a method of loading a microfluidic device in accordance with a fourth embodiment of the invention. 
         FIG. 6 b    is schematic diagram depicting a method of loading a microfluidic device in accordance with a fourth embodiment of the invention. 
         FIG. 6 c    is schematic diagram depicting a method of loading a microfluidic device in accordance with a fourth embodiment of the invention. 
         FIG. 6 d    is schematic diagram depicting a method of loading a microfluidic device in accordance with a fourth embodiment of the invention. 
         FIG. 7 a    is schematic diagram depicting a method of loading a microfluidic device in accordance with a fifth embodiment of the invention. 
         FIG. 7 b    is schematic diagram depicting a method of loading a microfluidic device in accordance with a fifth embodiment of the invention. 
         FIG. 7 c    is schematic diagram depicting a method of loading a microfluidic device in accordance with a fifth embodiment of the invention. 
         FIG. 7 d    is schematic diagram depicting a method of loading a microfluidic device in accordance with a fifth embodiment of the invention. 
         FIG. 8 a    is schematic diagram depicting a method of loading a microfluidic device in accordance with a sixth embodiment of the invention. 
         FIG. 8 b    is schematic diagram depicting a method of loading a microfluidic device in accordance with a sixth embodiment of the invention. 
         FIG. 8 c    is schematic diagram depicting a method of loading a microfluidic device in accordance with a sixth embodiment of the invention. 
         FIG. 8 d    is schematic diagram depicting a method of loading a microfluidic device in accordance with a sixth embodiment of the invention. 
         FIG. 8 e    is schematic diagram depicting a method of loading a microfluidic device in accordance with a sixth embodiment of the invention. 
         FIG. 9 a    is schematic diagram depicting a method of loading a microfluidic device in accordance with a seventh embodiment of the invention. 
         FIG. 9 b    is schematic diagram depicting a method of loading a microfluidic device in accordance with a seventh embodiment of the invention. 
         FIG. 9 c    is schematic diagram depicting a method of loading a microfluidic device in accordance with a seventh embodiment of the invention. 
         FIG. 9 d    is schematic diagram depicting a method of loading a microfluidic device in accordance with a seventh embodiment of the invention. 
         FIG. 10 a    is a graphical representation of a cartridge based around a microfluidic device. 
         FIG. 10 b    is an exploded view of the cartridge of  FIG. 10   a.    
         FIG. 11 a    is a graphical representation of a benchtop reader device to control the operation of a microfluidic device. 
         FIG. 11 b    is a graphical representation of a handheld reader device to control the operation of a microfluidic device. 
     
    
    
     DESCRIPTION OF EMBODIMENTS 
     To the accomplishment of the foregoing and related ends, the invention comprises the features hereinafter fully described and identified in the claims. The following description and the annexed drawings set forth in detail certain illustrative embodiments of the invention. These embodiments are indicative, however, of but a few of the various ways in which the principles of the invention may be employed. Other objects, advantages and novel features of the invention will become apparent from the following detailed description of the invention when considered in conjunction with the drawings. 
     Although the invention has been shown and described with respect to a certain embodiment or embodiments, equivalent alterations and modifications may occur to others skilled in the art upon the reading and understanding of this specification and the annexed drawings. In particular regard to the various functions performed by the above described elements (components, assemblies, devices, compositions, etc.), the terms (including a reference to a “means”) used to describe such elements are intended to correspond, unless otherwise indicated, to any element which performs the specified function of the described element (i.e., that is functionally equivalent), even though not structurally equivalent to the disclosed structure which performs the function in the herein exemplary embodiment or embodiments of the invention. In addition, while a particular feature of the invention may have been described above with respect to only one or more of several embodiments, such feature may be combined with one or more other features of the other embodiments, as may be desired and advantageous for any given or particular application. 
       FIG. 1  is a schematic diagram depicting a conventional AM-EWOD device  1  in cross-section. The AM-EWOD device  1  has a lower substrate  6 , for example a CG (“continuous grain”) silicon substrate and an upper substrate  2 , for example of indium tin oxide (ITO) coated glass. Electrodes  3  are disposed upon the upper and lower substrates  2 ,  6 . The electrodes  3  control the movement of liquid droplets  8  through the device  1 . A liquid droplet  8 , which may consist of any polar liquid and which typically may be ionic and/or aqueous, is enclosed between the lower substrate  6  and the top substrate  2 , although it will be appreciated that multiple liquid droplets  8  can be present. The content of the liquid droplet will be referred to herein as “assay fluid” for convenience but, as explained below, this does not mean that the invention is limited to use in performing an assay 
     A general requirement for the operation of the device is that the assay fluid comprises a polar fluid, typically a liquid, that may be manipulated by electro-mechanical forces, such as the electro-wetting force, by the application of electrical signals to the electrodes. Typically, but not necessarily, the assay fluid may comprise an aqueous material, although non-aqueous assay fluids (e.g. ionic liquids) may also be manipulated. Typically, but necessarily, the assay fluid may contain a concentration of dissolved salts, for example in the range 100 nM-100M or in the range 1 uM to 10M or in the range 10 uM to 1M or in the range 100 uM to 100 mM or in the range 1 mM to 10 mM. 
     The assay fluid may optionally comprise a quantity of a surfactant material. The addition of a surfactant may be beneficial for reducing the surface tension at the interface between the droplet and the filler fluid. The addition of a surfactant may have further benefits in reducing or eliminating unwanted physical or chemical interactions between the assay liquid and the hydrophobic surface. Non-liming examples of surfactants that may be used in electro-wetting on dielectric systems include Brij 020, Brij 58, Brij S100, Brij S10, Brij S20, Tetronic 1107, IGEPAL CA-520, IGEPAL CO-630, IGEPAL DM-970, Merpol OJ, Pluronic F108, Pluronic L-64, Pluronic F-68, Pluronic P-105, Tween-20, Span-20, Tween-40, Tween-60. 
     Whilst the term assay is generally taken to refer to some analytical procedure, method or test, the term assay fluid in the scope of this invention may be taken more widely to refer to a fluid involved in any chemical or biochemical processes as may be performed on the AM-EWOD device, for example, but not limited to the following: 
     (a) A laboratory test for testing for the presence, absence or concentration of some molecular or bio-molecular species, for example a molecule, a protein, a sequence of nucleic acid etc 
     (b) A medical or bio-medical test for testing for the presence, absence or concentration of some physiological fluid, species or substance, for example a medical diagnostic test 
     (c) A procedure for preparing a material sample, for example the extraction, purification and/or amplification of a biochemical species, including but not limited to, a nucleic acid, a protein from a sample, a single cell from a sample 
     (d) A procedure for synthesising a chemical or bio-chemical compound, including, but not limited to the examples of a protein, a nucleic acid, a pharmaceutical product or a radioactive tracer 
     A suitable gap between the two substrates may be realized by means of a spacer  9 , and a non-polar filler fluid  7 , which could be oil, for example dodecane, silicone oil or other alkane oil, or alternatively air, may be used to occupy the volume not occupied by the liquid droplet  8 . The inner surfaces of the upper  2  and lower substrates  6  may have a hydrophobic coating  4 . Non-limiting examples of materials that may be used to form the hydrophobic coating include Teflon AF1600, Cytop, Parylene C and Parylene HT. 
     The lower substrate  6  may further be provided with an insulator layer  5 . Here, and elsewhere, the invention has been described with regard to an Active Matrix Electro-wetting on dielectric device (AM-EWOD). It will be appreciated however that the invention, and the principles behind it, are equally applicable to a ‘passive’ EWOD device, whereby the electrodes are driven by external means, as is well known in prior art. Likewise, in this and subsequent embodiments the invention has been described in terms of an AM-EWOD device utilizing thin film electronics  74  to implement array element circuits and driver systems in thin film transistor (TFT) technology. It will be appreciated that the invention could equally be realized using other standard electronic manufacturing processes to realise Active Matrix control, e.g. Complementary Metal Oxide Semiconductor (CMOS), bipolar junction transistors (BJTs), and other suitable processes. 
       FIG. 2 a    is a schematic plan view of a microfluidic device in accordance with a first and exemplary embodiment of the invention. In this embodiment the device  100  is an electro-wetting on dielectric Active Matrix Electro-wetting on Dielectric (AM-EWOD) device comprising electrodes (not shown in  FIG. 2 a   ). As in  FIG. 1 , the device  100  comprises a lower substrate (not visible in  FIG. 2 a   ), an upper substrate  102  spaced from the lower substrate so that a fluid chamber  101  is formed between the upper and lower substrates, and a fluid barrier provided between the lower substrate and the upper substrate  102  to define a perimeter of the chamber  101 . The interior of the chamber  101  is at least partially coated with a hydrophobic coating. In this illustrated example, the fluid barrier is an adhesive track  106 . The adhesive track  106  adheres the upper substrate  102  (in this example comprising ITO coated glass) to the lower substrate (in this example comprising a TFT chip). 
     To manufacture the device of this embodiment, the substrates are prepared and a glue track is disposed on one substrate. A spacer, for example a Kapton spacer, having a thickness equal to the desired cell gap is placed between the substrates, and the substrates are pushed together until the spacer prevents them from being pushed closer together. The glue is then cured to make it hard and seal the device. The cured glue track thus serves both to adhere the substrates to one another and to form a fluid barrier that retains fluids within the device chamber  101 . Once the glue track has been cured, the spacer may be removed since the glue track is now the correct thickness or alternatively the spacer may be retained. The glue track may be formed of any suitable material that will adhere the substrates together and form a fluid seal. 
     As an alternative, a photoresist pattern having the same general shape as the adhesive track of  FIG. 3 a    may be formed on one substrate, for example by UV patterning. The photoresist pattern may then be used to bond the top and bottom substrates together, for example by heating the photoresist. No separate spacer is required, since the thickness of the photoresist pattern may be chosen to provide a desired cell gap between the substrates. 
     It should be understood that the invention is not limited to any particular implementation of the barrier. In principle a device of the invention could have a fluid barrier that does not adhere the substrates together. As a further example, the barrier could be a gap in the top substrate, for example a slot that is cut out of the top plate and that has a similar shape to the barrier of  FIG. 2 a   . When oil (or other filler fluid) is introduced into the chamber, the oil would not cross the slot, but would fill the region inside this slot in the same way that it fills around a hole in the top substrate. Alternatively, a groove may be provided in the lower surface of the upper substrate—provided that the groove were of sufficient depth, oil would again not cross the groove and would be contained in the region inside the groove. (It will be understood that, if a slot is provided in the upper substrate, gaps are preferably left in the slot so that the slot does not divide the substrate into two separate pieces.) 
     The chamber  101  has a plurality of inlet ports  111 ,  112  and a plurality of vents  110 . The inlet ports  111 ,  112  and vents  110  are provided in the upper substrate  102  of the device  100 . In this example, the inlet ports comprise assay fluid inlets ports  111  and an oil inlet port  112 . The inlet ports  111 ,  112  and the vents  110  are shown as (substantially) identical, comprising apertures in the upper substrate  102 . However the invention is not limited to this, the inlet ports may be formed to be of differing sizes to one another, to hold different volumes of assay fluid. The apertures may be produced using a variety of techniques, for example, laser drilling or HF (hydrofluoric acid) etching, CNC drilling, powderblasting and moulding (in examples where the top plate is made of a plastics material). The vents  110  are substantially located at the periphery of the chamber  101 . For example, at least one of the vents  110  is located in a corner of the chamber  101 . 
     The chamber  101  further comprises a vent area  105  which is in fluid communication with at least one of the vents  110 . The chamber  101  further comprises an active area  109  for carrying out one or more assays. The active area  109  is defined as the area over which fluid is loaded into the device and the assay is carried out. The vent area  105  and the active area  109  are defined by the adhesive track  106 . In addition to a vent  110  at the end of the vent area  105 , there is provided a further vent  110  at the end of the adhesive track  106 , which separates the vent area  105  from the active area  109 . This vent  110  is shown on the right hand side of  FIG. 2   a.    
     As noted, the device is provided with electrodes (not shown in  FIG. 2 a   ) in the active area, to allow manipulation of droplets of assay fluid within the active area. These electrodes may be considered as defining one or more “internal reservoirs” (not shown) in which fluid may be controlled by actuation of the electrodes of the device  100 . 
     The device  100  is configured to, when the chamber  101  contains a metered volume of a filler fluid such as oil (not shown here) that partially fills the chamber  101 , preferentially maintain the metered volume of the filler fluid in a part of the chamber  101 ; and to allow displacement of some of the filler fluid from the part of the chamber  101  when a volume of an assay fluid (not shown here) is introduced into one of the one or more inlet ports  111  enters the part of the chamber  101 , thereby causing a volume of a venting fluid to vent through at least one of the vents  110 . This is explained in more detail below. 
       FIG. 2 b    is a schematic diagram depicting a microfluidic device in accordance with a second embodiment of the invention. The device  100  of  FIG. 2 a    may be described as top-loading, whereas the device  200  of  FIG. 2 b    may be described as side-loading. An outer periphery of the spacer  204  and an outer periphery of the lower substrate  203  extend beyond an outer periphery of the upper substrate  202 , and the inlet ports  211 ,  212  are defined by respective indentations provided in an internal edge of the spacer  204  and which extend beyond the upper substrate so as to provide fluid communication between the chamber  101  and the exterior of the device. In other structural respects, the device  200  of  FIG. 2 b    is substantially identical to the device  100  of  FIG. 2 a   .  FIG. 2 c    shows a cross-section along the line X-X of  FIG. 2 b   . In  FIG. 2 b   , the larger indentation at the bottom left corner of the device may be used as the oil (or other filler fluid) inlet port. In practice it may be convenient for the oil inlet port to be larger than inlet ports for assay fluid, as a larger volume of oil is required to operate the device—and a large oil inlet port allows the use of a larger pipette tip. However, it isn&#39;t necessary for the oil inlet port to be larger than other ports, and in principle a small pipette could be used to dispense oil multiple times instead. 
     A method of introducing fluid into a microfluidic device  100  will now be described with reference to  FIGS. 3 a  to 3 d   .  FIGS. 3 a  to 3 d    are schematic diagrams which depict a microfluidic device in accordance with the first embodiment of the invention.  FIG. 3 a    depicts the device  100  as described with reference to  FIG. 2 a    above. In this example, the chamber  101  initially contains a venting fluid. In general the venting fluid may be any fluid. Typically the venting fluid may be air  115 . Other examples of possible venting fluids include any inert atmosphere such as nitrogen or argon. Alternatively the fluid could be a polar liquid, for example, water. Advantageously, but not necessarily, the venting fluid may be substantially free from moisture A combination of venting fluids, may also be utilised. 
       FIG. 3 b    indicates the introduction into the chamber  101  of a metered volume of filler fluid, in this case oil  107 . The filler fluid is typically selected to be a non-polar material, or a material of low polarity. The filler fluid is typically selected to have a low interfacial surface tension with the assay fluid. The filler fluid is typically selected to be immiscible, or substantially immiscible with the assay fluid. The filler fluid may typically, but not necessarily have a low viscosity in order to maximise the speed of movement of droplets of the assay fluid. The filler fluid may typically, but not necessarily, have a lower density than the assay fluid. The filler fluid may typically, but not necessarily, be chosen to have a low or relatively low toxicity. The filler fluid may typically, but not necessarily, be chosen to have little or low reactivity with the materials comprising the assay fluid. The filler fluid is typically, but not necessarily a liquid. 
     Non-limiting examples of suitable filler fluids commonly used in electro-wetting on dielectric systems and suitable for this invention include silicone oils, alkanes, e.g. n-dodecane. Non-limiting examples of surfactants that may optionally be dissolved or partially dissolved in the oil include Brij 52, Brij 93, Tetronic 70, IGEPAL CA-210, MERPOL-A, Pluronic L-31, Pluroni L-61, Pluronic L-81, Pluronic L-121, Pluronic P-123, Pluronic 31R1, polyethylene-block-poly(ethylene glycol), Span 80 and Span 40. Other suitable non-polar filler fluids may also be used. The oil  107  is introduced, for example pipetted, into the chamber  101  using the oil inlet port  112 . It will be appreciated that the metered volume of oil  107  may be introduced into the chamber  101  by other suitable means. The volume of oil  107  is metered such that enough oil  107  is introduced to cover a desired part of the chamber but not to completely fill the chamber. In this embodiment the part of the chamber that contains oil includes the active area  109  of the chamber  101 . As shown, the vent area  105  remains substantially filled with air  115  even after the metered volume of oil has been introduced. 
     The device  100  may be provided with an optical and/or an electrical sensor for metering the volume of the oil  107  introduced into the chamber. Alternatively, an optical and/or an electrical sensor may be provided separately. As a further example, the volume of oil  107  may be pre-measured before introduction to the chamber  101 . 
     It will be appreciated that as the oil  107  is introduced to the chamber  101 , air  115  in the active area  109  vents from the chamber until substantially all of the active area  109  is covered with oil  107 . Air may vent through any suitable aperture, and so may vent though the assay fluid ports as well as though the vent  110  in the vent area. Apertures suitable for venting are preferably located at the periphery of the chamber  101 , in particular in the corners of the chamber  101 , in order to facilitate venting and to ensure no air  115  is trapped within the active area  109 . 
     As shown in  FIG. 3 b   , when the oil is introduced into the chamber, the inlet ports and vents remain dry since the oil fills around the inlet ports and vents. 
     The device  100  is configured to preferentially maintain the metered volume of oil  107  in the desired part of the chamber  101 . In this example, the device  100  comprises a flow restriction element for this purpose. Due to the position of the adhesive track  106  and the vent  110  at the far right end of the vent area  115  (as noted, oil does not enter the vent area), a constriction  116  in a fluid flow path from the part of the chamber  101  to the vent area  105  is provided. This constriction  116  acts as an oil flow restriction element. The oil  107  therefore tends to reside in the active area  109  even when subject to marginal tilts of the chamber  101 . A volume or bubble of air  115  remains within the vent area  105 . 
     The dimensions of the constriction are determined based on the known properties of the filler fluid, for example its surface tension with the hydrophobic surface and with the assay fluid, its viscosity and its density. 
     A volume of an assay fluid  108  is now introduced to the chamber  101  by loading into a fluid input port  111 , as shown in  FIG. 3 c   . This may be done using a pipette, alternatively another input method, such as a capillary track or line, could be used. The assay fluid  108  is a polar fluid, for example, blood. Alternatively, the assay fluid may be a type of reagent. The fluid input electrodes (not shown here) defining an internal reservoir of the device  10  are first activated. The assay fluid  108  is then pipetted into a fluid input port  111  whereby it enters the chamber  101  via capillary forces. In other words, the assay fluid  108  is drawn onto the active area  109 . It will be appreciated that the assay fluid  108  enters the chamber  101  without the requirement for any pressure-actuated input means such as pistons, pumps or gravity wells or even for an electro-wetting force. 
     In this embodiment the fluid will draw into the device by capillary forces only. The direction in which fluid enters the chamber from a particular inlet port however will not be well controlled, and the fluid will likely occupy a circular region around the inlet port. Optionally, therefore, an electrowetting force may be applied to guide the direction of fluid fill—this is particularly advantageous if two or more different assay fluids are being introduced into the chamber via different inlet ports, and it is desired to control the manner in which different assay fluids contact one another. However, the electro-wetting force in this instance is used solely to control the position of the assay fluid  108  within the active area  109 . 
     Alternatively, the device may be configured such that the capillary force is not sufficient to draw assay fluid from an inlet port into the chamber. (How this may be done is described elsewhere). In this case, application of an electrowetting force would both draw the fluid into the chamber and control the position of the assay fluid in the chamber. As the assay fluid  108  enters the chamber  101 , substantially in the direction of arrow C, some of the oil  107  is laterally displaced from the active area  109  of the device  100 . It will be understood that the assay fluid  108  and the oil  107  are substantially immiscible. As the active area  109  is substantially full of oil  107 , the oil  107  is displaced into the vent area  105  through the constriction  116  in the direction indicted by arrow B of  FIG. 3 c   . This causes a volume of air  115  to vent out of the chamber  101  through the air vent  110  at the far left end of the vent area  105 . The volume of air  115  or air bubble moves towards the far left air vent  110 , substantially in the direction indicated by arrow A in  FIG. 3 c   . Hence, the air bubble decreases in size. 
     A further volume of assay fluid  108 ′ is now introduced to the active area  109  of the chamber  101  via a second fluid inlet port  111 . As described above, the assay fluid  108 ′ causes some of the oil  107  to displace into the vent area  105 , in turn causing a further volume of air  115  to vent through the air vent  110  at the far left of the vent area  105 . The air bubble hence decreases further in size. The further volume of assay fluid  108 ′ is controlled within an internal reservoir of the active area  109  by electro-wetting forces provided by fluid input electrodes in the lower substrate  103  of the device. The further volume of assay fluid  108 ′ may be substantially identical in composition to the first volume  108  or may have a different composition. For example, the first assay fluid  108  may be blood and the second assay fluid  108 ′ may be reagent. The further volume of assay fluid  108 ′ may have a substantially different volume, for example 2 ul (microlitres), or may have the same volume, for example 0.25 ul, as the first volume  108 . The internal reservoirs are configurable to accommodate a range of fluid volumes, for example 0.1 ul to 100 ul. The volume and shape of the internal reservoirs can be changed by controlling the size and number of electrodes that define an internal reservoir. Further volumes of assay fluid  108 ,  108 ′ may be loaded into the active area  109  of the device  100  until all required fluids have been loaded, or until the vent area  105  is substantially completely filled with oil  107  and substantially all of the air  115  has vented. Once the vent area  105  is full of oil  107  no further assay fluid  108 ,  108 ′ can be loaded unless some of the oil  107  is drained from the device  100 . Once all required assay fluids  108 ,  108 ′ have been loaded onto the active area, droplets can be formed from the internal reservoirs using standard EWOD operation. Fluid droplets may be dispensed from the internal reservoirs by electro-wetting function. Droplet size is easily adjusted, accurate and reproducible. 
     The configuration of the device  100  provides a simple method for inputting assay fluid into the device. Compared to the prior art, no external input pumps, input pistons or large gravity wells are required, and external moving parts are eliminated. The likelihood of leakage is therefore reduced, and a device of the invention is much simpler to manufacture. The lack of large pistons means that a larger number of fluid inputs can be provided in a given area. Furthermore, pre-determined volumes of assay fluid may be loaded onto the internal reservoirs, and the volumes of the internal reservoirs may be chosen to suit the desired amount of a particular assay fluid. 
     In the above example, the assay fluid  108  is introduced to the chamber  101  after the introduction of the oil  107 . In another example, not illustrated here, one or more assay fluids and one or more filler fluids may be introduced substantially at the same time as one another. The fluids may be introduced by pipette or by any other suitable input means, through a fluid input port or other input port in the device. The fluids may be substantially mixed at the point of input or may be substantially separated. In this case, the assay fluid  108  is controlled within the chamber  101  by actuation of the electrodes during the introduction of the assay fluid  108  and filler fluid  107 , so that the assay fluid is retained in the active area of the device. 
     In this example apertures  110   a ,  110   b  and  110   c  are provided to act solely as vents since the arrangement of fluid inlet ports  111  of  FIG. 3 a    may not provide adequate venting in the corners of the chamber  107 —but in principle it may not be necessary to provide apertures intended to act solely as vents if the arrangement of inlet ports provides adequate venting of the chamber. 
     Moreover, in this embodiment all ports are designed to be dry when oil is introduced into the chamber. In an alternative embodiment it would be possible for all inlet ports to stay dry but for oil to enter venting ports (except for venting port  110  in the air vent area  105 ) when oil is introduced into the chamber. To do this the diameter of the venting ports would be made small so that they capillary fill with oil. 
       FIGS. 4 a  to 4 d    are schematic diagrams depicting a method of loading a microfluidic device in accordance with the second embodiment of the invention. The device  200  in this embodiment may be described as side-loading, as discussed with reference to  FIG. 2 b    above. The method of loading a volume of assay fluid  208  into the device  200  is substantially the same method as described with reference to the first embodiment of  FIGS. 3 a  to 3 d    above. In this embodiment, the Kapton spacer  204  generally separates the vent area  205  and active area  209  of the device  200 , and defines a constriction (in this embodiment a narrow channel) between the vent area  205  and the active area  209 . In addition, the spacer  204  creates separate filling zones along the bottom edge of the device  200 . As previously discussed, the upper substrate  202  is smaller than the spacer  204  by a controlled amount to create small gaps around the perimeter of the device  200  through which fluid may be introduced or through which a venting fluid such as air may vent. 
     A metered volume of a filler fluid such as oil  207  is introduced, for example by pipette, into the chamber  201  through an aperture in the bottom left hand corner of the chamber  201 . The volume of oil  107  is carefully controlled such that there is enough oil to substantially cover the active area  209  but the vent area  205  remains predominantly filled with venting fluid, in this case, air. 
     Once the metered volume of oil  207  has been loaded and substantially all of the air  215  in the active area  209  has vented via the vents  210 , the fluid input electrodes (not shown here) of the internal reservoir are activated and a volume of assay fluid  208  is pipetted into one or more of the filling zones or fluid input ports  211  running along the bottom edge of the chamber  201 . It will be appreciate that these input ports  211  may be positioned anywhere along the periphery of the active area  209 . 
     The volume of assay fluid  208  enters the device  200  substantially in the direction of arrow C by capillary action and is controlled once it enters the chamber  201  by electro-wetting forces. As the assay fluid  208  enters the active area  209 , some of the oil  207  is displaced through the constriction  216  into the vent area  205 , substantially in the direction of arrow B. As some of the oil  207  enters the vent area  205 , a volume of air  215  vents through the vent  210  at the far left end of the vent area  205 , substantially in the direction of arrow A. The air bubble therefore decreases in size. 
     As described with reference to the first embodiment above, further volumes of assay fluid  208 ′ may now be introduced into the chamber  201  and more of the oil will be displaced into the vent area  205 , until such time as all of the required fluids have been loaded for an assay or all of the air  215  in the vent area  205  has vented. Further volumes of assay fluid  208 ,  208 ′ may be introduced if a volume of the oil  207  is extracted from the chamber  201 . Droplets for an assay may now be produced from the internal reservoirs of assay fluids  208 ,  208 ′ by electro-wetting forces. 
     As discussed with reference to the first embodiment above, in an alternate method of filling one or more assay fluids and one or more filler fluids may be introduced to the chamber  201  substantially at the same time as each other. 
       FIGS. 5 a  to 5 d    are schematic diagrams depicting a method of loading a microfluidic device in accordance with a third embodiment of the invention. In this embodiment, the vent area  305  is integral to the active area  309  of the device  300  unlike in the first and second embodiments above. While a separate vent area simplifies operation, it takes up valuable space on the TFT chip. 
     In this embodiment the metered volume of filler fluid (oil  307 ) is again preferentially maintained in a part of the chamber  301  using a flow restriction element. In this example, the flow restriction element comprises one or more physical walls and possibly a patterned hydrophobic coating  314  on an interior of the chamber  301 . The walls may for example be formed of adhesive or photoresist. If the device is tipped, the presence of walls alone may not be sufficient to contain the oil (or other filer fluid), and oil may escape the glue wall boundary. The hydrophobicity of the surface of one or both substrates may therefore be patterned to further retain the oil on areas within the wall. For example, the hydrophobic surface may be removed behind the inlet ports, so that oil will then preferentially go onto these areas. Then, if the device is tipped, the presence of walls and the patterned hydrophobic surface may be enough to keep the oil in the correct area for filling the assay fluid. 
     In alternative embodiments the physical walls alone may be sufficient, for example if the user is told not to tip the device. 
     This coating  314  provides “walls” which surround the fluid inputs  311 . 
     The device  300  shown in  FIG. 5 a    is substantially filled with a venting fluid, in this case, air. A metered volume of oil  307  is input into the zones surrounded by the walls  314 . The oil  307  is constrained by the walls  314  and the pattern of hydrophobicity, and tends to remain in the zones. A volume of assay fluid  308  is then introduced to the active area  309  via a fluid input port  311 . It will be noted that in this embodiment, fluid input ports  311  may be used to introduce oil  307  and assay fluid  308 . 
     As the assay fluid enters the active area  309 , it is constrained to enter the active area substantially in the direction indicated by arrow C by electro-wetting forces, provided by actuated electrodes (not shown). (As noted above, the capillary force may be sufficient to cause the assay fluid to enter the active region with the electro-wetting forces controlling the direction of fluid entry, or alternatively the electro-wetting forces may both cause the assay fluid to enter the active area and control the direction of fluid entry.) Some of the oil is displaced further into the active area  309 , substantially in the direction of arrow B. Some of the air is vented through vents  310  as the oil  307  is displaced. 
     As shown in  FIG. 5 d    and as described above for the first and second embodiments, further volumes of assay fluid  308 ′ may be introduced into the chamber  301  until all fluids required for the assay are loaded or until the active area  309  is substantially filled with oil  307  i.e. all of the air has vented through the vents  310 . Further volumes of assay fluid  308 ,  308 ′ may be introduced if a volume of oil  307  is extracted or drawn out of the chamber  301 . 
     As discussed for the embodiments above, one or more filler fluids and one or more assay fluids may be introduced to the device  300  substantially at the same time as each other rather than separately. 
       FIGS. 6 a  to 6 d    are schematic diagrams depicting a method of loading a microfluidic device in accordance with a fourth embodiment of the invention. The fourth embodiment of the microfluidic device  400  is structurally similar to the first embodiment discussed with reference to  FIG. 2 a    above, and again comprises one or more flow restriction elements that preferentially maintain the metered volume of filler fluid (oil  407 ) in a desired part of the chamber  401 . In this embodiment the flow restriction elements comprises one or more controllable flow restriction elements that can be controlled, for example electrically, to be in either an “open” state or a “closed state”.  FIG. 6 a    shows two electrically activated barriers  417  provided in series between the part of the chamber and the vent  410  at the far left end of the vent area  405 , but the invention is not limited to this specific arrangement. Each of the barriers  417  comprise a fluid immiscible with the filler fluid  407 . When a barrier is “closed”, the fluid extends across the width of the vent area  405  as shown in  FIG. 6   a.    
     In this example, barrier electrodes (not shown) are provided at locations where it is desired to provide a barrier  417 . A polar fluid (which may be an assay fluid), for example, water, is loaded into the vent area  405  to form one or more barriers or gates  417 . The polar fluid may be loaded via input ports  411  adjacent to the barriers. The polar fluid is held within the barrier location by electro-wetting forces provided by the electrodes at the barrier location. A metered volume of oil  407  is then introduced to the chamber  401  as described for the first embodiment above, such that the active area  409  is substantially covered with oil  307  while the separate vent area  405  is substantially filled with air  415 . It will be noted that oil  407  cannot fill the device  400  further than the first barrier  417 , since the oil  407  is immiscible with the non-polar barrier fluid and, when the barrier is closed, the barrier fluid extends over substantially the entire width of the vent area. 
     The provision of the barrier(s)  417  means that the constriction  116 ,  216  of  FIG. 3 b    or  4   b    2   a  is not needed in this embodiment and may be removed, as indicated by the larger gap  416  of  FIG. 6 b   . In principle however a constriction could be provided in the embodiment of  FIGS. 6 a   - 6   d.    
     One or more volumes of assay fluid  408 ,  408 ′ may then be loaded into one or more of the fluid input ports on the active area  409 . Since the oil  307  is prevented from being displaced into the vent area  405  by the barrier  417 , the volume or volumes of assay fluid  408 ,  408 ′ are unable to enter the chamber  401  and are therefore “stored” in the fluid inlet port  411 . This method is advantageous in that assay fluids may be “stored” until all fluids are loaded and the assay is ready to begin. 
     When a user is ready to load assay fluid  408 ,  408 ′ into the device  400 , the position of the barrier fluid in one or more of the barriers  417  may be changed by suitably controlling the barrier active electrode(s). The barrier fluid is reconfigured so that it no longer extends over the entire width of the vent area, so allowing some of the oil  407  to flow past the barrier  417  into the vent area  405 , as shown in  FIG. 6 d   . Oil may now be displaced from the active area substantially in the direction of arrow B into the vent area  405  and one or more volumes of assay fluid  408 ,  408 ′ stored in the inlet ports are drawn onto the active area, with the direction of fluid entry substantially in the direction indicated by arrow C as controlled by electro-wetting forces. Air  415  in the vent area  405  may vent through the fluid input ports  411  adjacent the barriers  417 . 
     It will be appreciated that multiple barriers  417  may be provided in order to allow staged introduction of one or more volumes of assay fluid  408 ,  408 ′ into the active area  409 . 
       FIGS. 7 a  to 7 d    are schematic diagrams depicting an alternative method of loading a microfluidic device in accordance with a fifth embodiment of the invention. In this embodiment, the device  500  does not comprise a vent area which is separated from an active area. In use, the device  500  is firstly substantially completely filled with filler fluid (eg oil  507 ) via oil input port  512 , as shown in  FIG. 7 b   . As the device  500  is filled with oil  507 , any venting fluid (air) present within the chamber  501  will vent out of the vent area  505  through vents  510 . The oil  507  will fill around the vents  510  and the fluid input ports  511  such that these apertures remain dry. 
     One or more volumes of assay fluid  508 ,  508 ′ are then loaded into fluid input ports  511 . The assay fluid  508 ,  508 ′ remains in the input ports since the oil  507  cannot be displaced as the chamber  501  is full, as shown in  FIG. 7   c.    
     Some of the oil  507  is now extracted via the oil outlet port  513 , and leaves the chamber  501  substantially in the direction indicated by arrow B. Extraction may comprise the use of a capillary line, pipette or absorbing pad, for example. As some of the oil  507  is removed from the active area  509 , assay fluid  508 ,  508 ′ is drawn into the chamber  501 , substantially in the direction of arrow C, by capillary forces and is controlled by electro-wetting into internal reservoirs. The volume of extracted oil  507  is carefully metered to match the volume(s) of assay fluid(s) which is required to be loaded into the device  500 . 
     In the above embodiments, the device is arranged such that assay fluid introduced into an inlet port would naturally be drawn into the chamber  101 , and is restrained from doing this solely because the active area of the device already contains fluid (either filler fluid or a combination of filler fluid and one or more previously introduced assay fluids). This may be arranged by choosing suitable values for the cell gap (that is the separation between the upper and lower substrate), the hydrophobic coating, and the properties of the assay fluid(s) such as viscosity, density and surfactant level. For example, the cell gap may be chosen based on knowledge of the assay fluid(s) to be used. The assay fluid may then be introduced into the chamber in a controlled manner according to any of the embodiments described above. 
     The invention is not however limited to this, and the device could alternatively be arranged such that assay fluid introduced into an inlet port would naturally remain in the inlet port.  FIGS. 8 a  to 8 e    are schematic diagrams depicting a method of loading a microfluidic device in accordance with a sixth embodiment of the invention, in which the device is configured in this way. In this embodiment, the device  600  provided with a vent area  605  which is integral to the active area  69  of the chamber. 
     One or more volumes of assay fluid  608 ,  608 ′ are introduced, for example by pipette, to the fluid input ports  611 . The active area  609  is substantially filled with venting fluid (air) only at this stage, such that the assay fluid  608 ,  608 ′ is not drawn onto the active area  609  by capillary action and remains in the input ports  611 . A metered volume of filler fluid such as oil  607  is now introduced to the device  600  via an input port  612 . As the oil  607  flows across the active area  609 , substantially in the direction indicated by arrow B, the assay fluid  608  is drawn out of the input port(s) onto the active area  609  by capillary forces. Once within the active area  609  the assay fluid  608  is held in position by electro-wetting forces provided by actuated electrodes. Air contained within the active area  609  vents through vents  610 . 
     Further metered volumes of oil  607  may now be introduced into the device  600  such that oil  607  moves further across the active area  609  and further volumes of assay fluid  608 ′ are drawn into the device  600 , as shown in  FIG. 8 d   . The process of loading assay fluids  608 ,  608 ′ and oil  607  may continue until all required fluids have been loaded or until the active area  609  is substantially filled with oil  607 . Oil  607  may then be extracted from the device  600  in order to load further assay fluids  608 ,  608 ′. 
     Optionally in this embodiment one or more of, and optionally all of, the fluid input ports  611  further comprise an upper well  618  in which a larger volume of assay fluid  608 ,  608 ′ may be held than in the fluid input ports  611  themselves. As shown in the cross section of  FIG. 8 e   , the wells  618  may comprise plastic slots in the ports  611  which are formed in the upper substrate  602  of the device  600 . 
     It will be understood that wells similar to the wells  618  may be provided in the devices used in other embodiments of the invention. This is of particular benefit in embodiments in which assay fluid is “pre-stored” in an inlet port, such as in the embodiment of  FIGS. 6 a    to  6   d.    
       FIGS. 9 a  to 9 d    are schematic diagrams depicting a method of loading a microfluidic device in accordance with a seventh embodiment of the invention. In this embodiment, a device  700  substantially identical to that of the first embodiment discussed with reference to  FIG. 2 a    is provided with 26 separate fluid input ports  711 . The ports  711  are positioned around a perimeter of the active area  709  of the device, however, their position may be varied as required. It will be appreciated that each port  711  may be used for a different assay fluid  708 ,  708 ′ as required. The internal reservoirs associated with each input port  711  may be varied with regard to shape and volume as discussed above in order to accommodate the volume of assay fluid required for an assay. 
     In this way, the device  700  provides a flexible, versatile and easy method of loading fluids for an assay. Although the structure of the device  700  is comparable with that of the first embodiment discussed above, it will be understood that any of the embodiments discussed herein may be provided with a similar number of fluid input ports. The number of ports is restricted only by the size of the device and hence may be varied to suit the requirements of the assay or assays to be carried out. The device may be configured such that assays may be carried out in parallel. In addition, the configuration of the fluid input ports of the various embodiments discussed above provide consistent heating of fluids within the device, since no large, tall fluid wells are required. 
     A number of potential applications for microfluidics devices require some form of thermal control. A further advantage of the present invention is that, by eliminating bulky input devices such as pistons, tubes or tall fluid wells, it is possible to obtain much better uniformity of temperature over the active area, even in embodiments where the ports and vents are provided by holes in the upper substrate. 
       FIG. 10 a    is a graphical representation of a cartridge  119  based around a microfluidic device. In this illustrated example, the device  100  shown is the device of the first embodiment, however, any of the embodiments discussed herein may be included in a similar cartridge  119 . The cartridge  119  in this example is configured to be disposable and/or recyclable and suitable for manufacture at large volumes (for example, millions of units a year) and at low cost. The cartridge acts as the interface for the fluids within the AM-WOD device and the outside world and may also provide heating for fluid droplets contained within the device.  FIG. 10 b    is an exploded view of the cartridge of  FIG. 10 a    in which the various components of the cartridge are displayed. 
       FIG. 11 a    is a graphical representation of a benchtop control/reader device  120  configured to control the operation of a microfluidic device contained within the cartridge and read out data as appropriate of  FIGS. 10 a  and 10 b   .  FIG. 11 b    is a graphical representation of a handheld control/reader device  120 ′ configured to control the operation of such a microfluidic device. The cartridge  119  containing the microfluidic device ( 100 ,  200 ,  300 ,  400 ,  500 ,  600 ,  700 ) is inserted or connected into the control/reader device  120 ,  120 ′, as is known in the art. 
     Although the invention has been shown and described with respect to a certain embodiment or embodiments, equivalent alterations and modifications may occur to others skilled in the art upon the reading and understanding of this specification and the annexed drawings. In particular regard to the various functions performed by the above described elements (components, assemblies, devices, compositions, etc.), the terms (including a reference to a “means”) used to describe such elements are intended to correspond, unless otherwise indicated, to any element which performs the specified function of the described element (i.e., that is functionally equivalent), even though not structurally equivalent to the disclosed structure which performs the function in the herein exemplary embodiment or embodiments of the invention. In addition, while a particular feature of the invention may have been described above with respect to only one or more of several embodiments, such feature may be combined with one or more other features of the other embodiments, as may be desired and advantageous for any given or particular application. 
     (Overview) 
     A first aspect of the invention provides a method of loading a microfluidic device with an assay fluid, the method comprising: introducing, into a chamber in the microfluidic device, the chamber having one or more inlet ports, a metered volume of a filler fluid such that the chamber is partially filled with the filler fluid, said device being configured to preferentially maintain the metered volume of the filler fluid in a part of the chamber; and introducing a volume of the assay fluid into the part of the chamber via one of the one or more inlet ports and thereby causing a volume of a venting fluid to vent from the chamber. 
     The chamber may have at least one vent in addition to the inlet port(s), so that the venting fluid vents from the chamber through the at least one vent. By “vent” is meant a port that is provided solely to allow venting fluid to vent from the chamber, and that is not used as an inlet port. Alternatively, the venting fluid may vent from the chamber through one or more of the inlet ports. 
     A second aspect of the invention provides a method of loading a microfluidic device with an assay fluid, the method comprising: substantially completely filling a chamber with a filler fluid or with a fluid mixture containing a filler fluid as one component, the chamber having one or more inlet ports and an outlet port for extracting the filler fluid; inserting a volume of the assay fluid into one of the one or more inlet ports; and extracting sufficient of the filler fluid through the outlet port to enable at least some of the volume of the assay fluid to enter the chamber from the one of the one or more inlet ports. In this aspect the chamber may be initially filled with filler fluid, and the method may then be used to enable the introduction of assay fluid. Alternatively, the chamber may initially be filled with a mixture of filler fluid and an assay fluid, and the method may then be used to enable the introduction of more assay fluid and/or of one or more different assay fluids. 
     The present invention allows assay fluid to be introduced easily into the device. There is no need to apply high pressure to force the assay fluid into the device, and problems associated with the use of pistons and pumps, such as the need to the provide a good, high-pressure seal at the inlet port in order to avoid sample loss and/or introduction of air bubbles are overcome. A device of the invention is simple, and hence cheap, to manufacture, and is simple to operate. A further advantage is that many inlet ports may easily be provided on a device, whereas the physical size of pumps/pistons or gravity wells used in the prior art means that it is difficult to accommodate them on a typical device. 
     In either aspect, the method may further comprise introducing the filler fluid and the assay fluid into the chamber at substantially the same time as one another. The wording “at substantially the same time as one another” is intended to cover a method in which the time period within which the filler fluid is introduced overlaps the time period within which assay fluid is introduced. Alternatively, in either aspect of the invention the filler fluid may be introduced into the chamber first, with the assay fluid being introduced into the chamber after the filler fluid has been introduced. As a further alternative, in either aspect of the invention the assay fluid may first be introduced into one or more of the inlet ports but the assay fluid remains in the inlet port(s). (Essentially this requires that the device and the assay fluid are arranged so that the capillary force tending to draw the assay fluid from the inlet port(s) into the chamber is not sufficient to overcome the repulsion of the fluid by the chamber—which is naturally hydrophobic.) When filler fluid is introduced into the chamber, it acts to draw assay fluid into the chamber. 
     The device may be an electro-wetting on dielectric (EWOD) device comprising electrodes. The method may further comprise controlling the assay fluid within the chamber by actuation of said electrodes. 
     Where the filler fluid and the assay fluid are introduced into the chamber at substantially the same time as one another, the method may comprise controlling the assay fluid within the chamber by actuation of said electrodes during the introduction of the filler fluid and the assay fluid into the chamber. 
     In a method of the first aspect, the volume of the assay fluid may be introduced into the chamber after the metered volume of filler fluid has been introduced into the chamber, whereby the assay fluid may enter the part of the chamber by displacing some of the filler fluid from the part of the chamber. 
     At least part of an interior of the chamber may be coated with a hydrophobic coating. 
     The device may be configured such that the one or more inlet ports are provided in an upper surface of the chamber. If one or more vents are present, this/they may also be provided in the upper surface of the chamber. 
     The device may be configured such that the one or more inlet ports are provided in one or more sides of the chamber. If one or more vents are present, this/they may also be provided in the sides of the chamber. 
     The device may be configured such that the chamber is provided with a vent area in fluid communication with at least one vent, said vent area configured to contain the venting fluid. 
     The device may be configured to have at least one vent that is substantially identical to the one or more inlet ports. 
     The device may be configured such that the chamber is provided with an active area for carrying out one or more assays. 
     The device may be configured such that the vent area is integral to the active area. 
     The device may be configured such that the vent area is partially separated from the active area by a fluid-impermeable barrier. 
     The method may further comprise preferentially maintaining the metered volume of the filler fluid in the part of the chamber using a flow restriction element. 
     The flow restriction element may be a patterned hydrophobic coating on an interior of the chamber. 
     The flow restriction element may be a constriction in a fluid path from the part of the chamber to the vent area. 
     The method may comprise maintaining the metered volume of the filler fluid in a part of the chamber using one or more electrically activated barriers between the part of the chamber and the vent, said one or more barriers comprising a fluid immiscible with the filler fluid. 
     The method may further comprise metering the metered volume of the filler fluid by one of volume measurement, optical sensing, and electrical sensing. 
     A vent may be provided substantially at a corner of the part of the chamber. This eliminates the risk of air being trapped at the corner when the filler fluid is introduced into the chamber. Preferably a vent is provided at every corner of the part of the chamber. 
     A method of the invention may further comprise introducing a second assay fluid, for example via another inlet port. This may be repeated until all desired assay fluids have been introduced into the chamber. 
     A third aspect of the invention provides a microfluidic device, comprising: a chamber having one or more inlet ports; said device being configured to, when the chamber contains a metered volume of a filler fluid that partially fills the chamber, preferentially maintain the metered volume of the filler fluid in a part of the chamber; and the device being configured to allow displacement of some of the filler fluid from the part of the chamber when a volume of an assay fluid introduced into one of the one or more inlet ports enters the part of the chamber, thereby causing a volume of a venting fluid to vent from the chamber. 
     A fourth aspect of the invention provides a microfluidic device, comprising: a chamber having one or more inlet ports and an outlet port for extracting a filler fluid; whereby in use the chamber is substantially completely filled with the filler fluid, and a volume of an assay fluid introduced into one of the one or more inlet ports is enabled to enter the chamber as sufficient of the filler fluid is extracted through the outlet. 
     In a device of the third or fourth aspect the chamber may have at least one vent in addition to the inlet port(s), so that the venting fluid vents from the chamber through the at least one vent. By “vent” is meant a port that is provided solely to allow venting fluid to vent from the chamber, and that is not used as an inlet port. Alternatively, the venting fluid may vent from the chamber through one or more of the inlet ports. 
     The device may be an electro-wetting on dielectric (EWOD) device comprising electrodes and the assay fluid may, in use, be controlled within the chamber by actuation of said electrodes. 
     An interior of the chamber may be is at least partially coated with a hydrophobic coating. 
     The device may comprise a lower substrate, an upper substrate spaced from the lower substrate, and a fluid barrier provided between the lower substrate and the upper substrate to define a perimeter of the chamber. 
     The fluid barrier may be provided by an adhesive track that adheres the lower substrate to the upper substrate. 
     The fluid barrier may be provided by a spacer that spaces the lower substrate from the upper substrate. 
     At least one of the one or more inlet ports may be provided in the upper substrate of the device. If one or more vents are present, this/they may also be provided in the upper substrate of the chamber. 
     At least one of the one or more inlet ports and/or the at least one vent may be provided in the fluid barrier. If one or more vents are present, this/they may also be provided in the fluid barrier. 
     An outer periphery of the spacer may extend beyond an outer periphery of the upper substrate, and at least one of the one or more inlet ports may be defined by respective indentations provided in an internal edge of the spacer. Alternatively, at least one of the one or more inlet ports may be defined by gaps in the spacer. If one or more vents are present, this/they may also be defined by indentations or gaps in the spacer. 
     The chamber may further comprise a vent area, said vent area in fluid communication with at least one vent and configured to contain the venting fluid. 
     The chamber may further comprise an active area for carrying out one or more assays. 
     The device may have at least one vent that is substantially identical to the one or more inlet ports. 
     The vent area may comprise the active area. 
     The fluid barrier may further define the vent area and the active area in the chamber. 
     The device may comprise a flow restriction element for preferentially maintaining the metered volume of the filler fluid in the part of the chamber. 
     The flow restriction element may comprise a patterned hydrophobic coating on an interior of the chamber. 
     The flow restriction element (feature) may comprise a constriction in a fluid flow path from the part of the chamber to the vent area. 
     The flow restriction element may comprise one or more electrically activated barriers between the part of the chamber and the vent, said one or more barriers comprising a fluid immiscible with the filler fluid. 
     The device may comprise an optical and/or an electric sensor for metering the volume of the filler fluid. 
     The chamber may comprise at least one vent substantially located in a corner of the part of the chamber. 
     A fifth aspect of the invention provides a microfluidic system comprising a microfluidic device of the third or fourth aspect, said device contained within a disposable cartridge, and a control and/or reader device configured to control and/or read the microfluidic device. 
     In a method of the first or second aspect, the filler fluid may comprise a non-polar fluid. It may comprise an oil. It may comprise a surfactant. 
     In a method of the first or second aspect, the assay fluid may comprise a-polar fluid. It may comprise an aqueous material. It may comprise a surfactant. 
     In a method of the first or second aspect, the venting fluid may comprise a gas. It may comprise air. It may comprise an inert gas. 
     CROSS-REFERENCE TO RELATED APPLICATIONS 
     This Nonprovisional application claims priority under 35 U.S.C. § 119 on Patent Applications Nos. 1516430.4 which is filed in United Kingdom of Great Britain and Northern Ireland on Sep. 16, 2015, the entire contents of which are hereby incorporated by reference. 
     INDUSTRIAL APPLICABILITY 
     An AM-EWOD device may be used for a number of digital microfluidic applications such as Point-of-Care (POC) diagnostics, disease detection, RNA testing and biological sample synthesis (e.g. DNA amplification). Mechanisms for sample and reagent loading are an important part of an integrated self-contained disposable system which can be used simply by the operator to carry out such tests. Ease of fluid loading is fundamental to a reliable device.