Microfluidic device and method for filling fluid therein

Abstract

A microfluidic AM‑EWOD device and a method of filling such a device are provided. The device includes a chamber having one or more inlet ports. The device is configured to preferentially retain the metered volume of filler fluid in a portion of the chamber when the chamber contains a metered volume of filler fluid that partially fills the chamber. The device is configured to allow some of the filler fluid to be removed from a portion of the chamber when a volume of assay fluid introduced into one of the one or more inlet ports enters a portion of the chamber, thereby causing a volume of exhaust fluid to be exhausted from the chamber.

Description

[0001] Description of the case

[0002] This application is a divisional application of an invention patent application with an application date of September 14, 2016, application number 201680066755.9, and invention name “Microfluidic device and method for filling fluid therein”. Technical Field

[0003] The present invention relates to a microfluidic device and to a method for loading a fluid into such a device. More specifically, the present invention relates to an active matrix electrowetting on dielectric (AM-EWOD) microfluidic device. Electrowetting on dielectric (EWOD) is a known technique for manipulating droplets of fluid on an array. Active matrix EWOD (AM-EWOD) refers to the implementation of EWOD in an active matrix array incorporating transistors, for example by using thin film transistors (TFTs). Background Art

[0004] Microfluidics is a rapidly expanding field that deals with the manipulation and precise control of fluids at smaller scales, typically dealing with sub-microlitre volumes. There is increasing interest in applying microfluidics to chemical or biochemical assays and syntheses, as well as to medical diagnostics ("labs on a chip"), both in research and in production. In the latter case, the small nature of such devices allows for rapid testing when much smaller clinical sample volumes than are used for traditional laboratory-based testing are required.

[0005] Microfluidic devices can be identified by the fact that microfluidic devices have one or more channels (or more generally referred to as gaps) of 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 various measurements of interest, including molecular diffusion coefficients, fluid viscosity, pH, chemical bonding coefficients, and enzyme reaction kinetics. Other applications of microfluidic devices include capillary electrophoresis, isoelectric focusing, immunoassays, enzymatic assays, flow cytometry, sample injection via mass spectrometry analysis of proteins, PCR amplification, DNA analysis, cell manipulation, cell separation, cell patterning, and chemical gradient formation. Many of these applications have been used for clinical diagnosis.

[0006] Many techniques are known for manipulating fluids at the submillimeter scale, characterized primarily by laminar flow and the dominance of surface forces over volume forces. Most techniques fall into the category of continuous flow systems, which typically employ bulky external plumbing and pumps. Systems employing discrete droplets instead have the advantage of greater functional flexibility.

[0007] Electrowetting on dielectric (EWOD) is a well-known technique for manipulating discrete fluid droplets by applying an electric field. Therefore, it is a candidate technology for microfluidics of lab-on-a-chip technology. An introduction to the basic principles of this technology can be found in the following literature: "Digital microfluidics: is a true lab-on-a-chip possible?" (RB Fair, Microfluid Nanofluid (2007) 3: 245-281). The review notes that: the method of introducing fluids into EWOD devices is not discussed in detail in the literature. It should be noted that the technology uses a hydrophobic inner surface. Therefore, in general, it is energy-unfavorable for aqueous fluids to be filled into such a device from the outside by means of capillary action alone. In addition, this may still be the case when a voltage is applied and the device is in an actuated state. Due to the lower surface tension at the liquid-solid interface, capillary filling of non-polar fluids (e.g., oil) may be energy-advantageous.

[0008] Some examples of small microfluidic devices in which fluid input mechanisms are described exist. U.S. Patent No. 5,096,669 (Lauks et al., published on March 17, 1992) shows such a device, which includes an inlet hole and an entry channel for sample input, which is coupled to an air bag that pumps fluid around the device when actuated. The patent does not describe how to input discrete fluid droplets into the system, nor does it describe a method for measuring or controlling the input volume of such droplets. This control of the input volume (called "metering") is important in avoiding overloading the device with excess fluid, and helps to improve the accuracy of measurements performed in situations where a known volume or volume ratio is required.

[0009] US20100282608 (Srinivasan et al.; published on November 11, 2010) describes an EWOD device comprising an upper portion of two parts having a hole through which a fluid can enter. The patent does not describe how the fluid can be forced into the device, nor does it describe a method of measuring or controlling the input volume of such a fluid. A related application US20100282609 (Pollack et al.; published on November 11, 2010) does describe a piston mechanism for inputting a fluid, but also does not describe a method of measuring or controlling the input volume of such a fluid.

[0010] US20100282609 describes the use of a piston to force a fluid into a reservoir contained in a device already filled with oil. US20130161193 describes a method of driving a fluid onto a device filled with oil by using, for example, a bistable actuator. Summary of the invention

[0011] A first aspect of the present invention provides a method for filling a microfluidic device with a measuring fluid, the method comprising: introducing a metered volume of filler fluid into a chamber having one or more inlet ports in the microfluidic device so that the chamber is partially filled with the filler fluid, the device being configured to preferentially retain the metered volume of filler fluid in a portion of the chamber; and introducing a certain volume of the measuring fluid into the portion of the chamber via one of the one or more inlet ports, thereby causing a certain volume of discharge fluid to be discharged from the chamber.

[0012] A second aspect of the present invention provides a method for filling a microfluidic device with a measurement fluid, the method comprising: substantially completely filling a chamber with a filler fluid or with a fluid mixture including the filler fluid as a component, the chamber having one or more inlet ports and an outlet port for extracting the filler fluid; inserting a certain volume of the measurement fluid into one of the one or more inlet ports; and extracting a sufficient amount of the filler fluid through the outlet port so that at least some volume of the measurement fluid can enter the chamber from one of the one or more inlet ports.

[0013] The third aspect of the present invention provides a microfluidic device comprising: a chamber having one or more inlet ports; the device being configured to: when the chamber contains a metered volume of filler fluid that partially fills the chamber, preferentially retain the metered volume of filler fluid in a portion of the chamber; and the device being configured to: when a certain volume of measurement fluid introduced into one of the one or more inlet ports enters the said portion of the chamber, allow some of the filler fluid to move out of the said portion of the chamber, thereby causing a certain volume of discharge fluid to be discharged from the chamber.

[0014] The fourth aspect of the present invention provides a microfluidic device, comprising: a chamber having one or more inlet ports and an outlet port for extracting a filler fluid; whereby when in use, the chamber is substantially completely filled with the filler fluid, and when a sufficient amount of the filler fluid is extracted through the outlet, a certain volume of measurement fluid introduced into one of the one or more inlet ports is able to enter the chamber. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 is a schematic diagram depicting a conventional AM-EWOD device in cross section.

[0016] Figure 2a is a schematic diagram depicting a plan view of a microfluidic device according to a first and exemplary embodiment of the present invention.

[0017] Figure 2b is a schematic diagram depicting a plan view of a microfluidic device according to a second embodiment of the present invention.

[0018] Figure 2c is a schematic diagram depicting a cross-sectional view of a microfluidic device according to a second embodiment of the present invention.

[0019] Figure 3a is a schematic diagram depicting a method for filling a microfluidic device according to a first embodiment of the present invention.

[0020] Figure 3b is a schematic diagram depicting a method for filling a microfluidic device according to a first embodiment of the present invention.

[0021] Figure 3c is a schematic diagram depicting a method for filling a microfluidic device according to a first embodiment of the present invention.

[0022] Figure 3d is a schematic diagram depicting a method for filling a microfluidic device according to a first embodiment of the present invention.

[0023] Figure 4a is a schematic diagram depicting a method for filling a microfluidic device according to a second embodiment of the present invention.

[0024] Figure 4b is a schematic diagram depicting a method for filling a microfluidic device according to a second embodiment of the present invention.

[0025] Figure 4c is a schematic diagram depicting a method for filling a microfluidic device according to a second embodiment of the present invention.

[0026] Figure 4d is a schematic diagram depicting a method for filling a microfluidic device according to a second embodiment of the present invention.

[0027] Figure 5a is a schematic diagram depicting a method for filling a microfluidic device according to a third embodiment of the present invention.

[0028] Figure 5b is a schematic diagram depicting a method for filling a microfluidic device according to a third embodiment of the present invention.

[0029] Figure 5c is a schematic diagram depicting a method for filling a microfluidic device according to a third embodiment of the present invention.

[0030] Figure 5d is a schematic diagram depicting a method for filling a microfluidic device according to a third embodiment of the present invention.

[0031] Figure 6ais a schematic diagram depicting a method for filling a microfluidic device according to a fourth embodiment of the present invention.

[0032] Figure 6b is a schematic diagram depicting a method for filling a microfluidic device according to a fourth embodiment of the present invention.

[0033] Figure 6c is a schematic diagram depicting a method for filling a microfluidic device according to a fourth embodiment of the present invention.

[0034] Figure 6d is a schematic diagram depicting a method for filling a microfluidic device according to a fourth embodiment of the present invention.

[0035] Figure 7a is a schematic diagram depicting a method for filling a microfluidic device according to a fifth embodiment of the present invention.

[0036] Figure 7b is a schematic diagram depicting a method for filling a microfluidic device according to a fifth embodiment of the present invention.

[0037] Figure 7c is a schematic diagram depicting a method for filling a microfluidic device according to a fifth embodiment of the present invention.

[0038] Figure 7d is a schematic diagram depicting a method for filling a microfluidic device according to a fifth embodiment of the present invention.

[0039] Figure 8a is a schematic diagram depicting a method for filling a microfluidic device according to a sixth embodiment of the present invention.

[0040] Figure 8b is a schematic diagram depicting a method for filling a microfluidic device according to a sixth embodiment of the present invention.

[0041] Figure 8c is a schematic diagram depicting a method for filling a microfluidic device according to a sixth embodiment of the present invention.

[0042] Figure 8d is a schematic diagram depicting a method for filling a microfluidic device according to a sixth embodiment of the present invention.

[0043] Figure 8e is a schematic diagram depicting a method for filling a microfluidic device according to a sixth embodiment of the present invention.

[0044] Figure 9a is a schematic diagram depicting a method for filling a microfluidic device according to a seventh embodiment of the present invention.

[0045] Figure 9bis a schematic diagram depicting a method for filling a microfluidic device according to a seventh embodiment of the present invention.

[0046] Fig.9c is a schematic diagram depicting a method for filling a microfluidic device according to a seventh embodiment of the present invention.

[0047] Figure 9d is a schematic diagram depicting a method for filling a microfluidic device according to a seventh embodiment of the present invention.

[0048] Fig.10a is a graphical representation of a microfluidic based cartridge.

[0049] Fig.10b yes Fig.10a Exploded view of the box.

[0050] Fig.11a is a graphical representation of a benchtop reader device used to control the operation of a microfluidic device.

[0051] Fig.11b is a pictorial representation of a handheld reader device for controlling the operation of a microfluidic device. DETAILED DESCRIPTION

[0052] To achieve the foregoing and related purposes, the present invention includes the features fully described below and identified in the claims. The following description and the accompanying drawings set forth in detail certain illustrative embodiments of the present invention. However, these embodiments indicate only some of the various ways in which the principles of the present invention may be employed. Other objects, advantages and novel features of the present invention will become apparent from the following detailed description of the invention when considered in conjunction with the accompanying drawings.

[0053] Although the present invention has been shown and described with respect to certain embodiments, equivalent alternatives and modifications may occur to those skilled in the art after reading and understanding this specification and the accompanying drawings. Specifically, with respect to the various functions performed by the elements (assemblies, assemblies, devices, compositions, etc.) described above, unless otherwise indicated, the terms used to describe these elements (including references to "devices") are intended to correspond to any element that performs the specified functions of the described elements (i.e., functionally equivalent), even if the structure is not equivalent to the disclosed structure that performs the functions in the exemplary embodiments herein of the present invention. In addition, although the above may have described specific features of the present invention only for one or more of several embodiments, such features may be combined with one or more other features that may be desirable and advantageous for any given or specific application in other embodiments.

[0054] Figure 1is a schematic diagram depicting a conventional AM-EWOD device 1 in cross-section. The AM-EWOD device 1 has a lower substrate 6 (e.g., a CG ("continuous grain") silicon substrate) and an upper substrate 2 (e.g., indium tin oxide (ITO) coated glass). Electrodes 3 are disposed on the upper substrate 2 and the lower substrate 6. The electrodes 3 control the movement of liquid droplets 8 through the device 1. The liquid droplets 8, which may be composed of any polar liquid and may typically be ionic and / or aqueous, are enclosed between the lower substrate 6 and the top substrate 2, but it will be understood that there may be multiple liquid droplets 8. For convenience, the contents of the liquid droplets will be referred to herein as "assay fluids", but as explained below, this does not mean that the present invention is limited to use in performing assays.

[0055] A general requirement for the operation of the device is that the assay fluid comprises a polar fluid, which is typically a liquid that can be manipulated by electromechanical forces (such as electrowetting forces) by applying an electrical signal to the electrodes. Typically, but not necessarily, the assay fluid may comprise an aqueous material, although non-aqueous assay fluids (e.g., ionic fluids) may also be manipulated. Typically, but not necessarily, the assay fluid may contain a concentration of dissolved salts, such as in the range of 100nM to 100M, or in the range of 1uM to 10M, or in the range of 10uM to 1M, or in the range of 100uM to 100mM, or in the range of 1mM to 10mM.

[0056] The measuring fluid can optionally include a certain amount of surfactant material. Adding a surfactant can help reduce the surface tension at the interface between the droplet and the filler fluid. Adding a surfactant can have further benefits in reducing or eliminating the physical or chemical interactions that are not expected between the measuring fluid and the hydrophobic surface. The non-limiting example of the surfactant that can be used for the electrowetting system on the medium includes Brij O20, 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.

[0057] Although the term assay is generally considered to refer to some analytical process, method or test, the term assay fluid within the scope of the present invention may be used more broadly to refer to a fluid that participates in any chemical or biochemical process that may be performed on an AM-EWOD device, such as but not limited to the following:

[0058] Laboratory tests used to test for the presence, absence or concentration of some molecular species or biomolecular species (e.g., molecules, proteins, nucleic acid sequences, etc.);

[0059] Medical or biomedical tests used to test for the presence, absence or concentration of some physiological fluid, species or substance, such as medical diagnostic tests;

[0060] · Processes for preparing a sample of material, such as extraction, purification and / or amplification of biochemical species (including, but not limited to, nucleic acids, proteins from a sample, single cells from a sample);

[0061] • A process for synthesizing a chemical or biochemical compound (including, but not limited to, examples of proteins, nucleic acids, pharmaceutical products, or radioactive tracers).

[0062] A suitable gap between the two substrates can be achieved with the aid of a spacer 9, and a non-polar filler fluid 7 (which can be an oil (e.g., dodecane, silicone oil or other alkane oil) or alternatively air) can be used to occupy the volume not occupied by the liquid droplets 8. The inner surfaces of the upper substrate 2 and the lower substrate 6 can have a hydrophobic coating 4. Non-limiting examples of materials that can be used to form the hydrophobic coating include Teflon AFF1600, perfluororesin (Cytop), polychloroparaxylene (Parylene C) and polyparaxylene HT (Parylene HT).

[0063] The lower substrate 6 may also be provided with an insulator layer 5. Here and elsewhere, the present invention has been described for an active matrix dielectric electrowetting device (AM-EWOD). However, it should be understood that the present invention and the principles behind it are equally applicable to "passive" EWOD devices, whereby the electrodes are driven by external devices as known in the prior art. Similarly, in this embodiment and subsequent embodiments, the present invention has been described in terms of an AM-EWOD device, wherein the AM-EWOD device utilizes thin film electronics 74 to implement array element circuits and driver systems in thin film transistor (TFT) technology. It should be understood that the present invention may also be implemented using other standard electronic manufacturing processes for implementing active matrix control, such as complementary metal oxide semiconductor (CMOS), bipolar junction transistor (BJT) and other suitable processes.

[0064] Figure 2a 1 is a schematic plan view of a microfluidic device according to a first and exemplary embodiment of the present invention. In this embodiment, the device 100 is a device including electrodes ( Figure 2a Active matrix on dielectric electrowetting (AM-EWOD) device with electrowetting on dielectric (not shown). Figure 1 As shown, the device 100 includes: a lower substrate (in Figure 2a101 ); an upper substrate 102 spaced apart from a lower substrate so as to form a fluid chamber 101 between the upper substrate and the lower substrate; and a fluid barrier disposed between the lower substrate and the upper substrate 102 to define the boundaries of the chamber 101. The interior of the chamber 101 is at least partially coated with a hydrophobic coating. In the illustrated example, the fluid barrier is an adhesive track 106. The adhesive track 106 bonds the upper substrate 102 (comprising ITO-coated glass in this example) to the lower substrate (comprising a TFT chip in this example).

[0065] In order to manufacture the device of the present embodiment, substrates are prepared and adhesive tracks are set on one substrate. A spacer (e.g., a polyimide 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 the substrates from being pushed closer together. Then, the adhesive is cured to harden and seal the device. Therefore, the cured adhesive track is used to bond the substrates to each other and form a fluid barrier that keeps the fluid in the device chamber 101. Once the adhesive track has been cured, the spacer can be removed, or the spacer can be kept alternatively, because the adhesive track now has a suitable thickness. The adhesive track can be formed by any suitable material that will bond the substrates together and form a fluid seal.

[0066] As an alternative, a substrate having a Figure 3a The adhesive track is generally the same shape as the photoresist pattern. The photoresist pattern can then be used to bond the top and bottom substrates together (e.g., by heating the photoresist). Because the thickness of the photoresist pattern can be selected to provide a desired cell gap between the substrates, no separate spacer is required.

[0067] It should be understood that the present invention is not limited to any specific implementation of the barrier. In principle, the device of the present invention can have a fluid barrier that does not bond the substrates together. As another example, the barrier can be a gap in the top substrate, for example cut out from the top plate and having the same Figure 2a When oil (or other filler liquid) is introduced into the chamber, the oil will not pass through the groove, but the oil will fill the area within the groove in the same way as it fills around the hole in the top substrate. Alternatively, a groove can be provided in the lower surface of the upper substrate, and assuming the groove is of sufficient depth, the oil will no longer pass through the groove and will be contained in the area within the groove. (It should be understood that if a groove is provided in the upper substrate, it is preferably left with a gap in the groove so that the groove does not divide the substrate into two separate pieces.)

[0068] The chamber 101 has a plurality of inlet ports 111, 112 and a plurality of outlet ports 110. The inlet ports 111, 112 and the outlet port 110 are arranged in the upper substrate 102 of the device 100. In this example, the inlet port includes a measuring fluid inlet port 111 and an oil inlet port 112. The inlet ports 111, 112 and the outlet port 110 are shown as being (substantially) the same, including the aperture in the upper substrate 102. However, the present invention is not limited thereto, and the inlet ports can be formed to have different sizes from each other to hold different volumes of measuring fluid. The aperture can be produced using various techniques, for example, laser drilling or HF (hydrofluoric acid) etching, CNC drilling, powder spraying and molding (in an example where the top plate is made of a plastic material). The outlet port 110 is substantially located at the periphery of the chamber 101. For example, at least one outlet port 110 is located in a corner of the chamber 101.

[0069] The chamber 101 also includes a drain region 105 in fluid communication with at least one drain port 110. The chamber 101 also includes an active region 109 for performing one or more assays. The active region 109 is defined as the region where fluid is loaded into the device and the assay is performed. The drain region 105 and the active region 109 are defined by an adhesive track 106. In addition to the drain port 110 at the end of the drain region 105, another drain port 110 is provided at the end of the adhesive track 106, which separates the drain region 105 from the active region 109. The drain port 110 is shown as being located at the end of the adhesive track 106. Figure 2a on the right side.

[0070] As indicated, the device is provided with electrodes in the active region ( Figure 2a 100) to allow manipulation of droplets of assay fluid within the active region. These electrodes can be considered to define one or more "internal reservoirs" (not shown) in which fluids can be controlled by the electrodes of the actuating device 100.

[0071] The device 100 is configured to preferentially retain the metered volume of filler fluid (e.g., oil) in a portion of the chamber 101 when the chamber 101 contains a metered volume of filler fluid (e.g., oil) (not shown here) that partially fills the chamber 101, and to allow some of the filler fluid to move out of the portion of the chamber 101 when a volume of a measured fluid (not shown here) is introduced into the portion of the chamber 101 through one of the one or more inlet ports 111, thereby causing a volume of exhaust fluid to be exhausted through at least one exhaust port 110. This is explained in more detail below.

[0072] Figure 2b is a schematic diagram depicting a microfluidic device according to a second embodiment of the present invention. Figure 2a The device 100 may be described as top-loaded, while Figure 2b The device 200 can be described as side-filled. The outer periphery of the spacer 204 and the outer periphery of the lower substrate 203 extend beyond the outer periphery of the upper substrate 202, and the entry ports 211, 212 are defined by corresponding notches provided in the inner edge of the spacer 204, and the entry ports 211, 212 extend beyond the upper substrate to provide fluid communication between the chamber 101 and the outside of the device. In other structural aspects, Figure 2b The device 200 and Figure 2a The devices 100 are substantially the same. Figure 2c Shown along Figure 2b The cross section is taken along line XX.

[0073] exist Figure 2b In the example of FIG. 1 , the larger notch at the lower left corner of the device can be used as an oil (or other filler fluid) entry port. In practice, it can be convenient for the oil entry port to be larger than the entry port for the assay liquid, because a larger amount of oil is required to operate the device, and a large oil entry port allows the use of larger pipette tips. However, the oil entry port does not necessarily have to be larger than the other ports, and in principle a small pipette could be used instead to dispense the oil multiple times.

[0074] Now refer to Figure 3a to Figure 3d A method of introducing fluid into the microfluidic device 100 is described. Figure 3a to Figure 3d is a schematic diagram depicting a microfluidic device according to a first embodiment of the present invention. Figure 3a Depicted as above reference Figure 2a The device 100 is described. In this example, the chamber 101 initially contains an exhaust fluid. Generally, the exhaust fluid can be any fluid. Generally, the exhaust fluid can be air 115. Other examples of possible exhaust fluids include any inert atmosphere (e.g., nitrogen or argon). Alternatively, the fluid can be a polar liquid (e.g., water). Advantageously, but not necessarily, the exhaust fluid can be substantially free of moisture. Combinations of exhaust fluids can also be utilized.

[0075] Figure 3b Instructions are given to introduce a metered volume of a filler fluid, in this case oil 107, into chamber 101. Typically, the filler fluid is selected to be a non-polar material or a low polar material. Typically, the filler fluid is selected to have a low interfacial surface tension with the measuring fluid. Typically, the filler fluid is selected to be immiscible or substantially immiscible with the measuring fluid. Typically, but not necessarily, the filler fluid may have a low viscosity so as to maximize the speed of movement of droplets of the measuring fluid. Typically, but not necessarily, the filler fluid has a lower density than the measuring fluid. Typically, but not necessarily, the filler fluid is selected to have low or relatively low toxicity. Typically, but not necessarily, the filler fluid is selected to have little or low reactivity with materials including the measuring fluid. Typically, but not necessarily, the filler fluid is a liquid.

[0076] Non-limiting examples of suitable filler fluids commonly used in electrowetting systems on dielectrics and suitable for use in the present invention include silicone oils, alkanes such as n-dodecane. Non-limiting examples of surfactants that can be optionally 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.

[0077] Other suitable non-polar filler fluids may also be used. Oil 107 is introduced (e.g., pipetted) into chamber 101 using oil entry port 112. It should be understood that a metered volume of oil 107 may be introduced into chamber 101 by other suitable means. The volume of oil 107 is metered so that enough oil 107 is introduced to cover a desired portion of the chamber, but not completely fill the chamber. In this embodiment, the portion of the chamber containing oil includes active area 109 of chamber 101. As shown, even after the metered volume of oil is introduced, exhaust area 105 is still substantially filled with air 115.

[0078] The device 100 may be provided with an optical and / or electrical sensor for metering the volume of the oil 107 introduced into the chamber. Alternatively, an optical and / or electrical sensor may be provided separately. As another example, the volume of the oil 107 may be measured in advance before the oil 107 is introduced into the chamber 101.

[0079] It will be appreciated that as oil 107 is introduced into chamber 101, air 115 in active area 109 is exhausted from the chamber until substantially all of active area 109 is covered with oil 107. The air may be exhausted through any suitable aperture, and thus may be exhausted through the assay fluid port and exhaust port 110 in the exhaust area. Apertures suitable for exhaust are preferably located at the periphery of chamber 101, particularly at the corners of chamber 101, to aid exhaust and ensure that no air 115 is trapped within active area 109.

[0080] like Figure 3b As shown, when oil is introduced into the chamber, the inlet port and the exhaust port remain dry because the oil fills around the inlet port and the exhaust port.

[0081] The device 100 is configured to preferentially retain a metered volume of oil 107 in a desired portion of the chamber 101. In this example, the device 100 includes a flow restriction element for this purpose. Due to the location of the adhesive rail 106 and the drain port 110 at the rightmost end of the drain area 115 (as described, the oil does not enter the drain area), a constriction 116 is provided in the fluid flow path from a portion of the chamber to the drain area 105. This constriction 116 acts as an oil flow restriction element. Therefore, even in the case of a sloping edge of the chamber 101, the oil 107 still tends to reside in the active area 109. A certain volume or a certain bubble amount of air 115 is retained in the drain area 105.

[0082] The size of the constriction is determined based on known properties of the filler fluid (eg, surface tension of the filler fluid with the hydrophobic surface and with the assay fluid, viscosity and density of the filler fluid).

[0083] like Figure 3c As shown, a certain volume of the measurement fluid 108 is now introduced into the chamber 101 by filling it into the fluid input port 111. This can be done using a pipette, alternatively it can be done using another input method (e.g., a capillary track or line). The measurement fluid 108 is a polar fluid (e.g., blood). Alternatively, the measurement fluid can be a type of reagent. First, the fluid input electrode (not shown here) that defines the internal reservoir of the device 10 is activated. Then, the measurement fluid 108 is pipetted into the fluid input port 111, whereby it enters the chamber 101 via capillary forces. In other words, the measurement fluid 108 is sucked onto the active area 109. It should be understood that the measurement fluid 108 enters the chamber 101 without any pressure-actuated input device (e.g., a piston, pump, or gravity trap) or even without electrowetting forces.

[0084] In this embodiment, the fluid will be sucked into the device only by capillary forces. However, the direction of the fluid entering the chamber from a specific entry port cannot be well controlled, and the fluid may occupy a circular area around the entry port. Therefore, optionally, an electrowetting force can be applied to guide the direction of fluid filling, which is particularly advantageous if two or more different assay fluids are to be introduced into the chamber via different entry ports, and it is desired to control the way in which the different assay fluids contact each other. However, the electrowetting force in this case is only used to control the position of the assay fluid 108 in the active area 109.

[0085] Alternatively, the device may be configured so that capillary forces are insufficient to draw the assay fluid from the inlet port into the chamber. (How this may be done is described elsewhere.) In this case, application of electrowetting forces draws the fluid into the chamber and controls the position of the assay fluid in the chamber.

[0086] When the assay fluid 108 enters the chamber 101 substantially in the direction of arrow C, some of the oil 107 is displaced laterally from the active area 109 of the device 100. It should be understood that the assay fluid 108 and the oil 107 are substantially immiscible. When the active area 109 is substantially filled with the oil 107, the oil 107 is displaced laterally from the active area 109 of the device 100. Figure 3c The air 115 is displaced into the exhaust region 105 through the constriction 116 in the direction indicated by the arrow B. This causes a volume of air 115 to be exhausted from the chamber 101 through the exhaust port 110 at the leftmost end of the exhaust region 105. The volume of air 115 or bubbles is substantially displaced along Figure 3c The direction indicated by the arrow A in FIG. 1 moves toward the leftmost exhaust port 110. Therefore, the size of the bubble decreases.

[0087] Another volume of assay fluid 108' is now introduced into the active area 109 of the chamber 101 via the second fluid inlet port 111. As described above, the assay fluid 108' causes some of the oil 107 to be displaced into the drain area 105, which in turn causes another volume of air 115 to be exhausted through the exhaust port 110 at the far left of the drain area 105. Thus, the bubble size is further reduced. The other volume of assay fluid 108' is controlled to be within the internal reservoir of the active area 109 by the electrowetting forces provided by the fluid input electrodes in the lower substrate 103 of the device.

[0088] The other volume of the assay fluid 108' may be substantially the same in composition as 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 a reagent. The other volume of the assay fluid 108' may have a substantially different volume than the first volume 108 (e.g., 2 μl (microliters)), or may have the same volume as the first volume 108 (e.g., 0.25 ul). The internal reservoir may be configured to accommodate a range of fluid volumes (e.g., 0.1 ul to 100 ul). The volume and shape of the internal reservoir may be changed by controlling the size and number of electrodes defining the internal reservoir.

[0089] Another volume of assay fluid 108, 108' can be loaded into the active area 109 of the device 100 until all the required fluid is loaded, or until the drain area 105 is substantially completely filled with oil 107 and all the air 115 is substantially exhausted. When the drain area 105 is full of oil 107, no more assay fluid 108, 108' can be loaded unless some oil 107 leaks out of the device 100. Once all the required assay fluid 108, 108' has been loaded onto the active area, droplets can be formed from the internal reservoir using standard EWOD operations. Fluid droplets can be dispensed from the internal reservoir by electrowetting. Droplet size is easily adjustable, accurate and reproducible.

[0090] The configuration of device 100 provides a simple method for inputting the assay fluid into the device. Compared to the prior art, no external input pump, input piston or large gravity trap is required, and external moving parts are eliminated. Therefore, the possibility of leakage is reduced, and the device of the present invention is much simpler to manufacture. The lack of a large piston means that a greater number of fluid inputs can be provided in a given area. In addition, a predetermined volume of assay fluid can be loaded onto an internal reservoir, and the volume of the internal reservoir can be selected to accommodate a desired amount of a specific assay fluid.

[0091] In the above example, the assay fluid 108 is introduced into the chamber 101 after the oil 107 is introduced. In another example, not shown here, one or more assay fluids and one or more filler fluids can be introduced substantially simultaneously with each other. The fluid can be introduced through a fluid input port or other input port in the device by a pipette or by any other suitable input device. The fluids can be substantially mixed at the input point or can be substantially separated. In this case, the assay fluid 108 is controlled in the chamber 101 by actuating electrodes during the introduction of the assay fluid 108 and the filler fluid 107 so that the assay fluid remains in the active area of ​​the device.

[0092] In this example, apertures 110a, 110b and 110c are provided to act only as exhaust ports because Figure 3a The arrangement of the fluid inlet ports 111 may not provide adequate drainage at the corners of the chamber 107, but if the arrangement of the inlet ports provides adequate drainage of the chamber then in principle there may be no need to provide an aperture that acts only as a drainage port.

[0093] Furthermore, in this embodiment, when oil is introduced into the chamber, all ports are designed to be dry. In an alternative embodiment, when oil is introduced into the chamber, all inlet ports may remain dry, but oil may enter the outlet ports (except outlet ports 110 in exhaust area 105). To this end, the diameter of the outlet ports should be made small so that these outlet ports are filled with oil by capillary action.

[0094] Figures 4a to 4d Schematic diagram of a method for filling a microfluidic device according to a second embodiment of the present invention. Figure 2b As discussed, the device 200 in this embodiment can be described as side-filled. The method of filling a volume of assay fluid 208 into the device 200 is similar to that described above with reference to Figure 3a to Figure 3d The method described in the first embodiment of the present invention is substantially the same. In this embodiment, the polyimide spacer 204 generally separates the drain region 205 and the active region 209 of the device 200 and defines a constriction (a narrow channel in this embodiment) between the drain region 205 and the active region 209. In addition, the spacer 204 forms a separate fill region along the bottom edge of the device 200. As previously described, the upper substrate 202 is smaller than the spacer 204 by a controlled amount to form a small gap around the periphery of the device 200 through which a fluid can be introduced or through which a drain fluid (such as air) can be discharged.

[0095] A metered volume of filler fluid (e.g., oil 207) is introduced into chamber 201, for example, by a pipette, through an aperture at the lower left corner of chamber 201. The volume of oil 107 is carefully controlled so that enough oil substantially covers active area 209, but exhaust area 205 remains primarily filled with exhaust fluid (in this case, air).

[0096] Once the metered volume of oil 207 has been filled and substantially all of the air 215 in the active area 209 has been expelled via the exhaust port 210, the fluid input electrode of the internal reservoir (not shown here) is activated and a volume of assay fluid 208 is pipetted into one or more fill areas or fluid input ports 211 extending along the bottom edge of the chamber 201. It should be understood that these input ports 211 may be located anywhere along the perimeter of the active area 209.

[0097] A volume of assay fluid 208 enters the device 200 substantially in the direction of arrow C by capillary action, and is controlled once the assay fluid 208 enters the chamber 201 by electrowetting forces. As the assay fluid 208 enters the active region 209, some of the oil 207 is displaced substantially in the direction of arrow B through the constriction 216 into the drain region 205. As some of the oil 207 enters the drain region 205, a volume of air 215 is exhausted substantially in the direction of arrow A through the drain port 210 at the leftmost end of the drain region 205. Thus, the bubble size is reduced.

[0098] As described with reference to the first embodiment above, further volumes of assay fluid 208' can now be introduced into the chamber 201 and further oil will be displaced into the drain region 205 until either the full required fluid charge for the assay is complete or all air 215 in the drain region 205 has been exhausted. Further volumes of assay fluid 208, 208' can be introduced if a volume of oil 207 is extracted from the chamber 201. Droplets for the assay can now be generated from the internal reservoir of assay fluid 208, 208' by electrowetting forces.

[0099] As discussed with reference to the first embodiment above, in an alternative method of filling with one or more assay fluids, one or more filler fluids may be introduced into the chamber 201 substantially simultaneously with one another.

[0100] Figure 5a to Figure 5d is a schematic diagram depicting a method of filling a microfluidic device according to a third embodiment of the present invention. In this embodiment, unlike the first and second embodiments described above, the drain region 305 is integrated with the active region 309 of the device 300. Although a separate drain region simplifies operation, a separate drain region takes up valuable space on the TFT chip.

[0101] In this embodiment, the flow restriction element is used to preferentially keep the metered volume of filler fluid (oil 307) in a part of chamber 301 again. In this example, the flow restriction element includes one or more physical walls and a patterned hydrophobic coating 314 that may be on the inside of chamber 301. The wall can be formed by, for example, an adhesive or a photoresist. If the device tipped over, only the wall may not be enough to contain the oil (or other filler fluid), and the oil may escape the adhesive wall boundary. Therefore, the hydrophobicity patterning of the surface of one or two substrates can be used to further retain the oil on the region in the wall. For example, the hydrophobic surface behind the entry port can be removed so that the oil will then preferentially enter these regions. Then, if the device tipped over, the presence of the wall and the patterned hydrophobic surface will be enough to keep the oil in the correct region for filling the assay fluid.

[0102] In alternative embodiments, for example if the user is advised not to tip the device over, a physical wall alone may be sufficient.

[0103] The coating 314 provides a “wall” around the fluid input 311 .

[0104] Figure 5a The device 300 is shown substantially filled with an exhaust fluid (in this case air). A metered volume of oil 307 is input into the region surrounded by the wall 314. The oil 307 is constrained by the wall 314 and the hydrophobic pattern and tends to remain in the region. Then, a volume of assay fluid 308 is introduced into the active region 309 via the fluid input port 311. It should be noted that in this embodiment, the fluid input port 311 can be used to introduce both the oil 307 and the assay fluid 308.

[0105] As the assay fluid enters the active area 309, the assay fluid is constrained to enter the active area substantially in the direction indicated by arrow C by electrowetting forces provided by the actuation electrodes (not shown). (As described above, capillary forces may be sufficient to cause the assay fluid to enter the active area, while the electrowetting forces control the direction of fluid entry, or alternatively the electrowetting forces may cause the assay fluid to enter the active area and control the direction of fluid entry.) Some of the oil is further displaced into the active area 309 substantially in the direction of arrow B. As the oil 307 is displaced, some air is exhausted through exhaust port 310.

[0106] like Figure 5d As shown and as described above for the first and second embodiments, further volumes of assay fluid 308' may be introduced into chamber 301 until all fluid required for the assay is loaded or until active area 309 is substantially filled with oil (i.e., all air is exhausted through exhaust port 310). If a volume of oil 307 is extracted or aspirated from chamber 301, further volumes of assay fluid 308, 308' may be introduced.

[0107] As discussed with respect to the above embodiments, one or more filler fluids and one or more assay fluids may be introduced into the device 300 substantially simultaneously with one another, rather than introducing the filler fluids and assay fluids separately.

[0108] Figures 6a to 6d FIG. 4 is a schematic diagram illustrating a method for filling a microfluidic device according to a fourth embodiment of the present invention. The fourth embodiment of the microfluidic device 400 is structurally similar to the above referenced Figure 2aThe first embodiment discussed is similar, and again includes one or more flow restriction elements that preferentially retain a metered volume of filler fluid (oil 407) in a desired portion of chamber 401. In this embodiment, the flow restriction elements include one or more controllable flow restriction elements that can be, for example, electrically controlled to be in an "open" state or a "closed" state. Figure 6a Two electrically activated barriers 417 are shown arranged in series between a portion of the chamber and the exhaust port 410 at the leftmost end of the exhaust region 405, but the present invention is not limited to this particular arrangement. Each barrier 417 comprises a fluid that is immiscible with the filler fluid 407. Figure 6a As shown, when the barrier is “closed,” the fluid extends across the width of the drain region 405 .

[0109] In this example, barrier electrodes (not shown) are provided at locations where it is desired to provide barriers 417. A polar fluid (which may be a determination fluid) (e.g., water) is loaded into the drain region 405 to form one or more barriers or gates 417. The polar fluid may be loaded via an input port 411 adjacent to the barrier. The polar fluid is held within the barrier location by electrowetting forces provided by the electrodes at the barrier location. A metered volume of oil 407 is then introduced into the chamber 401 as described in the first embodiment above, such that the active region 409 is substantially covered with oil 307, while the separated drain region 405 is substantially filled with air 415. It should be noted that since the oil 407 is immiscible with the non-polar barrier fluid and the barrier fluid extends substantially across the entire width of the drain region when the barrier is closed, the oil 407 cannot fill the device 400 further than the first barrier 417.

[0110] The provision of barrier 417 means Figure 3b or Figure 4b , Figure 2a The constrictions 116, 216 are not required in this embodiment and may be removed, as shown in FIG. Figure 6b The larger gap 416 is shown. However, in principle, Figures 6a to 6d In an embodiment of the present invention, a constriction is provided.

[0111] Then, a certain volume or more of the assay fluid 408, 408' can be loaded into one or more fluid input ports on the active area 409. Since the oil 307 is prevented from displacing into the exhaust area 405 by the barrier 417, a certain volume of the assay fluid 408, 408' cannot enter the chamber 401 and is thus "stored" in the fluid inlet port 411. The advantage of this method is that the assay fluid can be "stored" until all the fluid is loaded and the assay is ready to begin.

[0112] When the user is ready to load the assay fluid 408, 408' into the device 400, the position of the barrier fluid in one or more barriers 417 can be changed by appropriately controlling the barrier active electrodes. Figure 6d As shown, the barrier fluid is reconfigured to no longer extend across the entire width of the drain region, thereby allowing some of the oil 407 to flow through the barrier 417 into the drain region 405. The oil can now be displaced from the active region into the drain region 405 substantially in the direction of arrow B, and a volume or more of the assay fluid 408, 408' stored in the inlet port is drawn into the active region, with the direction of fluid entry being substantially in the direction indicated by arrow C as the assay fluid is controlled by electrowetting forces. The air 415 in the drain region 405 can be exhausted through the fluid input port 411 adjacent to the barrier 417.

[0113] It will be appreciated that a plurality of barriers 417 may be provided to allow for the staged introduction of a volume or more of the assay fluid 408 , 408 ′ into the active region 409 .

[0114] Figures 7a to 7d is a schematic diagram depicting an alternative method of filling a microfluidic device according to a fifth embodiment of the present invention. In this embodiment, the device 500 does not include a drain region separated from the active region. Figure 7b As shown, in use, the device 500 is first substantially completely filled with a filler fluid (e.g., oil 507) via the oil input port 512. When the device 500 is filled with the oil 507, any exhaust fluid (air) present in the chamber 501 will be exhausted out of the exhaust region 505 through the exhaust port 510. The oil 507 will fill around the exhaust port 510 and the fluid input port 511, so that these spaces remain dry.

[0115] Then, a certain volume or more of the measurement fluid 508, 508' is loaded into the fluid input port 511. Figure 7c As shown, since the oil 507 cannot be displaced when the chamber 501 is full, the assay fluid 508, 508' is retained in the input port.

[0116] Now, some oil 507 is extracted via the oil outlet port 513, and the oil 507 leaves the chamber 501 substantially in the direction indicated by arrow B. For example, the extraction may include the use of a capillary line, a pipette, or an absorbent pad. When some oil 507 is removed from the active area 509, the assay fluid 508, 508' is drawn into the chamber 501 substantially in the direction of arrow C due to capillary forces and is controlled into the internal reservoir by electrowetting. The volume of the extracted oil 507 is carefully metered to match the volume of the assay fluid that needs to be loaded into the device 500.

[0117] In the above embodiments, the device is arranged so that the assay fluid introduced into the entry port is naturally drawn into the chamber 101, and the assay fluid introduced into the entry port cannot be naturally drawn into the chamber 101 simply because the active area of ​​the device already contains fluid (a filler fluid or a combination of a filler fluid and one or more previously introduced assay fluids). This can be arranged by selecting appropriate values ​​for the cell gap (i.e., the spacing between the upper substrate and the lower substrate), the hydrophobic coating, and the properties of the assay fluid (e.g., viscosity, density, and surfactant level). For example, the cell gap can be selected based on an understanding of the assay fluid to be used. The assay fluid can then be introduced into the chamber in a controlled manner according to any of the above embodiments.

[0118] However, the present invention is not limited thereto, and the device may alternatively be arranged so that the assay fluid introduced into the inlet port is naturally retained in the inlet port. Figures 8a to 8e is a schematic diagram depicting a method of filling a microfluidic device according to a sixth embodiment of the present invention, wherein the device is configured in this manner. In this embodiment, the device 600 is provided with a drain region 605 that is integral with the active region 69 of the chamber.

[0119] A certain volume or more of the assay fluid 608, 608' is introduced into the fluid input port 611, for example, by a pipette. The active area 609 is only substantially filled with the exhaust fluid (air) at this stage, so that the assay fluid 608, 608' is not sucked onto the active area 609 due to capillary action, but remains in the input port 611. A metered volume of filler fluid (e.g., oil 607) is now introduced into the device 600 via the input port 612. When the oil 607 flows through the active area 609 substantially in the direction indicated by the arrow B, the assay fluid 608 is sucked out from the input port onto the active area 609 due to capillary forces. Once within the active area 609, the assay fluid 608 is held in place by the electrowetting force provided by the actuating electrode. The air contained within the active area 609 is exhausted through the exhaust port 610.

[0120] Now, another metered volume of oil 607 can be introduced into the device 600, causing the oil 607 to move further across the active area 609, and another volume of assay fluid 608' is drawn into the device 600, as shown in FIG. Figure 8d The process of loading the assay fluid 608, 608' and the oil 607 can continue until all the required fluids are loaded or until the active area 609 is substantially filled with the oil 607. The oil 607 can then be extracted from the device 600 in order to load another assay fluid 608, 608'.

[0121] Optionally, in this embodiment, one or more of the fluid input ports 611 and optionally all of the fluid input ports 611 further include an upper well 618 in which a larger volume of assay fluid 608, 608' can be held than the volume in the fluid input port 611 itself. Figure 8e As shown in cross-section in FIG. 6 , the well 618 may include a plastic groove formed in the port 611 in the upper substrate 602 of the device 600 .

[0122] It should be understood that a well similar to well 618 may be provided in devices used in other embodiments of the present invention. This may be useful in embodiments where the assay fluid is "pre-stored" in the inlet port (e.g., Figures 6a to 6d In the embodiment of ) is particularly beneficial.

[0123] Figures 9a to 9d Schematic diagram of a method for filling a microfluidic device according to a seventh embodiment of the present invention. Figure 2a The device 700, which is substantially identical to the device of the first embodiment discussed, is provided with 26 separate fluid input ports 711. The ports 711 are located around the perimeter of the active area 709 of the device, however, the location of the ports 711 can be varied as desired. It should be understood that each port 711 can be used for a different assay fluid 708, 708' as desired. The internal reservoir associated with each input port 711 can be varied with respect to shape and volume as described above in order to accommodate the volume of assay fluid required for the assay.

[0124] In this way, device 700 provides a flexible, versatile and simple method of loading fluids for determination. Although the structure of device 700 is comparable to that of the first embodiment discussed above, it should be understood that any of the embodiments discussed herein can be provided with a similar number of fluid input ports. The number of ports is limited only by the size of the device and can therefore be varied to accommodate the requirements of the determination to be performed. The device can be configured so that determinations can be performed in parallel. In addition, since no large, tall fluid trap is required, the configuration of the fluid input ports of the various embodiments discussed above provides consistent heating of the fluid within the device.

[0125] Many possible applications of microfluidic devices require some form of thermal control. Another advantage of the present invention is that by eliminating bulky input devices (such as pistons, tubes or high fluid traps), much better temperature uniformity can be obtained over the active area, even in embodiments where the ports and exhaust ports are provided by holes in the upper substrate.

[0126] Fig.10a1 is a diagrammatic representation of a box 119 based on a microfluidic device. In the illustrated example, the device 100 shown is a device of the first embodiment, however, any of the embodiments discussed herein may be included in a similar box 119. The box 119 in this example is configured to be disposable and / or recyclable and is suitable for manufacturing in large quantities (e.g., millions of units per year) at low cost. The box acts as an interface between the fluid within the AM-WOD device and the outside world, and may also provide heating for the fluid droplets contained within the device. Fig.10b yes Fig.10a An exploded view of the box showing the various components of the box.

[0127] Fig.11a is a diagrammatic representation of a benchtop control / reader device 120 configured to control the operation of the microfluidic device contained within the cartridge and to Fig.10a and Fig.10b The data is read appropriately according to the situation. Fig.11b is a diagrammatic representation of a handheld control / reader device 120' configured to control the operation of such a microfluidic device. As is known in the art, a cartridge 119 containing a microfluidic device (100, 200, 300, 400, 500, 600, 700) is inserted or connected to the control / reader device 120, 120'.

[0128] Although the present invention has been shown and described with respect to certain embodiments, equivalent alternatives and modifications may occur to those skilled in the art after reading and understanding this specification and the accompanying drawings. Specifically, with respect to the various functions performed by the elements (assemblies, assemblies, devices, compositions, etc.) described above, unless otherwise indicated, the terms used to describe these elements (including references to "devices") are intended to correspond to any element that performs the specified functions of the described elements (i.e., functionally equivalent), even if the structure is not equivalent to the disclosed structure that performs the functions in the exemplary embodiments herein of the present invention. In addition, although the above may have described specific features of the present invention only for one or more of several embodiments, such features may be combined with one or more other features that may be desirable and advantageous for any given or specific application in other embodiments.

[0129] (Overview)

[0130] A first aspect of the present invention provides a method for filling a microfluidic device with a measuring fluid, the method comprising: introducing a metered volume of filler fluid into a chamber having one or more inlet ports in the microfluidic device so that the chamber is partially filled with the filler fluid, the device being configured to preferentially retain the metered volume of filler fluid in a portion of the chamber; and introducing a certain volume of measuring fluid into the said portion of the chamber via one of the one or more inlet ports, thereby causing a certain volume of discharge fluid to be discharged from the chamber.

[0131] In addition to the inlet port, the chamber may have at least one exhaust port, so that exhaust fluid is discharged from the chamber through the at least one exhaust port. "Exhaust port" refers to a port that is provided only to allow exhaust fluid to be discharged from the chamber and does not serve as an inlet port. Alternatively, the exhaust fluid can be discharged from the chamber through one or more inlet ports.

[0132] A second aspect of the invention provides a method for filling a microfluidic device with an assay fluid, the method comprising: substantially completely filling a chamber with a filler fluid or with a fluid mixture including the filler fluid as a 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 a sufficient amount of the filler fluid through the outlet port so that at least some volume of the assay fluid can enter the chamber from one of the one or more inlet ports. In this regard, the chamber can be initially filled with the filler fluid, and the method can then be used to enable the introduction of the assay fluid. Alternatively, the chamber can be initially filled with a mixture of the filler fluid and the assay fluid, and the method can then be used to enable the introduction of more assay fluid and / or one or more different assay fluids.

[0133] The present invention allows the assay fluid to be easily introduced into the device. There is no need to apply high pressure to force the assay fluid into the device, and the problems associated with the use of pistons and pumps (e.g., the need to provide a good high pressure seal at the entry port to avoid sample loss and / or the introduction of bubbles) are overcome. The device of the present invention is simple and therefore inexpensive to manufacture and simple to operate. Another advantage is that many entry ports can be easily provided on the device, while the physical size of the pump / piston or gravity trap used in the prior art means that it is difficult to accommodate the pump / piston or gravity trap on a typical device.

[0134] In any aspect, the method may further include introducing a filler fluid and a measuring fluid into the chamber substantially simultaneously with each other. The phrase "substantially simultaneously with each other" is intended to encompass methods in which the time period for introducing the filler fluid overlaps the time period for introducing the measuring fluid. Alternatively, in any aspect of the invention, the filler fluid may be introduced into the chamber first, and the measuring fluid may be introduced into the chamber after the filler fluid is introduced. As another alternative, in any aspect of the invention, the measuring fluid may be introduced into one or more inlet ports first, but the measuring fluid is retained in the inlet port. (Basically this requires that the device and the measuring fluid are arranged so that the capillary forces tending to draw the measuring fluid from the inlet port into the chamber are insufficient to overcome the repulsion of the chamber, which is naturally hydrophobic, to the fluid.) When the filler fluid is introduced into the chamber, the filler fluid acts to draw the measuring fluid into the chamber.

[0135] The device may be an electrowetting on dielectric (EWOD) device comprising electrodes. The method may further comprise controlling the assay fluid within the chamber by actuating the electrodes.

[0136] Where the filler fluid and the assay fluid are introduced into the chamber substantially simultaneously with each other, the method may include controlling the assay fluid within the chamber by actuating the electrodes during the introduction of the filler fluid and the assay fluid into the chamber.

[0137] In the method of the first aspect, after a metered volume of filler fluid has been introduced into the chamber, a certain volume of measuring fluid can be introduced into the chamber, whereby the measuring fluid can enter a portion of the chamber by removing some filler fluid from the portion of the chamber.

[0138] At least a portion of the interior of the chamber may be coated with a hydrophobic coating.

[0139] The device may be configured such that one or more inlet ports are provided in the upper surface of the chamber. If one or more outlets are present, the outlets may also be provided in the upper surface of the chamber.

[0140] The device may be configured so that one or more inlet ports are provided in one or more sides of the chamber. If one or more outlets are present, the outlets may also be provided in the sides of the chamber.

[0141] The device may be configured such that the chamber is provided with a drain region in fluid communication with the at least one drain port, the drain region being configured to contain a drain fluid.

[0142] The device may be configured to have at least one exhaust port that is substantially identical to one or more inlet ports.

[0143] The device may be configured such that a chamber is provided with an active area for performing one or more assays.

[0144] The device may be configured such that the drain region is integral with the active region.

[0145] The device may be configured such that the drain region is partially separated from the active region by a fluid-impermeable barrier.

[0146] The method may also include preferentially retaining the metered volume of filler fluid in a portion of the chamber using a flow restriction element.

[0147] The flow restriction element may be a patterned hydrophobic coating on the interior of the chamber.

[0148] The flow restriction element may be a constriction in the fluid path from a portion of the chamber to the exhaust region.

[0149] The method may include retaining a metered volume of filler fluid in a portion of the chamber using one or more electrically activated barriers between a portion of the chamber and an exhaust port, the one or more barriers comprising a fluid that is immiscible with the filler fluid.

[0150] The method may further include metering the metered volume of filler fluid by one of volume measurement, optical sensing, and electrical sensing.

[0151] The exhaust port may be located substantially at a corner of a portion of the chamber. This eliminates the risk of air being trapped at the corner when the filler fluid is introduced into the chamber. Preferably, the exhaust port is located at each corner of a portion of the chamber.

[0152] The method of the invention may also comprise introducing a second assay fluid, for example via another inlet port.This may be repeated until all of the desired assay fluid has been introduced into the chamber.

[0153] The third aspect of the present invention provides a microfluidic device comprising: a chamber having one or more inlet ports; the device being configured to: when the chamber contains a metered volume of filler fluid that partially fills the chamber, preferentially retain the metered volume of filler fluid in a portion of the chamber; and the device being configured to: when a certain volume of measurement fluid introduced into one of the one or more inlet ports enters a portion of the chamber, allow some of the filler fluid to move out of a portion of the chamber, thereby causing a certain volume of discharge fluid to be discharged from the chamber.

[0154] The fourth aspect of the present invention provides a microfluidic device comprising: a chamber having one or more inlet ports and an outlet port for extracting a filler fluid; whereby when in use, the chamber is substantially completely filled with the filler fluid, and when a sufficient amount of filler fluid is extracted through the outlet, a certain volume of measurement fluid introduced into one of the one or more inlet ports is able to enter the chamber.

[0155] In the device of the third aspect or the fourth aspect, in addition to the inlet port, the chamber may have at least one outlet port, so that the exhaust fluid is discharged from the chamber through the at least one outlet port. "Exhaust port" refers to a port provided only to allow the exhaust fluid to be discharged from the chamber and not used as an inlet port. Alternatively, the exhaust fluid can be discharged from the chamber through one or more inlet ports.

[0156] The device may be an electrowetting on dielectric (EWOD) device comprising electrodes and, in use, may control an assay fluid within a chamber by actuating the electrodes.

[0157] The interior of the chamber may be at least partially coated with a hydrophobic coating.

[0158] The device may include a lower substrate, an upper substrate spaced apart from the lower substrate, and a fluid barrier disposed between the lower substrate and the upper substrate for defining a perimeter of the chamber.

[0159] The fluid barrier may be provided by an adhesive track adhering the lower substrate to the upper substrate.

[0160] The fluid barrier may be provided by a spacer separating the lower substrate from the upper substrate.

[0161] At least one of the one or more inlet ports may be provided in the upper substrate of the device.If one or more exhaust ports are present, the exhaust ports may also be provided in the upper substrate of the chamber.

[0162] At least one of the one or more inlet ports and / or the at least one outlet port may be provided in the fluid barrier. If one or more outlet ports are present, the outlet port may also be provided in the fluid barrier.

[0163] The outer periphery of the spacer may extend beyond the outer periphery of the upper substrate, and at least one of the one or more inlet ports may be defined by a corresponding notch provided in the inner edge of the spacer. Alternatively, at least one of the one or more inlet ports may be defined by a gap in the spacer. If one or more outlet ports are present, the outlet ports may also be defined by a notch or gap in the spacer.

[0164] The chamber may also include a drain region in fluid communication with the at least one drain port and configured to contain drain fluid.

[0165] The chamber may also include an active area for performing one or more assays.

[0166] The device may have at least one exhaust port that is substantially identical to the one or more inlet ports.

[0167] The exhaust region may include an active region.

[0168] The fluid barrier may also define drainage areas and active areas within the chamber.

[0169] The device may include a flow restriction element for preferentially retaining a metered volume of filler fluid in a portion of the chamber.

[0170] The flow restriction element may include a patterned hydrophobic coating on the interior of the chamber.

[0171] The flow restriction element (feature) may include a constriction in the fluid flow path from a portion of the chamber to the exhaust region.

[0172] The flow restriction element may include one or more electrically activated barriers between a portion of the chamber and the exhaust port, the one or more barriers comprising a fluid that is immiscible with the filler fluid.

[0173] The device may include an optical and / or electrical sensor for metering the volume of fill fluid.

[0174] The chamber may include at least one exhaust port located substantially in a corner of a portion of the chamber.

[0175] The fifth aspect of the present invention provides a microfluidic system, comprising: the microfluidic device of the third aspect or the fourth aspect, the device being contained in a disposable box; and a control and / or reader device configured to control and / or read the microfluidic device.

[0176] In the method of the first aspect or the second aspect, the filler fluid may include a non-polar fluid. The filler fluid may include oil. The filler fluid may include a surfactant.

[0177] In the method of the first aspect or the second aspect, the assay fluid may include a polar fluid, an aqueous material, or a surfactant.

[0178] In the method of the first aspect or the second aspect, the exhaust fluid may include a gas. The exhaust fluid may include air. The exhaust fluid may include an inert gas.

[0179] (CROSS-REFERENCE TO RELATED APPLICATIONS)

[0180] This nonprovisional application claims priority under 35 USC §119 to Patent Application No. 1516430.4 filed on September 16, 2015 in the United Kingdom of Great Britain and Northern Ireland, the entire contents of which are incorporated herein by reference.

[0181] Industrial Applicability

[0182] AM-EWOD devices can be used in many digital microfluidics applications such as point-of-care (POC) diagnostics, disease detection, RNA testing, and biosample synthesis (e.g., DNA amplification). Sample and reagent loading mechanisms are an important part of an integrated, self-contained, disposable system that can be easily used by operators to perform such tests. Ease of fluid loading is critical for a reliable device.

Claims

1. A method for filling an electrowetting on dielectric (EWOD) device using a measuring fluid, the EWOD device comprising: a) a first substrate and a second substrate, the first substrate and the second substrate being spaced apart from each other to define a chamber therebetween, wherein the chamber has an active area and a drain area integral with each other, and a patterned hydrophobic coating serving as a flow restriction element is coated on the interior of the chamber; b) at least one inlet port configured to input an assay fluid and / or a filler fluid into the chamber; c) at least one discharge port configured to discharge from the chamber; as well as d) electrodes configured to control the assay fluid within the chamber by stimulating the electrodes, The method comprises: introducing a metered volume of filler fluid into the chamber of the EWOD device via at least one first of the at least one inlet port, the EWOD device being configured to retain the metered volume of filler fluid in a region surrounded by the patterned hydrophobic coating using the flow restriction element; as well as A certain volume of the measurement fluid is introduced into the area of ​​the chamber surrounded by the patterned hydrophobic coating via at least one second inlet port among the at least one inlet port, whereby due to the electrowetting force provided by the actuating electrode, the measurement fluid enters the active area of ​​the chamber by causing some of the filler fluid to shift from the area surrounded by the patterned hydrophobic coating to the discharge area of ​​the chamber, thereby causing a certain volume of discharge fluid to be discharged from the chamber.

2. The method of claim 1, further comprising introducing the filler fluid and the assay fluid into the chamber simultaneously with each other.

3. The method according to claim 2, comprising: During the introduction of the filler fluid and the assay fluid into the chamber, the assay fluid within the chamber is controlled by energizing the electrodes.

4. The method according to claim 1, 2 or 3, wherein: The EWOD device is configured such that the at least one inlet port is disposed in an upper surface of the chamber or in one or more side surfaces of the chamber.

5. The method according to claim 1, 2 or 3, wherein: The discharge region is in fluid communication with at least one discharge port.

6. The method according to claim 1, 2 or 3, wherein: The EWOD device comprises at least one exhaust port having the same size as the at least one inlet port, and / or wherein the EWOD device comprises an exhaust port disposed at a corner of a portion of the chamber.

7. The method of claim 1, 2 or 3, further comprising metering the metered volume of filler fluid by one of volume measurement, optical sensing and electrical sensing.

8. The method of claim 1 or 2 or 3, further comprising introducing a second assay fluid via at least one third inlet port.

9. The method according to claim 1, 2 or 3, wherein: The filler fluid includes a non-polar fluid, oil or a surfactant.

10. The method according to claim 1, 2 or 3, wherein: The measurement fluid includes a polar fluid, an aqueous material or a surfactant.

11. The method according to claim 1, 2 or 3, wherein: The exhaust fluid includes a gas.

12. The method according to claim 1, wherein: The exhaust fluid includes air or an inert gas.

13. A microfluidic device comprising: a chamber having one or more inlet ports, wherein the chamber has an active area and an exhaust area integral with each other, and a patterned hydrophobic coating serving as a flow restriction element is coated on the interior of the chamber; electrodes configured to control the assay fluid within the chamber by stimulating the electrodes, The microfluidic device is configured to: when the chamber contains a metered volume of filler fluid that partially fills the chamber, retain the metered volume of filler fluid in an area surrounded by the patterned hydrophobic coating using the flow restriction element, and The microfluidic device is configured to allow some of the filler fluid to shift from the area surrounded by the patterned hydrophobic coating to the discharge area of ​​the chamber when a certain volume of assay fluid introduced into one of the one or more inlet ports enters the active area of ​​the chamber due to the electrowetting force provided by the actuating electrode, thereby causing a certain volume of discharge fluid to be discharged from the chamber.

14. A microfluidic system comprising: The microfluidic device according to claim 13, wherein the microfluidic device is contained in a disposable box; and a reader device configured to control the microfluidic device.

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