TECHNICAL FIELD

The present invention relates to droplet microfluidic devices, and more specifically to Active Matrix Electrowetting-On-Dielectric (AM-EWOD) devices, and to methods of operating such devices for manipulating multiple filler fluids having different properties to achieve a desired fluid interaction.

BACKGROUND ART

Electrowetting on dielectric (EWOD) is a well-known technique for manipulating droplets of fluid by application of an electric field. 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). It is thus a candidate technology for digital 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).

FIG. 1is a drawing depicting an exemplary EWOD based microfluidic system. In the example ofFIG. 1, the microfluidic system includes a reader32and a cartridge34. The cartridge34may contain a microfluidic device, such as an AM-EWOD device36, as well as (not shown) fluid input ports into the device and an electrical connection as are conventional. The fluid input ports may perform the function of inputting fluid into the AM-EWOD device36and generating droplets within the device, for example by dispensing from input reservoirs as controlled by electrowetting. As further detailed below, the microfluidic device includes an electrode array configured to receive the inputted fluid droplets.

The microfluidic system further may include a control system configured to control actuation voltages applied to the electrode array of the microfluidic device to perform manipulation operations to the fluid droplets. For example, the reader32may contain such a control system configured as control electronics38and a storage device40that may store any application software and any data associated with the system. The control electronics38may include suitable circuitry and/or processing devices that are configured to carry out various control operations relating to control of the AM-EWOD device36, such as a CPU, microcontroller or microprocessor.

In the example ofFIG. 1, an external sensor module35is provided for sensor droplet properties. For example, optical sensors as are known in the art may be employed as external sensors for sensing droplet properties, which may be incorporated into a probe that can be located in proximity to the EWOD device. Suitable optical sensors include camera devices, light sensors, charged coupled devices (CCD) and similar image sensors, and the like. A sensor additionally or alternatively may be configured as internal sensor circuitry incorporated as part of the drive circuitry in each array element. Such sensor circuitry may sense droplet properties by the detection of an electrical property at the array element, such as impedance or capacitance.

FIG. 2is a drawing depicting additional details of the exemplary AM-EWOD device36in a perspective view. The AM-EWOD device36has a lower substrate assembly44with thin film electronics46disposed upon the lower substrate assembly44. The thin film electronics46are arranged to drive array element electrodes48. A plurality of array element electrodes48are arranged in an electrode or element two-dimensional array50, having N rows by M columns of array elements where N and M may be any integer. A liquid droplet52which may include any polar liquid and which typically may be aqueous, is enclosed between the lower substrate44and a top substrate54separated by a spacer56, although it will be appreciated that multiple liquid droplets52can be present.

FIG. 3is a drawing depicting a cross section through some of the array elements of the exemplary AM-EWOD36device ofFIG. 2. In the portion of the AM-EWOD device depicted inFIG. 3, the device includes a pair of the array element electrodes48A and48B that are shown in cross section that may be utilized in the electrode or element array50of the AM-EWOD device36ofFIG. 3. The AM-EWOD device36further incorporates the thin-film electronics46disposed on the lower substrate44, which is separated from the upper substrate54by the spacer56. The uppermost layer of the lower substrate44(which may be considered a part of the thin film electronics layer46) is patterned so that a plurality of the array element electrodes48(e.g. specific examples of array element electrodes are48A and48B inFIG. 3) are realized. The term element electrode48may be taken in what follows to refer both to the physical electrode structure48associated with a particular array element, and also to the node of an electrical circuit directly connected to this physical structure. A reference electrode58is shown inFIG. 3disposed upon the top substrate54, but the reference electrode alternatively may be disposed upon the lower substrate44to realize an in-plane reference electrode geometry. The term reference electrode58may also be taken in what follows to refer to both or either of the physical electrode structure and also to the node of an electrical circuit directly connected to this physical structure.

In the AM-EWOD device36, a non-polar fluid60(e.g. oil) may be used to occupy the volume not occupied by the liquid droplet52. An insulator layer62may be disposed upon the lower substrate44that separates the conductive element electrodes48A and48B from a first hydrophobic coating64upon which the liquid droplet52sits with a contact angle66represented by θ. The hydrophobic coating is formed from a hydrophobic material (commonly, but not necessarily, a fluoropolymer). On the top substrate54is a second hydrophobic coating68with which the liquid droplet52may come into contact. The reference electrode58is interposed between the top substrate54and the second hydrophobic coating68.

The contact angle θ for the liquid droplet is defined as shown inFIG. 3, and is determined by the balancing of the surface tension components between the solid-liquid (γSL), liquid-gas (γLG) and non-ionic fluid (γSG) interfaces, and in the case where no voltages are applied satisfies Young's law, the equation being given by:

cos⁢⁢θ=γSG-γSLγLG(equation⁢⁢1)

In operation, voltages termed the EW drive voltages, (e.g. VT, V0and V00inFIG. 3) may be externally applied to different electrodes (e.g. reference electrode58, element electrodes48A and48B, respectively). The resulting electrical forces that are set up effectively control the hydrophobicity of the hydrophobic coating64. By arranging for different EW drive voltages (e.g. V0and V00) to be applied to different element electrodes (e.g.48A and48B), the liquid droplet52may be moved in the lateral plane between the two substrates, for example from being positioned over48A to being positioned over48B.

FIG. 4Ashows a circuit representation of the electrical load70A between the element electrode48and the reference electrode58in the case when a liquid droplet52is present. The liquid droplet52can usually be modeled as a resistor and capacitor in parallel. Typically, the resistance of the droplet will be relatively low (e.g. if the droplet contains ions) and the capacitance of the droplet will be relatively high (e.g. because the relative permittivity of polar liquids is relatively high, e.g. ˜80 if the liquid droplet is aqueous). In many situations the droplet resistance is relatively small, such that at the frequencies of interest for electrowetting, the liquid droplet52may function effectively as an electrical short circuit. The hydrophobic coatings64and68have electrical characteristics that may be modelled as capacitors, and the insulator62may also be modelled as a capacitor. The overall impedance between the element electrode48and the reference electrode58may be approximated by a capacitor whose value is typically dominated by the contribution of the insulator62and hydrophobic coatings64and68contributions, and which for typical layer thicknesses and materials may be on the order of a pico-Farad in value.

FIG. 4Bshows a circuit representation of the electrical load70B between the element electrode48and the reference electrode58in the case when no liquid droplet is present. In this case the liquid droplet components are replaced by a capacitor representing the capacitance of the non-polar fluid60which occupies the space between the top and lower substrates. In this case the overall impedance between the element electrode48and the reference electrode58may be approximated by a capacitor whose value is dominated by the capacitance of the non-polar fluid and which is typically small, on the order of femto-Farads.

For the purposes of driving and sensing the array elements, the electrical load70A/70B overall functions in effect as a capacitor, whose value depends on whether a liquid droplet52is present or not at a given element electrode48. In the case where a droplet is present, the capacitance is relatively high (typically of order pico-Farads), whereas if there is no liquid droplet present the capacitance is low (typically of order femto-Farads). If a droplet partially covers a given electrode48then the capacitance may approximately represent the extent of coverage of the element electrode48by the liquid droplet52.

U.S. Pat. No. 7,163,612 (Sterling et al., issued Jan. 16, 2007) describes how TFT based thin film electronics may be used to control the addressing of voltage pulses to an EWOD array by using circuit arrangements very similar to those employed in active matrix display technologies. The approach of U.S. Pat. No. 7,163,612 may be termed “Active Matrix Electrowetting on Dielectric” (AM-EWOD). There are several advantages in using TFT based thin film electronics to control an EWOD array, namely:Electronic driver circuits can be integrated onto the lower substrate.TFT-based thin film electronics are well suited to the AM-EWOD application. They are cheap to produce so that relatively large substrate areas can be produced at relatively low cost.TFTs fabricated in standard processes can be designed to operate at much higher voltages than transistors fabricated in standard CMOS processes. This is significant since many EWOD technologies require electrowetting voltages in excess of 20V to be applied.

FIG. 5is a drawing depicting an exemplary arrangement of thin film electronics46in the exemplary AM-EWOD device36ofFIG. 2. The thin film electronics46is located upon the lower substrate44. Each array element51of the array of elements50contains an array element circuit72for controlling the electrode potential of a corresponding element electrode48. Integrated row driver74and column driver76circuits are also implemented in thin film electronics46to supply control signals to the array element circuit72. The array element circuit72may also contain a sensor capability for detecting the presence or absence of a liquid droplet in the location of the array element. Integrated sensor row addressing78and column detection circuits80may further be implemented in thin film electronics for the addressing and readout of the sensor circuitry in each array element.

A serial interface82may also be provided to process a serial input data stream and facilitate the programming of the required voltages to the element electrodes48in the array50. A voltage supply interface84provides the corresponding supply voltages, top substrate drive voltages, and other requisite voltage inputs as further described herein. A number of connecting wires86between the lower substrate44and external control electronics, power supplies and any other components can be made relatively few, even for large array sizes. Optionally, the serial data input may be partially parallelized. For example, if two data input lines are used the first may supply data for columns 1 to X/2, and the second for columns (1+X/2) to M with minor modifications to the column driver circuits76. In this way the rate at which data can be programmed to the array is increased, which is a standard technique used in liquid crystal display driving circuitry.

FIG. 6is a drawing depicting an exemplary arrangement of the array element circuit72present in each array element51, which may be used as part of the thin film electronics ofFIG. 5. The array element circuit72may contain an actuation circuit88, having inputs ENABLE, DATA and ACTUATE, and an output which is connected to an element electrode48. The array element circuit72also may contain a droplet sensing circuit90, which may be in electrical communication with the element electrode48. Typically, the read-out of the droplet sensing circuit90may be controlled by one or more addressing lines (e.g. RW) that may be common to elements in the same row of the array, and may also have one or more outputs, e.g. OUT, which may be common to all elements in the same column of the array.

The array element circuit72may typically perform the functions of:(i) Selectively actuating the element electrode48by supplying a voltage to the array element electrode. Accordingly, any liquid droplet present at the array element51may be actuated or de-actuated by the electro-wetting effect.(ii) Sensing the presence or absence of a liquid droplet at the location of the array element51. The means of sensing may be capacitive or impedance, optical, thermal or some other means. Capacitive or impedance sensing may be employed conveniently and effectively using an integrated impedance sensor circuit as part of the array element circuitry.

Various methods of controlling an AM-EWOD device to sense droplets and perform desired droplet manipulations have been described. For example, US 2017/0056887 (Hadwen et al., published Mar. 2, 2017) describes the use of capacitance detection to sense dynamic properties of reagents as a way for determining the output of an assay. Such disclosure incorporates an integrated impedance sensor circuit that is incorporated specifically into the array element circuitry of each array element. Accordingly, attempts have been made to optimize integrated impedance sensing circuitry into the array element structure, and in particular as part of the array element circuitry. Examples of AM-EWOD devices having integrated actuation and sensing circuits are described, for example, in Applicant's commonly assigned patent documents as follows: U.S. Pat. No. 8,653,832 (Hadwen et al., issued Feb. 18, 2014); US2018/0078934 (Hadwen et al., published Mar. 22, 2018); US 2017/0076676 (Hadwen, published Mar. 16, 2017); and U.S. Pat. No. 8,173,000 (Hadwen et al., issued May 8, 2012). The enhanced method of operation described in the current application may be employed in connection with any suitable array element circuitry.

The description above demonstrates advantages of using a TFT configuration to make the backplane of the AM-EWOD device. This permits a large area for droplet manipulations that is achieved at relatively low cost. Example materials for manufacturing TFT based AM-EWOD devices could be any suitable materials for manufacturing active matrix displays, including for example low temperature polysilicon (LTPS), amorphous-silicon (a-Si), and indium gallium zinc oxide (IGZO), and any suitable related manufacturing processes may be employed. Even with the advantages of TFT based AM-EWOD devices, analytical challenges remain. In particular, it may be desirable to control or dictate the interface between a polar, aqueous liquid droplet and the non-polar fluid to achieve a desired fluidic operation or interaction.

EP 2 616 854 (Mallard et al., published Jul. 24, 2013) describes certain “desirable” characteristics of a non-polar fluid that might be utilized within an EWOD device to achieve a desired fluidic interaction. Such patent document, however, does not teach or suggest any ways of manipulating multiple non-polar fluids to achieve a desired fluidic interaction at different stages or stages of a multi-step reaction protocol.

Tao He et al. (BIOMICROFLUIDICS 10, 011908 (2016)) describe two-phase microfluidics in electrowetting displays and relates effects on optical performance. The article discloses a display device that comprises an array of micropixels having walls that separate the pixels. The article discloses: “The pixels were 150 um×150 um with grid height and width about 6 and 15 um, respectively. Coloured oil and conductive liquid were then filled and sealed with a cover plate to form an electrowetting display device.” As a microfluidic display device, there is nothing in He et al. to suggest operations that include transferring fluid from one pixel to another, as such operation would not be useful in a display device.

U.S. Pat. No. 8,658,111 (Srinivasan et al., issued Feb. 24, 2004) describes an EWOD device divided spatially into multiple zones that are designed to separate different oils within their respective zones, and a means of moving droplets between the zones. The difference zones are generated employing different actuation voltages to different portions of the device.

Applicant has previously attempted to control fluidic interactions through the use of electrowetting forces to generate reconfigurable barrier regions formed of a polar fluid. The barrier regions, for example, may control the flow of filler fluids (oil) that are inputted into the device, or may separate regions of the device for use in different reaction steps. Examples of such operations are described in Applicant's application Ser. No. 15/759,685 filed on Mar. 13, 2018, and application Ser. No. 16/147,964 filed Oct. 1, 2018, the contents of which are incorporated here by reference.

Liquid droplets to which manipulation operations are to be performed are typically polar, aqueous fluids that are commonly surrounded by a non-polar filler fluid (typically an oil) within which the polar liquid droplets are immiscible. Examples of the non-polar filler fluid include (without limitation) silicone oil, fluorosilicone oil, pentane, hexane, octane, decane, dodecane, pentadecane, hexadecane, which generally may be referred to as oil. Although less typical, in certain applications the filler fluid may simply be air or another gas.

A non-polar oil filler fluid may perform various functions, which may include (without limitation) the following. The oil filler fluid lowers the surface tension around the boundaries of the polar liquid droplets (as compared to having the droplets in air) so that the polar fluids can be inputted into the device more readily and/or be manipulated more easily by electrowetting operations. In some applications, a surfactant may be employed to enhance the lowering of the surface tension of the polar liquid droplets. When used, the surfactant may be dissolved in the filler fluid (although in some applications the surfactant alternatively may be dissolved in the polar fluid). The filler fluid also prevents the polar liquid droplets from reducing in size due to evaporation.

Attempts have been made to perform EWOD operations using multiple and different filler fluids for different reactions or different phases of a reaction protocol on a single EWOD device. For example, U.S. Pat. No. 7,439,014 (Pamula et al., issued Oct. 21, 2008) describes the sequential use of different filler fluids. To avoid cross-mixing or contamination, the differ filler fluids are inputted into separate physical chambers that are separated by walls or other comparable structural barriers. The need for structural barriers built onto the EWOD device limits spatial flexibility for performing reaction steps.

SUMMARY OF INVENTION

There is a need in the art for improved systems and methods of operating an EWOD or AM-EWOD device that can accommodate multiple filler fluids having different characteristics that may be employed within a single EWOD device. The requirements of the filler fluid may differ depending upon the particular application for which the EWOD device is to be used. Properties or characteristics of a filler fluid also may need to be different at different stages within a specific assay, sample preparation, or reaction protocol that is to be performed within an EWOD device. The present invention provides a system and methods that accommodate the need to use filler fluids of different properties or characteristics by facilitating the use of multiple and different filler fluids within a single EWOD device.

In exemplary embodiments, a polar fluid source may be dispensed into an EWOD device array by any suitable mechanism. Electrowetting forces are employed to modify the polar fluid to form an aqueous barrier across the EWOD device array that separates the EWOD device array into fluidly separated regions or zones. First and second non-polar filler fluids are then dispensed respectively into the EWOD device on opposites sides of the aqueous barrier, such that the aqueous barrier prevents intermixing between the filler fluids. Additional polar fluid constituting one or more sample and/or reagent polar liquid droplets are dispensed onto the EWOD device. The liquid droplets may be transferred between the different device regions having the different polar fluids by employing electrowetting operations to: reconfigure the aqueous barrier, such as by opening a passage in the aqueous barrier, transfer one or more liquid droplets through the reconfigured aqueous barrier from a first region to a second region of the EWOD device, and reconstituting the aqueous barrier to re-separate the first and second regions. By employing such an aqueous barrier, intermixing of the different filler fluids and any constituents thereof is minimized.

In another embodiment, different polar fluids may be employed sequentially in time. In such device operation, a first non-polar filler fluid is dispensed into an EWOD device, and a polar fluid constituting one or more sample and/or reagent polar liquid droplets are dispensed onto the EWOD device array. Following the performance of any desired droplet manipulation operations, the first filler fluid is extracted while electrowetting forces are applied to the polar liquid droplet(s) to maintain the droplet positioning on the EWOD device array. A second non-polar filler fluid is then dispensed into the EWOD device, again while electrowetting forces are applied to the polar liquid droplet(s) to maintain the droplet positioning on the EWOD device array during the filler fluid exchange.

An aspect of the invention, therefore, is a method of operating an electrowetting on dielectric (EWOD) device that performs electrowetting operations on fluids dispensed into the EWOD device, which provides enhanced operation for using multiple non-polar filler fluids. In exemplary embodiments, the method of operating includes the steps of: dispensing a polar fluid source into the EWOD device; performing an electrowetting operation to generate an aqueous barrier from the polar fluid source, wherein the aqueous barrier separates the EWOD device into a first region and a second region that are fluidly separated from each other by the aqueous barrier; inputting a non-polar first filler fluid into the first region; inputting a non-polar second filler fluid into the second region; dispensing a polar liquid droplet into the first region; transferring the polar liquid droplet from the first region to the second region by performing an electrowetting operation to reconfigure the aqueous barrier, and performing an electrowetting operation to move the polar liquid droplet from the first region to the second region through the reconfigured aqueous barrier; and performing an electrowetting operation to reconstitute the aqueous barrier to fluidly separate the first region from the second region. The methods of the present invention may be performed by an EWOD control system executing program code stored on a non-transitory computer readable medium.

Reconfiguring the aqueous barrier may include performing an electrowetting operation to open a passage through the aqueous barrier, and reconstituting the aqueous barrier may include performing an electrowetting operation to close the passage. Reconfiguring the aqueous barrier may include forming a double walled section of the aqueous barrier enclosing a third region of the EWOD device that is fluidly separated from the first region and the second region by said double walled section. The polar liquid droplet is then transferred from the first region to the second region through the third region using a double gated transference operation by which passages are formed sequentially through different limbs of the double walled section.

Another method of operating an EWOD device may include the steps of: inputting a non-polar first filler fluid into the EWOD device; dispensing a polar liquid droplet into the EWOD device, wherein the polar liquid droplet is surrounded by the first filler fluid; performing an electrowetting operation to perform a droplet manipulation operation on the polar liquid droplet; extracting the first filler fluid from the EWOD device while actuating a portion of array elements of the EWOD device to maintain a position of the polar liquid droplet within the EWOD device; and inputting a non-polar second filler fluid into the EWOD device while actuating a portion of array elements of the EWOD device to maintain a position of the polar liquid droplet within the EWOD device.

These and further features of the present invention will be apparent with reference to the following description and attached drawings. In the description and drawings, particular embodiments of the invention have been disclosed in detail as being indicative of some of the ways in which the principles of the invention may be employed, but it is understood that the invention is not limited correspondingly in scope. Rather, the invention includes all changes, modifications and equivalents coming within the spirit and terms of the claims appended hereto. Features that are described and/or illustrated with respect to one embodiment may be used in the same way or in a similar way in one or more other embodiments and/or in combination with or instead of the features of the other embodiments.

