A method of manipulating droplets

Abstract

A method and device for manipulating droplets using adjacent pixel actuation for differential electrowetting and particularly for forming an array of droplets via splitting of an aqueous liquid volume using differential electrowetting within the aqueous liquid volume to enhance the accuracy of droplet operations.

Description

A METHOD OF MANIPULATING DROPLETSFIELD OF THE INVENTIONThis invention is in the field of fluid electrokinetics: Electrowetting-on-dielectric (EWoD) and Dielectrophoresis (DEP); and the methods and devices using these phenomena. The invention relates to methods for moving droplets and efficiently forming arrays of evenly sized droplets via splitting of an aqueous liquid volume using differential electrowetting within the aqueous liquid volume to enhance the accuracy of droplet operations.BACKGROUNDThe manipulation of droplets by the application of electrical potential can be achieved on electrodes covered with an insulator or a dielectric or a series of insulators or dielectrics. Droplet manipulation as a result of an applied electrical potential is known as electrowetting. Electrokinesis occurs as result of a non-uniform electric field that influences the hydrostatic equilibrium of a dielectric liquid (dielectrophoresis or DEP) or a change in the contact angle of the liquid on solid surface (electrowetting-on-dielectric or EWoD). DEP can also be used to create forces on polarizable particles to induce their movement. The electrical signal can be transmitted to a discrete electrode, a transistor, an array of transistors, or a sheet of semiconductor film whose electrical properties can be modulated by an optical signal.EWoD phenomena occur when droplets are actuated between two electrodes covered with a hydrophobic insulator or dielectric. The electric field at the electrode-electrolyte interface induces a change in the surface tension, which results in droplet motion as a result of a change in droplet contact angle. The electrowetting effect can be quantitatively treated using Young-Lippmann equation:cosQ - cos0o= (1 / 2yLG) c.V2where 0o is the contact angle when the electric field across the interfacial layer is zero, yLG is the liquid-gas tension, c is the specific capacitance (given as £r. Eo / t, where £ris dielectric constant of the insulator / dielectric, £o is permittivity of vacuum, t is thickness) and V is the applied voltage or electrical potential. The change in contact angle (inducing droplet movement) is thus a function of surface tension, electrical potential, dielectric thickness, and dielectric constant.When a droplet is actuated by EWoD, there are two opposing sets of forces that act upon it: an electrowetting force induced by electric field and resistant forces that include the dragforces resulting from the interaction of the droplet with filler medium and the contact line friction. The minimum voltage applied to balance the electrowetting force with the sum of all drag forces (threshold voltage) is variably determined by the thickness-to-dielectric contact ratio of the insulator / dielectric, (t / er)1 / 2. Thus, to reduce actuation voltage, it is required to reduce (t / er)1 / 2(i.e., increase dielectric constant or decrease insulator / dielectric thickness). To achieve low voltage actuation, thin insulator / dielectric layers must be used. However, the deposition of high quality thin insulator / dielectric layers is a technical challenge, and these thin layers are easily damaged before the desired electrowetting contact angle is large enough to drive the droplet is achieved. Most academic studies thus report the use of much higher voltages >100 V on easily fabricated, thick dielectric films (>3 pm) to effect electrowetting.High voltage EWoD-based devices with thick dielectric films, however, have limited industrial applicability largely due to their limited droplet multiplexing capability. The use of low voltage devices including thin-film transistors (TFT) and optically-activated amorphous silicon layers (a-Si) have paved the way for the industrial adoption of EWoD-based devices due to their greater flexibility in addressing electrical signals in a highly multiplex fashion. The driving voltage for TFTs or optically-activated a-Si are low (typically <15 V). The bottleneck for fabrication and thus adoption of low voltage devices has been the technical challenge of depositing high quality, thin film insulators / dielectrics. Hence there has been a particular need for improving the fabrication and composition of thin film insulator / dielectric devices and methods for driving droplets thereon.The inventors wished to implement a method for producing an ordered array having a large number of evenly sized droplets. Previous approaches involve using TFT high-density arrays and individually placing each desired droplet at the desired locations, thereby requiring dispensing of multiple droplets to exact locations. Previously reported methods of splitting droplets involved splitting droplets individually and then moving them to exact location. Alternative methods involve splitting droplets in sequence rather than simultaneously, or repeatedly dispensing multiple droplets from a larger reservoir. Such methods are inefficient at producing large numbers of droplets and the droplets vary in size.W02007 / 147568 / US8,409,417B2 discloses conventional methods of splitting droplets by pulling the ends apart to form an increasingly narrow neck, for example Fig 8A-8C. Such methods split droplets along the longest axis and does not give an evenly distributed volume of material between the two droplets. Such methods can only split droplets into two volumes.EP1643231 discloses methods for manipulating liquids on devices. Certain embodiments disclosed involve the stretching of droplets in order to promote mixing, for example as shown on

[0065] Fig 27, which shows a divided electrode pattern having a stirring region. The application does not describe the preparation or handling of large numbers of droplets.W02021041709 describes further systems for droplet manipulation. The application describes many potential applications that could be performed on droplets, but does not describe specific details of how droplets are moved and handled.EP2884272 describes the known method of producing a train of droplets from a larger reservoir. Such a method of forming an array of droplets by removal of single droplets is inefficient in both time, space and reagent use.CN111545259 describes using electrodes in rows and columns for driving droplets on an electrowetting device. The droplets are smaller than the size of the electrodes such that a droplet does not cover multiple electrodes.EP2931425 recites in

[0057] -

[0065] as below

[0057] The control unit 23 is capable to define a path for a guided movement of a liquid droplet 19 by the selection of a series of subsequent drive electrodes 35’. Thereby at least one of these selected drive electrodes 35’ is subsequently provided with a drive voltage pulse along said path, under the control of the control unit 23. Furthermore the control unit 23 is accomplished to essentially simultaneously provide at least one electrode 35", which is adjacent to the pulsed drive electrode 35’ and different to the selected drive electrode 35’ of the path, with a ground voltage pulse.

[0058] Such a path for a guided movement of the liquid droplet is each shown in the Figures 3A to 3D. The subsequent selected drive electrodes 35’ are indicated. The actual drive electrode 35’ is shown to be that electrode 35, upon which the liquid droplet 19 is positioned. The direction of the planned guided movement of the liquid droplet 19 is indicated with an arrow. In that direction, subsequent electrodes 35’ along the path will be provided with a drive voltage pulse.

[0059] Preferably, the size, respectively the diameter of the liquid droplet 19 slightly exceeds the diameter of an electrode 35. Most preferably, for the guided movement by electrowetting, the liquid droplet touches not only the actual drive electrode 35’ but slightly touches simultaneously the subsequent electrode 35’ which will become the next actual drive electrode 35’. However, the adjustment of electrode size in relationto liquid droplet sizes is within the knowledge of the person skilled in the art and should not be repeated here. However, the actual size and design of the electrodes and the desired size of the liquid droplets 19 must be in accordance with each other and with the praxis of electrowetting.

[0060] According the second inventive aspect, the presence of at least one ground electrode 35" adjacent to the liquid droplet 19 to be moved provides a stabilizing effect to its movement. The Figures 3A-3D indicate those electrodes 35" that might be provided with a ground voltage pulse. Those ground electrodes 35" are preferably adjacent to the pulsed drive electrodes 35’ and different or identical to the selected drive electrodes 35’ of the path. The provision with the ground voltage pulse is preferably carried out essentially simultaneously to the provision with the drive voltage pulse. Alternatively, the provision with the ground voltage pulse is to be carried out simultaneously to the provision with the drive voltage pulse.

[0061] According to a preferred variant of the liquid droplet manipulation instrument 20, the control unit 20 is accomplished to provide at least two electrodes 35" which are adjacent to the pulsed drive electrode 35’ and different to the selected drive electrode 35’ of the path with a ground voltage pulse. Preferably, these at least two selected ground electrodes 35" are subsequent electrodes 35 on the same side of the path.

[0062] As shown in the Figures 3A-3B, ground electrodes 35" may be selected from electrodes 35 along the path, adjacent to the path and adjacent to the liquid droplet 19. Preferably, the selected ground electrodes 35" are on the same side of the path. When a group of three or more electrodes 35" is to be provided with a ground voltage potential essentially simultaneously, at least two first electrodes 35" are preferably selected from one side of the path. The remaining electrodes 35" of that group may however be selected from that side of the path being opposite to the first two ground electrodes 35" of that group. However, even when a group of ground electrodes 35" are selected from two sides of the path, they are provided with the ground voltage pulse essentially simultaneously or simultaneously to the pulsed drive electrode 35’. If a group of electrodes 35" is simultaneously provided with a ground voltage pulse, the other electrodes 35" may be adjacent to the path and ahead of the liquid droplet 19, adjacent to the path and behind the liquid droplet 19 or both.

[0063] In one variant of the liquid droplet manipulation instrument 20, a group of 2 or more electrodes 35 may be provided with a drive voltage pulse essentially simultaneously. In this case, a liquid droplet 19’ of a larger volume may be moved. However in this variant it is preferred that essentially simultaneously or simultaneously a group of 2 or more electrodes 35 are provided with a ground voltage pulse to sufficiently support the liquid droplet 19’ with the larger volume.

[0064] In a preferred variant of the liquid droplet manipulation instrument 20, the control unit is accomplished to provide at least one selected electrode with a stop voltage pulse for generating a stop electrode 35"’. Preferably, the provided stop voltage pulse is different to the drive voltage pulse and the ground voltage pulse.

[0065] The voltage pulses for defining a selected electrode 35 as a drive electrode preferably are between 20 and 100 V. The voltage pulses for defining a selected electrode 35 as a stop electrode preferably are between -50 V and +50 V. As shown in the Figures 3A-3D, selected stop electrodes 35’" are adjacent to the path, different to the selected drive electrode 35’ of the path and different to the at least one selected ground electrode 35" adjacent to the path.Furthermore, stop electrodes 35’" are selected from such electrodes 35 adjacent to the path, where the path provides a change of direction for the liquid droplet 19 movement. A stop electrode 35’” supports the direction change of the liquid droplet movement along the path.The disclosure in EP2931425 therefore uses droplets that are similar in size to the electrodes (the diameter of the liquid droplet 19 slightly exceeds the diameter of an electrode 35.). The electrodes outside the droplets are the ‘path’ electrodes towards which the droplets move. The path electrodes are not underneath the droplets.W02024028590 discloses methods for forming arrays of droplets on electrowetting devices. The disclosure does not use variable actuation underneath the droplets, the actuation electrodes are either on or off.Wang et al 2022 Flex. Print. Electron. 7023004 describes thin-film arrays transistor arrays for biological sensing systems and provides a review of devices used for biosensing applications.WO2014036915 describes dielectrophoresis based apparatuses and methods for the manipulation of particles in liquids. The device uses electrodes in rows to move droplets in channels. Figure 5A-E shows the separation of a droplets by switching on the electrodes outside the ends of the droplet, which splits in two as the ends are pulled apart. Whilst different voltages are used under each droplet, this is not significantly different to the on / off routine typically used to split droplets. In many instances of prior art microfluidics, droplets are pulled apart by actuating beyond opposing droplet edges and switching off the central electrodes.Alternatively, in traditional microfluidics literature, physical structures such as wells or beads are usually needed to immobilize / anchor the droplets. Other non-flow applications involve various microstructures or meshes, which would greatly increase the cost of device manufacturing.Conventional closed-configuration devices require an array of electrodes on one face of the device and a common electrode on an opposing face in order to form a circuit for droplet propulsion. Described herein are simplified devices that can be used for droplet movement without requiring the opposing common electrode.SUMMARY OF THE INVENTIONThe invention is defined in the independent claims below to which reference should now be made. Optional features are set forth in the dependent claims.Described herein are improved methods for performing droplets operations using two-dimensional arrays of electrodes. Droplet operations may be optimized using actuation patterns for all operations such as reagent loading, droplet movement, dispense and splitting. However the optimisation based on actuation patterns is limited to spatial constraints and cannot be used to address the failure modes seen in splitting / dispense or reagent loading such as uneven droplet sizes. Driving different droplet compositions using different driving pulse sequences (reagent specific driving) gives another dimension of control to vary the actuation forces / change in contact angle by varying the number of, length of and strength of bias pulses and optionally interphased zero pulses. The inventors herein have appreciated that this control in the effective voltage applied to pixels in the array of electrodes allows a gradual change to the contact angle from one pixel to the neighbouring pixel and thus provides a tailored flow of aqueous phases. This enhanced means for fluidic control can be exploited for droplet movement, reagent loading, dispense and split operations on aqueous liquid volumes.For traditional splitting, droplets are split by pulling the aqueous phases in opposite directions. When the voltage at the electrodes in the middle is turned off abruptly, the fluid volume occupying this region becomes hydrodynamically unstable, without any confining forces to hold it in place. This unconfined liquid is able to migrate to any of the adjacent electrodes and is thus unpredictable, giving rise to higher variation in split volumes and for undersized aqueous droplets, a binary operation in the split success, leading to missing droplets in an array. Using a gradient of electrowetting forces in the middle of droplets as they are being separated (rather than abruptly turning off the pixel electrodes) helps migration of thisaqueous phase; resulting in lower CV of split droplets and increases tolerance against droplet volumes that do not fully cover the actuated pixel area. For example the droplet volume before the split may cover between 60% and 90% of the actuated area.For device loading operations reagents are pulled into the cell gap using a given waveform (pulse sequence) and the excess is scooped back from the defined aqueous volume (aka reservoir) using the same actuation sequence in order to control the size of the aqueous volume loaded. The propensity of the liquid to go into the more hydrophilic plastic ports can sometimes lead to “back flow” which results in underloaded reservoirs. The use of electrowetting gradients as the reagents are loaded helps in the accurate loading of reagents.A single pixel dispense (i.e. droplet matching the size of a pixel electrode) can be achieved by extending a long yet thin (single pixel wide) aqueous phase; which is then chopped into single pixel sized droplets using gradient of ferees between the resulting droplets rather than abruptly turning the electrodes off. Thus a large number of evenly sized single pixel sized droplets can be produced. The method can also be used for droplet movement. The use of edge actuation can help reduce fouling.Described herein are methods for generating small droplets, for example single pixel droplets. Described is a method comprising using a droplet with an elongated neck and controlling the center of the elongated neck to create 2 necks and a central bulge, wherein the central bulge forms a droplet which is smaller than could be generated with elongation of a single neck. The starting droplet pixel width and length may be for example at least 3x the pixel width and length of the final droplet.Described herein are methods for generating volumes of evenly sized reagents. Disclosed is a method for manipulating an aqueous liquid volume on a digital microfluidic device having an array of electrodes, the method comprising using different electronic pulse sequences in different electrodes underneath the area covered by the aqueous liquid.Disclosed is a method for manipulating an aqueous liquid volume on a digital microfluidic device having an array of electrodes, the method comprising simultaneously supplying different electronic pulse sequences to different electrodes underneath the area covered by a single aqueous liquid volume, wherein the different electronic pulse sequences comprise either supplying stronger and weaker voltage pulses or sequences to adjacent electrodes or supplying positive and negative sequences of pulses to adjacent electrodes.Disclosed is a method for manipulating an aqueous liquid volume on a digital microfluidic device having an array of electrodes, the method comprising simultaneously supplying different electronic pulse sequences to different electrodes underneath the area covered by a single aqueous liquid volume, wherein the different electronic pulse sequences comprise either supplying stronger and weaker voltage pulses or sequences to adjacent electrodes or supplying positive and negative sequences of pulses to adjacent electrodes.The term different electronic pulse sequence applies to voltage driving patterns supplied to adjacent electrodes. For example the pulse sequences may be stronger and weaker, either due to having varying voltages or by the use of rest pulses to make a weaker sequence. Alternatively the pulse sequences may be of opposite polarity in adjacent pixel electrodes.The method can be used for moving droplets on the device. The method can be used for loading a reservoir from an external port or for splitting a loaded reservoir into smaller volumes. The method can be used to split an aqueous liquid volume into smaller droplets. The splitting can be performed when the aqueous liquid volume is split into two or more smaller droplets by creating a neck of fluid between two parts of the aqueous liquid volume, wherein the electrodes under the neck region are actuated with a weaker pulse sequence than the electrodes under the two resulting droplets.Optionally splitting the aqueous liquid volume into multiple evenly sized smaller droplets can be performed by moving the aqueous liquid volume in opposing lateral directions, wherein the lateral movement is at 90 degrees to the longer axis such that the ends of the aqueous liquid volume move perpendicular to the longer axis. When splitting across the longest axis, the aqueous liquid volume is typically rectangular. Once rectangular volumes are split, the droplets may be held as squares. In some embodiments, the electrodes under the trailing edges of the laterally moved volumes may be actuated with a weaker pulse sequence than the electrodes under the rest of the volumes. In some embodiments, the electrodes under the outer edges of the laterally moved volumes may be actuated with a weaker pulse sequence than the electrodes under the rest of the volumes. In some embodiments, the electrodes under the inner edges of the laterally moved volumes where the neck forms may be actuated with a weaker pulse sequence than the electrodes under the rest of the volumes.Alternatively splitting may be performed by moving opposing ends of the droplets further apart (i.e. by further extending the longest axis). Again typically rectangular volumes are split into squares. A rectangular shape can be split into multiple squares, for example 2, 3 or 4 smaller droplets or squares may be formed from a first reagent volume. The droplets may be splitsimultaneously or serially to produce multiple droplets from a starting reservoir. An array of multiple droplets may be formed. The splitting may generate an array having equal spacing Sv along one axis and equal spacing S2 along a second axis. The splitting process may be repeated. The aqueous liquid volume may be elongated and split into evenly sized smaller droplets, and the split droplets then elongated and further split into smaller droplets. The splitting may occur in multiple directions. For example each first droplet may be split at least twice in order to make at least 4 smaller evenly sized droplets from each of the first droplets.Any suitably sized droplets or reservoirs may be used. For example the final droplets may be less than 250 nL in volume. The volume of the reservoirs and droplets may be defined by the size of the area covered by liquid, for example in terms of the number of pixels. For example a reservoir may cover 100 pixels in a 10 x 10 pattern. The actual liquid volume varies with the height of the microfluidic gap. Such 10 x 10 reservoir may be split for example into two 7 x 7 reservoirs (i.e. 49 pixels per droplet). Each 7 x 7 reservoir may be split into three 4 x 4 droplets (16 pixels each).The dispensed droplets may be used for biochemical manipulations and assays, for example for performing droplet based cell-free expression of peptides or proteins, wherein the droplets contain a cell-free system having components for protein expression and / or a nucleic acid template. The droplets may be mixed with further droplets such that the cell-free system having components for protein expression and the nucleic acid template can be brought together by droplet merging.The device may have electrodes of any particular shape, for example square, hexagonal or triangular.Described herein is a method for the rapid generation of ordered droplet arrays using repeated droplet division to ensure evenly sized droplets. The droplets are split using a differential electrowetting pattern where the neck of the droplets has a weaker actuation than the resultant droplets. The neck meniscus thereby breaks in a controlled fashion ensuring an even distribution of liquid between the two droplets. The controlled actuation in the neck particularly prevents underfilled or missing droplets.The method can rapidly generate arrays of large numbers of droplets with a minimal level of variation in size. One benefit of the present invention is that it greatly reduces the time to create a large ordered array of uniform droplets by using an initial large droplet dispense followed by several steps of splitting. Such techniques reduce the physical area needed tosplit a large number of droplets. By carefully spacing the droplets initially it is possible to reduce movement beyond the split operation, allowing for the array to be generated in the shortest time possible and within the original space occupied by the larger droplets. Multiple starting volumes may be split using different numbers of steps and sizes in order to create a large number of discrete reagent volumes of even size rapidly on the device.An important criterion for splitting a droplet requires a large contact angle change (function of dielectric thickness, voltage applied, and surface tension), a small channel gap, or a large radius at the end regions of the droplet (dependent on the size of the electrode). Previous droplet splitting methods involve pulling on the droplet such that a neck is formed as the two ends are pulled apart. The splitting occurs via a mechanism of pulling to form a ‘figure of 8’ when eventually splits into two parts. This stretching does not result in an even split as the surface tension of the droplet tries to pull the droplet back together. The uneven splitting is cured if the electrodes under the neck portion are weakly actuated rather than switched off.The splitting is particularly uneven where the liquid shapes are square or rectangular shapes caused by the electrodes on the array being square. Rather than stretching the droplet, the inventors herein have appreciated that a more even split is obtained if the droplet is moved laterally (i.e. the ends slide along an axis perpendicular to the longest axis of the droplet). The lateral movement increases the radius of curvature at the distal end of the laterally moving droplet edges and thereby promotes an evenly sized split.The present application addresses the shortcomings of the prior art by providing an alternate architecture for a digital microfluidic device. When an aqueous droplet is present on the device the droplet can be used to complete a circuit between adjacent electrodes on an array of electrodes without the need for a top plate with an opposing separate common electrode.Described herein is a digital microfluidic device, comprising a bottom plate including an electrode array comprising a plurality of digital microfluidic propulsion electrodes. A first dielectric layer covers the bottom electrode array. The bottom layer has an upper hydrophobic layer. An optional top plate comprises a hydrophobic layer to control the size of the droplet, but no electronic circuitry or dielectric layers are required. Due to the methods of driving disclosed herein no top plate is required. The top plate may be used simply as a cover, and not be in contact with the liquid layer on the bottom plate.The device also includes a controller operatively coupled to the bottom electrode array, wherein the controller is configured to provide propulsion voltages between adjacentelectrodes at the updating frame of the bottom electrode array. The droplet operations may be performed without a top plate, or with a top plate not in contact with the droplets. The optional top plate and the bottom plate may be provided in a spaced relationship to provide a gap defining a microfluidic region therebetween to permit droplet motion within the microfluidic region under application of propulsion voltages between the separately voltage addressable digital microfluidic propulsion electrodes on the bottom electrode array. The different voltages to adjacent pixels are provided in the same frame, i.e at the same time or simultaneously. Prior art devices require a connected top plate and a plurality of electrodes on the opposing plate. Whilst such electrodes are individually controllable, electrical connections are through the top plate. Inventors herein have appreciated that an electrically connected top plate is not required.Prior art devices include open systems without a top plate at all. Such devices allow substantial evaporation and are unsuitable for handling small volumes of aqueous droplets. Open systems change the surface energy and droplet contact angle using electrode actuation, thereby allowing droplets to move to areas with a higher surface energy or lower contact angle. Prior art open systems do not use simultaneous adjacent pixel actuation with different voltages in the same frame, they use consecutive actuation of different pixels.Similarly prior art devices, such as for example as described in WO2021 / 255481 can use sub-pixel actuation of the area under droplets in order to reduce the number of pixels activated. Again this does not use simultaneous adjacent pixel actuation with different voltages in the same frame, they use consecutive actuation of different pixels. The invention herein provides adjacent pixels simultaneously having different voltages, some of which may be grounded to zero volts (but are not closed or ‘off’). The method may charge the adjacent electrodes charged with the same opposing voltages in the same frame. The method may charge the adjacent electrodes with +15 V and -15 V in the same frame.Disclosed herein is a digital microfluidic device for manipulating aqueous droplets, comprising:(a) a bottom plate comprising:a bottom electrode array comprising a plurality of digital microfluidic propulsion electrodes; a first dielectric layer covering the bottom electrode array; anda hydrophobic upper layer;(b) a controller operatively coupled to the bottom electrode array, wherein the controller is configured to simultaneously provide different propulsion voltages between adjacent electrodes of the bottom electrode array; and(c) a hydrophobic base fluid layer on top of the bottom electrode array containing the aqueous droplets;wherein the device manipulates aqueous droplets under application of propulsion voltages by providing different propulsion voltages between adjacent electrodes of the bottom electrode array. The different propulsion voltages are underneath the same liquid volume in order to provide an electrical circuit through the aqueous reagent. No top-plate electrical connection is thus required.Disclosed herein is a digital microfluidic device, comprising:(a) a bottom plate comprising:a bottom electrode array comprising a plurality of digital microfluidic propulsion electrodes; a first dielectric layer covering the bottom electrode array; anda hydrophobic upper layer;(b) a top plate comprising a lower hydrophobic layer;(c) a controller operatively coupled to the bottom electrode array, wherein the controller is configured to provide propulsion voltages between adjacent electrodes of the bottom electrode array;wherein the top plate and the bottom plate are not in an electrical connection and are provided in a spaced relationship to provide a gap defining a microfluidic region therebetween to permit motion of aqueous droplets within the microfluidic region under application of propulsion voltages between the separately voltage addressable digital microfluidic propulsion electrodes. The different propulsion voltages are underneath the same liquid volume in order to provide an electrical circuit through the aqueous reagent. No top-plate electrical connection is thus required.Disclosed herein is a digital microfluidic device for manipulating aqueous droplets, comprising:(a) a bottom plate array comprising:a thin-film transistor matrix, wherein each transistor of the matrix is operably connected to a gate line, a data line, and a separate propulsion electrode;a plurality of gate lines, wherein each gate line is operably connected to a gate driver; a plurality of data lines, wherein each data line is operably connected to a data driver; a first dielectric layer covering the bottom electrode array; anda hydrophobic upper layer;(b) a controller operably connected to the gate driver and the data driver; and(c) a processing unit operably connected to the controller and programmed to perform a microfluidic driving method, the method comprising:receiving input instructions in the processing unit, the input instructions relating to a droplet operation to be performed by the digital microfluidic system;outputting gate line and data line selecting instructions from the processing unit to the controller;outputting gate line signals from the controller to the gate driver, to drive a first gate line and an adjacent second gate line, andoutputting data line signals from the controller to the data driver, to drive a first data line and an adjacent second data line to simultaneously provide different signals to adjacent electrodes under a single droplet. The different propulsion voltages are underneath the same liquid volume in order to provide an electrical circuit through the aqueous reagent. No topplate electrical connection is thus required.Also disclosed is a method of driving a digital microfluidic system of active matrix TFTs, wherein a bottom plate comprises a thin film transistor (TFT) active matrix backplane, with each transistor of the backplane being operably connected to a gate driver, a data line driver, and one of the digital microfluidic propulsion electrodes, and moving an aqueous liquid using adjacent electrodes which are charged with the opposite polarity in the same frame. The circuit formed through the droplet which covers the electrodes of opposing polarity is sufficient to cause electrowetting operations without requiring an opposing common electrode.The method includes: receiving input instructions in the processing unit, the input instructions relating to a droplet operation to be performed by the digital microfluidic system; outputting gate line and data line selecting instructions from the processing unit to the controller; outputting gate line signals from the controller to the gate driver, to drive a first gate line and an adjacent second gate line, and outputting data line signals from the controller to the data driver, to drive a first data line and an adjacent second data line, thereby moving an aqueous droplet covering the first and second gate lines and the first and second data lines. Adjacent electrodes receive different signals from the first and second gate lines and data lines, thereby supplying a different voltage to adjacent electrodes and allowing droplet holding or movement. No electrical signals are sent to the top plate, which may be electrically disconnected or even absent entirely.Droplets are held in place by supplying different voltages to adjacent electrodes located under the droplets. In order to move droplets, electrodes located outside the droplets are actuated, and electrodes under the droplets are switched off. Typically electrodes are deactuated on the opposing / trailing edge from the leading edge which moves towards the actuatedelectrodes outside the droplet area. The droplets thereby move to the areas having a higher surface energy or lower droplet contact angle.Disclosed is a method for manipulating an aqueous liquid volume on a digital microfluidic device having an array of electrodes, the method comprising simultaneously using different electronic pulse sequences in different electrodes underneath the area covered by the aqueous liquid. Particular pulse sequences may be selected from pulse sequences: 1,-1; 1 ,0,-1 ; 1,0, 0,-1; 1 ,0,0, 0,-1 ;1 ,0,-1 ,-1 ; 1,-1, 0,-1; 1,1, 0,0,0, -1,-1; or 1,-1, 0, 1,-1.Disclosed is a method comprising:providing an electrokinetic device, including:a first substrate having a matrix of electrodes, wherein each of the matrix electrodes is coupled to a thin film transistor, and wherein the matrix electrodes are overcoated with a functional coating comprising:one or more dielectric layer(s) comprising silicon nitride, hafnium oxide or aluminium oxide in contact with the matrix electrodes,a conformal layer comprising parylene in contact with the dielectric layer, anda hydrophobic layer in contact with the conformal layer;a voltage source operatively coupled to the matrix electrodes;a hydrophobic base fluid layer on top of the bottom electrode array containing an aqueous volume covering at least first, second and third matrix electrodes; andproviding a series of electrical pulses to the first and second electrodes and a second weaker series of electrical pulses to the third electrodes such that the aqueous volume is split into droplets which cover the first and second electrodes and fail to cover the third electrode.The system and method can operate with just a layer of hydrophobic base fluid. The system and method can use a top plate with no electronic elements or layers such as dielectric coatings. The top plates can be glass or polymer coated with hydrophobic layers such as TeflonRTM. The top plates can be hydrophobic polymers. The top plates can be plastic top plates, for example polymethylmethacrylate (PMMA) or polycarbonate (PC), which may be optionally further coated. The electronic circuitry to the top plate may be absent or disconnected. Where the electrical signal is disconnected, optionally a switching mechanism may be used if desired in order to restore the connection.DEFINITIONSUnless otherwise noted, the following terms have the meanings indicated.“Actuate” with reference to one or more electrodes means effecting a change in the electrical state of the one or more electrodes which, in the presence of a droplet, results in a manipulation of the droplet."Droplet” means a volume of liquid that electrowets a hydrophobic surface and is at least partially bounded by carrier fluid. For example, a droplet may be completely surrounded by carrier fluid or may be bounded by carrier fluid and one or more surfaces of an EWoD device. Droplets may take a wide variety of shapes; non-limiting examples include generally disc shaped, slug shaped, truncated sphere, ellipsoid, spherical, partially compressed sphere, hemispherical, ovoid, cylindrical, and various shapes formed during droplet operations, such as merging or splitting or formed as a result of contact of such shapes with one or more working surface of an EWoD device. Droplets may include typical polar fluids such as water, as is the case for aqueous or non-aqueous compositions, or may be mixtures or emulsions including aqueous and non-aqueous components. The specific composition of a droplet is of no particular relevance, provided that it electrowets a hydrophobic working surface. In various embodiments, a droplet may include a biological sample, such as whole blood, lymphatic fluid, serum, plasma, sweat, tear, saliva, sputum, cerebrospinal fluid, amniotic fluid, seminal fluid, vaginal excretion, serous fluid, synovial fluid, pericardial fluid, peritoneal fluid, pleural fluid, transudates, exudates, cystic fluid, bile, urine, gastric fluid, intestinal fluid, fecal samples, liquids containing single or multiple cells, liquids containing organelles, fluidized tissues, fluidized organisms, liquids containing multi-celled organisms, biological swabs and biological washes. Moreover, a droplet may include one or more reagent, such as water, deionized water, saline solutions, acidic solutions, basic solutions, detergent solutions and / or buffers. Other examples of droplet contents include reagents, such as a reagent for a biochemical protocol, a nucleic acid amplification protocol, an affinity-based assay protocol, an enzymatic assay protocol, a gene sequencing protocol, a protein sequencing protocol, and / or a protocol for analyses of biological fluids. Further example of reagents include those used in biochemical synthetic methods, such as a reagent for synthesizing oligonucleotides finding applications in molecular biology and medicine, and / or one more nucleic acid molecules. The oligonucleotides may contain natural or chemically modified bases and are most commonly used as antisense oligonucleotides, small interfering therapeutic RNAs (siRNA) and their bioactive conjugates, primers for DNA sequencing and amplification, probes for detecting complementary DNA or RNA via molecular hybridization, tools for the targeted introduction of mutations and restriction sites in the context of technologies for gene editing such as CRISPR-Cas9, and for the synthesis of artificial genes by “synthesizing and stitching together” DNA fragments.“Droplet operation” means any manipulation of one or more droplets on a microfluidic device. A droplet operation may, for example, include: loading a droplet into the microfluidic device; dispensing one or more droplets from a source droplet; splitting, separating or dividing a droplet into two or more droplets; transporting a droplet from one location to another in any direction; merging or combining two or more droplets into a single droplet; diluting a droplet; mixing a droplet; agitating a droplet; deforming a droplet; retaining a droplet in position; incubating a droplet; heating a droplet; vaporizing a droplet; cooling a droplet; disposing of a droplet; transporting a droplet out of a microfluidic device; other droplet operations described herein; and / or any combination of the foregoing. The terms “merge,” “merging,” “combine,” “combining” and the like are used to describe the creation of one droplet from two or more droplets. It should be understood that when such a term is used in reference to two or more droplets, any combination of droplet operations that are sufficient to result in the combination of the two or more droplets into one droplet may be used. For example, “merging droplet A with droplet B,” can be achieved by transporting droplet A into contact with a stationary droplet B, transporting droplet B into contact with a stationary droplet A, or transporting droplets A and B into contact with each other. The terms “splitting,” “separating” and “dividing” are not intended to imply any particular outcome with respect to volume of the resulting droplets (i.e., the volume of the resulting droplets can be the same or different) or number of resulting droplets (the number of resulting droplets may be 2, 3, 4, 5 or more). The term “mixing” refers to droplet operations which result in more homogenous distribution of one or more components within a droplet. Examples of “loading” droplet operations include microdialysis loading, pressure assisted loading, robotic loading, passive loading, and pipette loading. Droplet operations may be electrode-mediated. In some cases, droplet operations are further facilitated by the use of hydrophilic and / or hydrophobic regions on surfaces and / or by physical obstacles.The ’’Gate driver” is connected to driver lines and accepts a voltage input from a controller, for instance a microcontroller integrated circuit (IC), and controls the open and closed (ON or OFF, saturated or cutoff) states of each individual TFT electrode, allowing these electrodes to be driven by a Source driver. The “Source driver” is connected to driver lines and controls the input voltage and current flow to each individual TFT electrode.When a liquid in any form (e.g., a droplet or a continuous body, whether moving or stationary) is described as being “on”, “at”, or “over an electrode, array, matrix or surface, such liquid could be either in direct contact with the electrode / array / matrix / surface, or could be in contact with one or more layers or films that are interposed between the liquid and the electrode / array / matrix / surface.When a droplet is described as being “on” or “loaded on” a microfluidic device, it should be understood that the droplet is arranged on the device in a manner which facilitates using the device to conduct one or more droplet operations on the droplet, the droplet is arranged on the device in a manner which facilitates sensing of a property of or a signal from the droplet, and / or the droplet has been subjected to a droplet operation on the droplet actuator."Each," when used in reference to a plurality of items, is intended to identify an individual item in the collection but does not necessarily refer to every item in the collection. Exceptions can occur if explicit disclosure or context clearly dictates otherwise.The terms ‘top’ and ‘bottom’ merely refer to two opposing surfaces. Whilst the devices are herein described in terms of a ‘bottom’ plate of electrodes and a ‘top’ plate which forms a sealed volume but has no electrical connections, the terms ‘top’ and ‘bottom’ are merely relative such that the ‘top’ plate could be the electrodes and the bottom plate may be disconnected. The terms refer to a sealed device having two opposing surfaces, not whether the device is placed in a certain orientation such that the plate having the electrodes is ‘above’ or ‘below’ the other plate. The claims herein refer to the device regardless of whether the device is rotated through 180 degrees.DETAILED DESCRIPTIONTraditional devices feature a top plate as a single continuous electrode, allowing for only one potential to be set for the top plate. As a result, propulsion electrodes that require a different voltage than their neighbors can only rely on their respective bottom plate electrode potentials for this purpose. Moreover, a defect encountered in the course of a droplet operation may induce current flows which force the top plate potential to change -sometimes, to corrode-, thereby affecting all the pixels in the array. In such instances, a microfluidic device and / or a microfluidic operation may be ruined by only a few defects in a bottom array of propulsion electrodes.Benefits of a lack of electrical connection through the top plate are therefore; a reduction in corrosion of the top plate as no electrical signal goes though the top plate; a reduction in driving voltages between adjacent pixels, reducing corrosion of the bottom plate (the distance between adjacent electrodes is less than the cell gap, hence voltages can be reduced within the circuit), and an increase in the speed of droplet motion for the same applied voltage. A further benefit comes from an ease of loading. Where aqueous reagents are loaded through holes in the top plate, clearly where the holes are present there is no top plate electrode. Therefore loading via top plate actuation is not possible. Where the electrodes are all in thelower plate, the loading can be assisted using the pixel electrodes immediately under the entry holes.As depicted herein, a bottom plate is fitted with an array of elements, where each element includes a digital microfluidic actuation electrode, to propel various droplets through the microfluidic region. The top plate of this aspect of the invention includes a top plate that is not a permanent electrode (i.e., it not always part of AM-EWoD circuit). Either no circuitry is coupled to the top electrode or the circuitry contains a switch allowing temporary electrical disconnection. The propulsion of droplets is formed by charging adjacent pixels on the device and using the presence of the droplets to form a circuit. This novel architecture allows for multiple bottom plane potentials to be set independently underneath or in the vicinity of a single droplet in order to enable the droplet to move towards the actuated pixel electrodes. Multiple simultaneous voltage ranges may be achieved on neighbouring pixels, a functionality beneficial for driving different types of droplets or different types of functional zones, for instance reservoirs vs. main array pixels.The use of “top” and “bottom” is merely a convention as the locations of the two plates can be switched, and the devices can be oriented in a variety of ways, for example, the top and bottom plates can be roughly parallel while the overall device is oriented so that the plates are normal to a work surface (as opposed to parallel to the work surface as shown in the figures). The top or the bottom plates may include additional functionalities, such as heating by commercial micro-heaters and thermocouples that are integrated with the microfluidic platform and / or temperature sensing.In a representative embodiment, the bottom plate of the device includes an active matrix electrowetting on dielectric (AM-EWoD) device featuring a plurality of array elements, each array element including a propulsion electrode, although other configurations for driving the bottom plate electrodes are also contemplated. The AM-EWoD matrix may be in the form of a transistor active matrix backplane, for example a thin film transistor (TFT) backplane where each propulsion electrode is operably attached to a transistor and capacitor actively maintaining the electrode state while the electrodes of other array elements are being addressed.A propulsion voltage is defined by a voltage difference between a first and second array electrode. By adjusting the frequency and amplitude of the signals driving the array electrodes, the propulsion voltage of each pixel of the array may be controlled to operate the AM-EWoD device at different modes of operation in accordance with different dropletmanipulation operations to be performed. In one embodiment, the TFT array may be implemented with amorphous silicon (a-Si), thereby reducing the cost of production to the point that the devices can be disposable.The fundamental operation of a traditional EWoD device is illustrated in the sectional image of Figure 22. The EWoD 100 includes microfluidic region filled with an oil 102 and at least one aqueous droplet 104. The microfluidic region gap depends on the size of droplets to be handled and is typically in the range 50 to 200 pm, but the gap can be smaller or larger. In a basic configuration, as shown in Figure 22, a plurality of propulsion electrodes 105 are disposed on one substrate and a singular top electrode 106 is disposed on the opposing surface. The device additionally includes hydrophobic coatings 107 on the top and bottom surfaces, i.e., where they contact the oil layer, in addition to a dielectric layer 108 between the propulsion electrodes 105 and the hydrophobic coating 107. (The upper substrate may also include a dielectric layer, but it is not shown in Figure 22). The hydrophobic layer prevents the aqueous droplet from wetting the surface. When no voltage differential is applied between adjacent electrodes, the droplet will maintain a spheroidal shape to minimize contact with the hydrophobic surfaces (oil and hydrophobic layer). Because the droplets do not wet the surface, they are less likely to contaminate the surface or interact with other droplets except when that behavior is desired.While it is possible to have a single layer for both the dielectric and hydrophobic functions, such layers typically require thick inorganic layers (to prevent pinholes) with resulting low -capacitance, thereby requiring more than 100 V for droplet movement. To achieve low voltage propulsion, it is often better to have a thin inorganic layer for high capacitance, topped by a thin organic hydrophobic layer to be pinhole free. With this combination it is possible to have electrowetting operation with voltages in the range + / -10 to + / -50 V, which is in the range that can be supplied by conventional TFT arrays built upon amorphous silicon.Hydrophobic layers may be manufactured from hydrophobic materials formed into coatings by deposition onto a surface via suitable techniques. Depending on the hydrophobic material to be applied, example deposition techniques include spin coating, molecular vapor deposition, and chemical vapor deposition. Hydrophobic layers may be more or less wettable as usually defined by their respective contact angles. Unless otherwise specified, angles are herein measured in degrees (°) or radians (rad), according to context. For the purpose of measuring the hydrophobicity of a surface, the term “contact angle” is understood to refer to the contact angle of the surface in relation to deionized (DI) water. If water has a contact angle between 0° < 0 < 90°, then the surface is classed as hydrophilic, whereas a surfaceproducing a contact angle between 90° < 0 < 180° is considered hydrophobic. Usually, moderate contact angles are considered to fall in the range from about 90° to about 120°, while high contact angles are typically considered to fall in the range from about 120° to about 150°. In instances where the contact angle is 150° < 0 then the surface is commonly known as superhydrophobic or ultrahydrophobic. Surface wettabilities may be measured by analytical methods well known in the art, for instance by dispensing a droplet on the surface and performing contact angle measurements using a contact angle goniometer. Anisotropic hydrophobicity may be examined by tilting substrates with gradient surface wettability along the transverse axis of the pattern and examining the minimal tilting angle that can move a droplet.Hydrophobic layers of moderate contact angle typically include one or a blend of fluoropolymers, such as PTFE (polytetrafluoroethylene), FEP (fluorinated ethylene propylene), PVF (polyvinylfluoride), PVDF (polyvinylidene fluoride), PCTFE (polychlorotrifluoroethylene), PFA (perfluoroalkoxy polymer), ETFE (polyethylenetetrafluoroethylene) and ECTFE (polyethylenechlorotrifluoroethylene). Commercially available fluoropolymers include Cytop® (AGC Chemicals, Exton, PA) and Teflon® AF (Chemours, Wilmington, DE). An advantage of fluoropolymer films is that they can be highly inert and can remain hydrophobic even after exposure to oxidizing treatments such as corona treatment and plasma oxidation.When a voltage differential is applied between adjacent electrodes, the voltage on one electrode attracts opposite charges in the droplet at the dielectric-to-droplet interface, and the droplet moves toward this electrode, also as illustrated in Figure 22. The voltages needed for acceptable droplet propulsion depend on the properties of the dielectric and hydrophobic layers. AC driving is used to reduce degradation of the droplets, dielectrics, and electrodes by various electrochemistries. Operational frequencies for EWoD can be in the range 100 Hz to 1 MHz, but lower frequencies of 1 kHz or lower are preferred for use with TFTs that have limited speed of operation.Returning to Figure 22, the top electrode 106 for traditional prior art devices is a single conducting layer normally set to zero volts or a common voltage value (VCOM) to take into account offset voltages on the propulsion electrodes 105 due to capacitive kickback from the TFTs that are used to switch the voltage on the electrodes. The top electrode can also have a square wave applied to increase the voltage across the liquid. Such an arrangement allows lower propulsion voltages to be used for the TFT connected propulsion electrodes 105 because the top plate voltage 106 is additional to the voltage supplied by the TFT. In manyinstances, the top electrode is constructed from a light-transmissive material, such as indium tin oxide (ITO) sputtered on a film of polyethylene terephthalate (PET), however, the top electrode does not have to be light-transmissive for most electrowetting on dielectric applications. Other simple conductive films, such as metal films, may be used, provided that they are sufficiently protected from the materials of the droplet layer, e.g., the oil and the aqueous droplet.As illustrated in Figure 23, an active matrix of propulsion electrodes can be arranged to be driven with data and gate (select) lines much like an active matrix in a liquid crystal display. The gate (select) lines are scanned for line-at-a time addressing, while the data lines carry the voltage to be transferred to propulsion electrodes for electrowetting operation. If no movement is needed, or if a droplet is meant to move away from a propulsion electrode, then 0 V will be applied to that (non-target) propulsion electrode. If a droplet is meant to move toward a propulsion electrode, an AC voltage will be applied to that (target) propulsion electrode.The dielectric must be thin enough and have a dielectric -property compatible with low voltage AC driving, such as available from conventional image controllers for LCD displays. For example, the dielectric layer may comprise a layer of approximately 20-40 nm SiC>2 overcoated with 200-400 nm plasma-deposited silicon nitride. Alternatively, the dielectric may comprise atomic-layer-deposited AI2O3 between 2 and 100 nm thick, preferably between 20 and 60 nm thick. The TFT may be constructed by creating alternating layers of differently-doped a-Si structures along with various electrode lines, with methods known to those of skill in the art. The hydrophobic layer can be constructed from materials such as Teflon® AF (Sigma-Aldrich, Milwaukee, Wl) and FlurorPel™ coatings from Cytonix (Beltsville, MD), which can be spin coated over the dielectric layer.The top and bottom plates are not in electrical connection. The top electrode array does not use or require conducting materials such as indium-doped tin oxide (ITO), or other transparent conductive oxide (TOO) to achieve a top electrode layer or dielectric layer. The top layer may be made of any material, for example glass or plastic. The coatings need to be hydrophobic in order to form confined aqueous droplets. The top layer may be Teflon coated glass. Where the electrical layers are retained on the top plate a switch may be employed in order to prevent electrical connection whilst the lower electrodes are being driven. The top plate may be absent such that the oil layer in the device is open to atmosphere. Alternatively the top plate may not be in contact with the oil layer and merely acting as a cover.Figure 24 shows an actuation pattern of the prior art. The entire area under each droplet (paler squares) is actuated using the same pattern. In order to move the droplet, the electrodes outside the droplet are activated and the electrodes under the droplet are switched off, usually in the form of a leading edge and trailing edge. The droplet moves towards the activated electrodes in order to centre over the areas of surface charge. The circuit is formed from a common top electrode; one voltage polarity per frame.In the pattern of the invention, as shown in Figure 25, no top electrode is needed. By forming actuation patterns (different patterns of paler squares) under each droplet the droplet can be steered across the surface. The charge is conducted through the droplet causing the droplets to move. Actuation patterns on the bottom plate can be chosen to activate adjacent electrodes in sequence, for example as stripes or as a checkerboard.Figures 26 and 27 show the simplification of the circuitry achieved by not needing an electrically connected top plate. The prior art devices achieve propulsion by activating bottom electrodes and generating a voltage potential across the cell gap using a common top electrode (Figure 26). In Figure 27, the potential is generated between adjacent electrodes on the bottom layer and no top plate connection is needed.Figures 28 (start) and 29 (end) show droplet generation using the driving patterns of the invention in relation to the prior art sequence (top). A reservoir on the right should dispense multiple evenly sized droplets. The 5 droplets on the left should be split in two. The top row (prior art) shows 3 droplets, meaning two were not dispensed. The 5 droplets did not split. The lower three rows, showing actuation patterns of the invention showed accurate dispensing of 5 droplets from each reservoir, and splitting of the 5 left hand droplets.Figure 30 shows construction of a device with a coated plastic top plate. Combining top plate / cartridge ports into a single plastic piece makes production easier and reduces cost. The device contains a PMMA sheet coated with TeflonRTMas the top plate. The top plate has no electronic components or connections. The bottom TFT array can be sealed into a device using the plastic top plate which has reagent entry ports. Figure 31 shows droplet generation from reservoirs on the device with a coated plastic top plate. The use of adjacent electrode driving allows droplets to be formed. Where the driving is performed using patterns without adjacent pixels being activated no droplets are formed.Figure 33 shows optical microscopy images from the top plate of an electrowetting device. The images compare the same device and aqueous buffer driven by adjacent electrodedriving (+15 V / -15 V) with no top plate driving vs using the top plate as part of the driving circuit and no adjacent driving of the TFT electrodes. The top plate corrodes when used as part of the circuit (R2). The use of adjacent TFT electrodes to form the driving circuit lowers the level of top plate corrosion as no current flows through the top plate electrode.The device can be used with no top plate at all, just a layer of oil to prevent evaporation of the droplets, or can be used having just a top plate not in contact with the layer of oil.Disclosed is a digital microfluidic device for manipulating aqueous droplets, comprising: (a) a bottom plate comprising:a bottom electrode array comprising a plurality of digital microfluidic propulsion electrodes; a first dielectric layer covering the bottom electrode array; anda hydrophobic upper layer;(b) a controller operatively coupled to the bottom electrode array, wherein the controller is configured to simultaneously provide different propulsion voltages between adjacent electrodes of the bottom electrode array; and(c) a hydrophobic base fluid layer on top of the bottom electrode array containing the aqueous droplets;wherein the device manipulates aqueous droplets under application of propulsion voltages by providing different propulsion voltages between adjacent electrodes of the bottom electrode array.Droplets and reservoirs of any size can be manipulated. The droplets should cover multiple electrodes on the device. In prior art devices the cell gap is smaller than the electrodes as there is no benefit in having smaller pixels. In the device and methods herein, driving is better with more pixels under the droplet, hence the arrays herein allow for the cell gap to be greater than the size of the electrodes. For example the cell gap may be greater than 100 .m and each electrode smaller than 80 .m.The adjacent electrodes are actuated in repeating patterns. The population voltages may be in the form of a checkerboard. The propulsion voltages may take the form of providing voltages to the electrodes in parallel stripes. The stripes may be parallel with or perpendicular to the direction of droplet propulsion.The droplets should cover multiple pixels. The droplets should cover at least 2 electrodes.One of the adjacent electrodes can be grounded to a voltage of 0 V. Alternatively the adjacent electrodes may be charged with the opposite polarity. Note that the application of zero volts is different to the pixel being off. The act of being open to ground provides a discharge route from adjacent pixels which are actuated. Where a pixel is closed, or off, as in the prior art, no circuit can be formed due to the lack of grounding. Thus an open connection of zero volts is not the same as a closed connection. The invention herein provides adjacent pixels simultaneously having different voltages, some of which may be grounded to zero volts.The dielectric may have a barrier layer such as an insulator material, produced by a vacuum deposition technique, of which there are several types including physical vapor deposition (PVD) and chemical vapor deposition (CVD) in one of its variants: low-pressure chemical vapor deposition (LPCVD), plasma-enhanced chemical vapor deposition (PECVD), and plasma-assisted CVD (PACVD). In several instances, a combination of PVD and CVD are used in the same or connected processing chambers. In one exemplary embodiment, the insulator material is parylene, the name for a variety of chemical vapor deposited poly(p-xylylene) polymers used as moisture and dielectric barriers. Among them, Parylene C, bearing one chlorine atom substituent to the phenyl moiety per repeat polymeric unit, is the most popular due to its combination of barrier properties, cost, and other processing advantages. Parylene N and C have a low degree crystallinity and are therefore usually suitable as optical materials. Other optically transparent or at least partially transparent polymeric and ceramic insulators may also be used.Circuitry for connecting and / or controlling the voltages may be housed in the bottom plate, for example on the edges of the electrode array, or elsewhere in the device depending on the needs and constraints of the application at hand. In another embodiment, the device is fitted with a connection interface with the bottom electrode array, for example connectors from the fan out on the edge. In this instance, filled vias are made in the top electrode segments and are fanned out to a location where a connection can be made.Electrode driving sequences for specific bottom plate electrodes can be calculated and implemented based on the requirements of a droplet operation to be performed on the EWoD device. The flow chart of Figure 32 illustrates an example embodiment of this process. In step 110, a user inputs a desired droplet operation in the form of instructions which are stored in a computer-readable medium that is accessed by a processing unit of the device. The processing unit is programmed to perform driving sequences featuring multiple bottom electrode potentials. The instructions cause the processing unit to calculate the polarity, frequency, and amplitude of each pulse applied to the bottom plate propulsion electrodeswhich are to take part in the droplet operation (112). Then, the processing unit outputs instructions to the controller (114), and the controller outputs signals to the drivers of the bottom plate propulsion electrodes (116). In instances where the bottom plate includes an array of TFT electrodes, the controller outputs gate line signals to the drivers of gate lines and data line signals to data line drivers, thereby driving the intended propulsion electrodes. The selected bottom plate propulsion electrodes are then driven to perform the droplet operation (118).The inorganic dielectric layer may comprise silicon dioxide, silicon oxynitride, silicon nitride, hafnium oxide, yttrium oxide, lanthanum oxide, titanium dioxide, aluminium oxide, tantalum oxide, hafnium silicate, zirconium oxide, zirconium silicate, barium titanate, lead zirconate titanate, strontium titanate, or barium strontium titanate. The dielectric layer may be between 10 nm and 100 pm thick. Combinations of more than one material may be used, and the dielectric layer may comprise more than one sublayer that may be of different materials.The conformal layer may comprise a parylene, a siloxane, or an epoxy. It may be a thin protective parylene coating in between the inorganic dielectric and the hydrophobic coating. Typically, parylene is used as a dielectric layer on simple devices. In this invention, the rationale for deposition of parylene is not to improve insulation / dielectric properties such as reduction in pinholes, but rather to act as a conformal layer between the dielectric and hydrophobic layers. The inventors find that parylene, as opposed to other similar insulating coatings of the same thickness such as PDMS (polydimethylsiloxane), prevent contact angle hysteresis caused by high conductivity solutions or solutions deviating from neutral pH for extended hours. The conformal layer may be between 10 nm and 100 pm thick. The conformal layer may be between 100 nm and 200 nm thick.The hydrophobic layer may comprise a fluoropolymer coating, fluorinated silane coating, manganese oxide polystyrene nanocomposite, zinc oxide polystyrene nanocomposite, precipitated calcium carbonate, carbon nanotube structure, silica nanocoating, or slippery liquid-infused porous coating.The elements may comprise one or more of a plurality of array elements, each element containing an element circuit; discrete electrodes; a thin film semiconductor in which the electrical properties can be modulated by incident light; and a thin film photoconductor whose properties can be modulated by incident light.The functional coating may include a dielectric layer comprising silicon nitride, a conformal layer comprising parylene, and a hydrophobic layer comprising an amorphous fluoropolymer.The electro kinetic device may include a controller to regulate a voltage provided to the individual matrix electrodes. The electro kinetic device may include a plurality of scan lines and a plurality of gate lines, wherein each of the thin film transistors is coupled to a scan line and a gate line, and the plurality of gate lines are operatively connected to the controller. This allows all the individual elements to be individually controlled.The method is particularly suitable for aqueous droplets with a volume of 1 pL or smaller.Described herein are electrokinetic devices, including:a first substrate having a matrix of electrodes, wherein each of the matrix electrodes is coupled to a thin film transistor, and wherein the matrix electrodes are overcoated with a functional coating comprising:a dielectric layer in contact with the matrix electrodes,a conformal layer in contact with the dielectric layer, anda hydrophobic layer in contact with the conformal layer;a second substrate comprising a top plate which is not in electrical connection with the first substrate;a spacer disposed between the first substrate and the second substrate and defining an electrokinetic workspace; anda voltage source operatively coupled to the matrix electrodes.Described herein is an electrokinetic device, including:a first substrate having a matrix of electrodes, wherein each of the matrix electrodes is coupled to a thin film transistor, and wherein the matrix electrodes are overcoated with a functional coating comprising:one or more dielectric layer(s) comprising silicon nitride, hafnium oxide and / or aluminum oxide in contact with the matrix electrodes,a conformal layer comprising parylene in contact with the dielectric layer, anda hydrophobic layer in contact with the conformal layer;a second substrate comprising a top plate which is not in electrical connection with the first substrate;a spacer disposed between the first substrate and the second substrate and defining an electrokinetic workspace; anda voltage source operatively coupled to the matrix electrodes.The electrokinetic devices as described may be used with other elements, such as for example devices for heating and cooling the device or reagent cartridges for the introduction of reagents as needed.The device can be an active-matrix thin film transistor (AM-TFT) based device. Also disclosed is an active-matrix thin film transistor (AM-TFT) device having a substrate bearing a plurality of electrodes, the device comprising multiple fluidic inlet ports on at least two sides of the device, wherein the inlet ports on each side of the device are evenly spaced and wherein the device is connected to a syringe pump.The device may comprise two substrates, wherein at least one substrate has a plurality of electrodes, and the two substrates define parallel plates that are separated by a spacer to define a volume.The fluidic entry may come via holes in the upper plate or through the spacer. The entry holes may be in the top substrate. The plurality of electrodes may be on a bottom substrate. The top substrate be of glass or polymer and may have a thickness ranging from 0.5 mm to 20 mm.The spacer may comprise an adhesive with beads of a defined size distribution. The spacer may comprise a polymer material of a defined thickness. The spacer may comprise glass, in which case the layers can be fused together. The spacer gap and therefore height of fluid in the device may be between 50 pm and 250 pm. The spacer gap and therefore height of fluid in the device may be between 100 pm and 150 pm.The enclosed volume may contain aqueous droplets in a hydrophobic filler fluid. The filler liquid may be a hydrophobic or non-ionic liquid. For example the filler liquid may be decane or dodecane. The filler fluid may be a silicone oil such as dodecamethylpentasiloxane (DMPS). The filler liquid may contain a surfactant, for example a sorbitan ester such as Span 85.The filler liquid may be moved via an automated manner, or may be moved under gravity. A hydrostatic head of pressure can be used to move the filler liquid within the device. The aqueous phase may be loaded via wells on the device. The wells may be at least partially filled with filler fluid before the aqueous reagents are loaded. The filler fluid may be less dense than the aqueous phase such that the aqueous phase sinks in the wells.The device may be connected to a pump, for example a syringe pump, a peristaltic pump, a disc pump, a diaphragm pump, or a pneumatic pump. The pump enables filling of the device with filler liquid in an automated manner. Once filled, the pump enables partial withdrawal of the filler fluid to create a negative pressure in the device which draws in reagents from the wells. Thus the filling and withdrawal of fluid may be performed in an automated manner to allow largely ‘hands-free’ loading of the aqueous reagents. An automated filler liquid filling and withdrawal method may be integrated into an instrument that provides other functions relating to the digital microfluidic device, including heating, cooling, optical, sensing, mechanical, and magnetic functions.The wells of the device may be at 90 degrees to each other. The inlets may be at 180 degrees to each other. The inlets may be on 4 sides of the device. Each side may have at least 4, 8 or 12 ports. Each side may have 8 ports. The device may have 4 sets of 8 ports. The number of ports may vary on different sides of the device, for example one side may have 8 ports and one side 4 ports. The device may have 8 ports on 3 sides and 16 ports on a fourth side. The device may have 16 ports on 3 sides and 8 ports on a fourth side. The ports may be offset to give multiple rows of linear ports on one side, for example a first and second row where the second row is behind by offset from the first row such that the source liquid can flow between the ports of the first row. The rows may be a zig-zag fashion.The pitch between inlet ports may be 9 mm. The pitch between inlet ports may be 4.5 mm. The inlet ports have a pitch of 4.5 mm or a multiple of thereof. This would cover 24 well, 48 well, 96 well, 384 well ports. The pitch of the ports may be the same on each side of the device, or may be different sized. In this context the pitch refers to the distance between the centre of each inlet.The volume of aqueous reagents loaded per inlet port may be between 1 microlitre and 100 microlitres. The volume of aqueous reagents loaded per inlet port may be between 1 microlitre and 50 microlitres. The volume may be between 1 microlitre and 20 microlitres. The aqueous liquid may be introduced to the wells by a pipette, a multichannel pipette, a syringe, a blister pack, an acoustic dispenser, or a robotic liquid handler. The aqueous liquids may be loaded simultaneously from multiple wells, which may be on the same side or multiple sides of the devices. Each well is a separate liquid, and can be the same or different to the contents of the aqueous volume in other wells. The volume of aqueous liquid loaded in each port can be the same or can be different.The automated filling and / or withdrawing of filler fluid may be controlled by software. The device may be part of a larger instrument system that provides environmental control such as temperature control or light control and may have analytical capabilities such as optical systems for fluorescence or luminescence assay detection.The location of the aqueous layer is controlled by the actuation of electrodes to form reservoirs in defined areas. A plurality of electrodes is actuated to control the location of the aqueous liquid once it has been drawn onto the substrate bearing a plurality of electrodes. Multiple reservoirs may be formed on the device. The device described herein allows improved loading compared to prior art devices having entry holes in the top plate. The devices herein can actuate electrodes immediately under the entry holes, thereby enabling a hydrophilic surface at the point of entry. Where the electrical top plate is required, holes in the top plate mean that the pixels directly under the entry point can not be driven as there is no opposing electrode.Where there is no top plate, reagents can be loaded into the open device. Any suitable method for reagents dispensing may be used, for example automated or manual liquid handling, inkjet dispensing etc. Loading of aqueous reagents may be performed before or after application of an oil layer. After loading, reagents can be split or moved as described herein. For example a large number of small droplets can be generated from a single reagent applied as a reservoir. Arrays of reagents can be applied and combined. For example n reservoirs each having different reagents can be split and combinatorially combined to produce n x n number of droplets having different reagent combinations.In an exemplary driving sequence, a first electrode segment is driven to a first voltage, and a second electrode segment is driven to a second voltage differing from the first voltage, at the same frame, in at least one of polarity, frequency, and amplitude.Disclosed is a method for manipulating an aqueous liquid volume on a digital microfluidic device having an array of electrodes, the method comprising simultaneously using different electronic pulse sequences in different electrodes underneath the area covered by the aqueous liquid.Disclosed is a method for forming a plurality of droplets on an electro kinetic device, the method comprising taking first droplets and splitting the droplets into multiple evenly sized smaller droplets by moving the droplets in opposing lateral directions. The splitting occurs by moving the droplets along an axis perpendicular to the longest axis of the droplets. The lateralmovement is at 90 degrees to the elongated axis such that at least one of the ends of the droplet move perpendicular to the elongated axis.Disclosed is a method for forming a plurality of droplets on an electro kinetic device, the method comprising taking first droplets and splitting the droplets into multiple evenly sized smaller droplets by moving the droplets in opposing lateral directions, wherein the first droplets are elongated before splitting and the lateral movement is at 90 degrees to the elongated axis such that the ends of the droplet move perpendicular to the elongated axis. In each case the splitting is performed using controlled actuation in the neck portion between the two droplets.The droplets may be rectangular. Once split, the droplets can be split into smaller rectangles or squares. The rectangles may be split into two, three or four smaller droplets of equal size. The rectangles may be split into three or four smaller droplets of equal size. The rectangles may be split into three smaller droplets of equal size. The rectangles may be split into four smaller droplets of equal size. The droplets may be square (in which case there are two longest axes and the square may be split into rectangles).The droplets may be circular. Once elongated to ovals, the droplets can be split into smaller circles.The droplets can be elongated prior to splitting providing the elongation does not split the droplet. Having the split droplets elongated prior to splitting makes splitting of the droplets more even as it increases tolerance against starting volume variations and compensates for reagent that is not actually actuated as it is part of the meniscus during the split.The liquid volumes in the rectangles may be formed using an actuated meniscus, which acts to control the liquid volume prior to the split. The approach for example consists of describing (in the form of actuation patterns) a continuous transformation from an initial shape into one or more final shapes while keeping the actuated area constant throughout the process. The intermediate steps of the transformation are chosen such that they closely approximate (pixel-limited) the outline naturally assumed by a droplet deformed in such a way taking into account the tendency of the droplet to minimise its surface energy. This level of control allows keeping the pinch-off point consistent; making the process reproducible within and amongst devices and droplets. Disclosed is a method wherein multiple first droplets are formed, each using an actuated meniscus in order to normalise the volume of liquid between the first droplets to be split, and thereby the final droplets after splitting. The actuated meniscus isparticularly beneficial when moving viscous or difficult to move reagents such as those containing beads.Multiple droplets may be split simultaneously to form an array of droplets. The arrays may be generated by splitting such that the array has equal spacing s? along one axis and equal spacing S2 along a second axis.The split droplets may be further elongated and split into order to generate further, smaller droplets. Droplets may be split at least twice in order to make at least 4 smaller evenly sized droplets from each of the first droplets.The final droplets may be for example less than 250 nL in volume. The final droplets may occupy fewer than 10 pixels of the electrokinetic device per droplet. The final droplets may occupy fewer than 4 pixels of the electrokinetic device per droplet. The invention is particularly beneficial when splitting small droplets, which otherwise would tend to split in a way that is not evenly distributed.The first starting droplets may occupy at least 10x10 pixels of the electrokinetic device per droplet. Droplets may be any particular size. For example a droplet 64x18 pixels may be split into 2X 32x18 pixels. A droplet of 9x2 pixels may be split into 2X 3x3 pixels.After splitting the droplets may be manipulated, for example by combining with other droplets. The droplets may contain reagents for assays, for example a nucleic acid template or a cell-free system having components for protein expression.Described is a method for forming a plurality of droplets on an electrokinetic device, the method comprising taking one or more first droplets and splitting the one or more first droplets n times into smaller droplets, wherein n >= 2 and the splitting occurs in at least two directions, x and y, on the electrokinetic device.Described is a method for forming a plurality of droplets on an electrokinetic device, the method comprising taking two or more first droplets and splitting each of the first droplets n times into smaller droplets, wherein n >= 2 and the splitting occurs in at least two directions, x and y, on the electrokinetic device to form a regular two-dimensional array with drops having equal spacing s? along one axis and equal spacing S2 along a second axis.Droplet splitting in this manner has many advantages over repeatedly dispensing droplets from a larger reservoir. The method is efficient in the use of space on the device. An array of droplets as a grid can be formed in a similar area to the size of the original droplet. The method is also efficient in the use of reagents, as all the material dispensed onto the device can be used to form droplets. The method is also efficient in the use of time, as a large number of droplets can be formed in the shortest time when compared to the single dispense operations. Splitting in this manner also reduces the variability between the sizes of different droplets. Where each volume is repeatedly halved, the variability in volume between the final droplets is less than where droplets are repeatedly dispensed from a reservoir. Large numbers of areas of differing liquid volumes can be rapidly produced on the device.The droplet can be split at least twice in each direction to make at least 16 smaller droplets from each of the one or more first droplets.The splitting can occur using a repeating pattern. The splitting can be recursive such that the array is formed with an even distribution of droplets on the surface of the electro kinetic device. The splitting can be iterative.The splitting can reduce the volume of the droplet in defined proportions. Each droplet can be halved by each splitting. Each of the splits can half the volume of the droplet. The split can pull the droplet into three smaller droplets. The split can pull the droplet into four smaller droplets. Each splitting does not have to be the same size. Thus the droplets can be split into 3 along one axis, then halved on the other axis, thus making 6 droplets from the first droplet.A large number of droplets can be split simultaneously. For example 96 first droplets can be split at the same time. Thus 96 droplets can be split in half to form 192 droplets. Each of the 192 can be further split into 384.The two directions x and y are typically at 90° to each other in order to form the array within the smallest area.The final droplets can be less than 1 pL in volume. The final droplets can be less than 500 nL in volume. The final droplets can be less than 250 nL in volume. The final droplets can be less than 100 nL in volume. The final droplets typically occupy only a few pixels on the array, for example less than 10x10 pixels. The final droplets may occupy 4x4 pixels on the array. The final droplets may occupy less than 4x4 pixels on the array.The initial droplets are typically in the range of 0.5 to 1 pL in volume. The first droplets typically occupy at least 14x14 pixels of the electrokinetic device per droplet.Waveforms or drive sequences used to manipulate the liquid may include one or more rest pulses. The term ‘weaker’ pulse sequence refers to a sequence having for example rest pulses or lower voltages than other sequences. The waveforms used to manipulate the liquid may include different voltages. The two or more waveforms are simultaneously applied within the same aqueous volume in order to enhance manipulation operations on the aqueous liquid.The pulse sequences used to manipulate the liquid may be selected from can include at least one of 1,0,-1; 1,0, 0,-1; 1 ,0,0, 0,-1 ;1 ,0,-1 ,-1 ; 1,-1, 0,-1; 1,1, 0,0,0, -1,-1; or 1, -1,0, 1,-1. The ‘stronger’ sequence may be for example 1,-1 without rest pulses (represented by 0).The weaker sequence may use a voltage of less than 15 V. The stronger sequence may use a voltage of 15 V or greater. Both voltages are used simultaneously within the same volume of aqueous liquid. The sequences may involve the use of positive and negative pulses to adjacent electrodes.The droplets can be used in a variety of assays, for example in assays where dilution to single molecules or single cells per reaction volume is desirable. For example the method can be used to obtain single cells per droplet or single nucleic acid templates per droplet. The single nucleic templates can be amplified in order to produce isolated amplicons. The droplets can be used to express proteins, for example using a cell-free expression system, wherein the droplets contain nucleic acid templates and a cell-free system having components for protein expression.Assays can be performed on the droplets, for example to determine the presence of or sequence of nucleic acids in a sample or for performing protein expression assays.During operation on a high density electrode array, droplets and locations can be defined in a discreet fashion based on the number of pixels, e.g. droplet size of 10 x 10 pixels and x / y droplet location of (100, 200) pixels. The first step to quickly generate a large ordered array of droplets is to dispense a small number of large droplets into an ordered array. These are the first volumes of aqueous liquid (aka reservoirs). In order to generate the large array quickly, the reservoirs are split multiple times along both the horizontal and vertical axes. Thereason for this is it will avoid droplet collisions and the requirement of having to move droplets around after splitting them.The splitting of reservoirs into droplets is shown in Figure 1. A rectangular reservoir is optimally split by moving the opposing ends in opposite directions ‘up and down’ rather than by pulling the ends apart. In Figure 1, two squares are produced, but the split can produce shapes that are still rectangular (when the length of the starting shape is more than twice the width).Figure 2 shows a longer rectangular shape which can be split into 4. The four sections are produced by ‘sliding’ sections of the liquid past each other. Four squares can be produced if the four sections are move up, down, up, down.The reservoir may be split into 3 sections if the two ends are moved in the same direction and the centre is moved in the opposite direction.The droplets held on the device may be square, square droplets (or as square as possible) are dispensed onto the array from reservoirs along the edges of the device (as many as desired). The droplets are organized into rows and columns, equally spaced apart, though the spacing need not be identical along each axis.The droplets being split can take any shape. A square droplet can be split along any central axis to make multiple droplets of substantially equal volume. The droplets can be split horizontally or vertically to make two rectangles, or along both axes to make 4 squares. The shape of the droplets are governed by the pixel activation on the array.Where the droplets are square, the droplets may be elongated to rectangular shapes prior to splitting. The aspect ratio (length to width) may be greater than 2 to split. The aspect ratio may be greater than 4. A longer aspect ratio is preferred in order to maximise the radius of curvature.Alternatively the droplets can be round in shape.Where the droplets contain the same reagents, the droplets may be split, remerged and resplit in order to promote reagent mixing and reduce inhomogeneity between droplets.The procedure can be carried out with multiple reagents sourcing the initial droplets, and is performed on a high density electrode grid (i.e. > 100x100 electrodes). A digital microfluidics cell comprised of a TFT and an ITO / glass top plate may be used,The droplets and reservoirs are aqueous droplets. The droplets may be within an infilling oil. The oil can be mineral oil, silicone oil such as dodecamethylpentasiloxane (DMPS), an alkyl-based solvent such as decane or dodecane, or a fluorinated oil. The droplets and / or oil may contain surfactants to adjust surface tension.The splitting involves turning off one or more of the electrodes under the existing droplet whilst turning on pixel electrodes adjacent to one or both sides of the droplet. Thus one end or both ends of the droplet move in lateral directions until the join between them breaks. Thus the droplets are slid apart using the electrodes on the device. The sliding split, along with the controlled actuation under the neck region generally makes even sized droplets. The lateral flow (i.e. flow in opposite directions on the device) across the longest axis increases the homogeneity of droplet sizing when compared to droplets that are pulled apart along the longest axis.Typically, the electrodes (or the array elements) used for EWoD are covered with (i) a hydrophilic insulator / dielectric and a hydrophobic coating or (ii) a hydrophobic insulator / dielectric. Commonly used hydrophobic coatings comprise of fluoropolymers such as Teflon AF 1600 or CYTOP. The thickness of this material as a hydrophobic coating on the dielectric is typically <100 nm and can have defects in the form of pinholes or a porous structure; hence, it is particularly important that the insulator / dielectric is pinhole free to avoid electrical shorting. Teflon has also been used as an insulator / dielectric, but it has higher voltage requirements due to its low dielectric constant and the thickness required to make it pinhole free. Other hydrophobic insulator / dielectric materials can include polymer-based dielectrics such as those based on siloxane, epoxy (e.g. Sll-8), or parylene (e.g., parylene N, parylene C, parylene D, or parylene HT). Due to minimal contact angle hysteresis and a higher contact angle with aqueous solutions, Teflon is still used as a hydrophobic topcoat on these insulator / dielectric polymers. However, there are difficulties in reliably producing <1 micron pinhole-free coatings of parylene or Sll-8; thus, the thickness of these materials is typically kept at 2-5 microns at the cost of increased voltage requirements for electrowetting. It has also been reported that traditional EWoD devices with parylene C are easily broken and unstable for repeated droplet manipulation with cell culture medium. Multi-layer insulator devices deposited with metal-oxide and parylene C films have been used to produce a more robust insulator / dielectric and enable operations with lower applied voltages. Inorganicmaterials, such metal oxides and semiconductor oxides, commonly used in the CMOS industry as “gate dielectrics”, have been used as insulator / dielectric for EWoD devices. They offer the advantage of utilizing standard cleanroom processes for thin film depositions (<100 nm). These materials are inherently hydrophilic, requiring an additional hydrophobic coating, and can be prone to pinhole formation as a result of thin film layer deposition process. Together with the need for lower voltage operations of EWoD, recent developmental work has focused on (1) using materials with improved dielectric properties (e.g., using high-dielectric constant insulators / dielectrics), (2) optimizing the fabrication process to make the insulator / dielectric pinhole free to avoid dielectric breakdown.The electrokinetic device may include a first substrate having a matrix of electrodes, wherein each of the matrix electrodes is coupled to a thin film transistor, and wherein the matrix electrodes are overcoated with a functional coating comprising: a dielectric layer in contact with the matrix electrodes, a conformal layer in contact with the dielectric layer, and a hydrophobic layer in contact with the conformal layer.The devices may comprise a second substrate comprising a top plate; a spacer disposed between the first substrate and the second substrate and defining an electrokinetic workspace; and a voltage source operatively coupled to the matrix electrodes. The method further comprises disposing an aqueous droplet on a first matrix electrode; and providing a differential electrical potential between the first matrix electrode and a second matrix electrode with the voltage source, thereby moving the aqueous droplet in multiple directions in order to repeatedly split the droplet.Alternatively the devices may be open, without a second opposing substrate. Alternatively the devices may have a second substrate which has no electrical connection to the first substrate. The second substrate may be in contact with the aqueous droplets or may not contact the aqueous droplets, and may therefore act as a ‘cover’ to prevent contamination of the droplets but not be in connection thereto.The inventors discovered that contact angle hysteresis arising from high conductivity solutions or solutions deviating from neutral pH can be mitigated by depositing a conformal layer. The method and device can be used when the ionic strength is over 0.1 M and over 1.0 M.The inventors have discovered that contact angle hysteresis on EWoD-based devices arising from high conductivity solutions or solutions deviating from neutral pH can be mitigated bydepositing a thin protective parylene coating in between the insulating dielectric and the hydrophobic coating.The ability to robustly actuate high ionic strength solutions for extended periods of time offers great utility to those wishing to conduct certain biochemical processes and experiments. High ionic strength solutions are commonly used as wash buffers to disrupt the interaction of nucleic acids and proteins, for example in the commonly performed chromatin immunoprecipitation (ChIP) assay. High ionic strength solutions can also be used for osmotic cell lysis. Additionally, the culture of marine algae is typically performed in media isotonic with seawater, with an ionic strength of 600-700 mM. A further application of high ionic strength solutions is for the elution of proteins from affinity matrices following purification. High ionic strength buffers are also used in enzymatic nucleic acid synthesis. Multiple high ionic strength solutions (1000 mM monovalent or greater) can be used in enzymatic DNA synthesis processes during both washing and deprotection steps.The dielectric layer may comprise silicon dioxide, silicon oxynitride, silicon nitride, hafnium oxide, yttrium oxide, lanthanum oxide, titanium dioxide, aluminium oxide, tantalum oxide, hafnium silicate, zirconium oxide, zirconium silicate, barium titanate, lead zirconate titanate, strontium titanate, or barium strontium titanate. The dielectric layer may be between 10 nm and 100 pm thick. Combinations of more than one material may be used, and the dielectric layer may comprise more than one sublayer that may be of different materials.Exemplary layers can be seen in applications WO2020226985 and WO2022096882. Dielectric layers of the invention can be deposited on a substrate, for example a substrate including a plurality of electrodes disposed between the substrate and the layered dielectric. In some embodiments, the electrodes are disposed in an array and each electrode is associated with a thin film transistor (TFT). In some embodiments, a hydrophobic layer is deposited on the third layer, i.e., on top of the dielectric stack. In some embodiments, the hydrophobic layer is a fluoropolymer, which can be between 10 and 50 nm thick, and deposited with spin-coating or another coating method. Also described herein is a method for creating a layered dielectric of the type described above. The method includes providing a substrate, depositing a first layer using atomic layer deposition (ALD), depositing a second layer using sputtering, and depositing the third layer using ALD. (The first layer is deposited on the substrate, the second layer is deposited on the first layer, and the third layer is deposited on the second layer). The first ALD layer typically includes aluminium oxide or hafnium oxide and has a thickness between 9 nm and 80 nm. The second sputtered layer can include tantalum oxide or hafnium oxide and has a thickness between 40 nm and 250nm. The third ALD layer typically includes tantalum oxide or hafnium oxide and has a thickness between 5 nm and 60 nm. In some embodiments, the atomic layer deposition comprises plasma-assisted atomic layer deposition. In some embodiments, the sputtering comprises radio-frequency magnetron sputtering. In some embodiments, the method further includes spin coating a hydrophobic material on the third layer.Optionally the dielectric ‘layer’ may include multiple layers. The first layer may include aluminium oxide or hafnium oxide, and have a thickness between 9 nm and 80 nm. The second layer may include tantalum oxide or hafnium oxide, and have a thickness between 40 nm and 250 nm. The third layer may include tantalum oxide or hafnium oxide, and have a thickness between 5 nm and 60 nm. The second and third layers may comprise different materials, for example, the second layer can comprise primarily hafnium oxide while the third layer comprises primarily tantalum oxide. Alternatively, the second layer can comprise primarily tantalum oxide while the third layer comprises primarily hafnium oxide. In some embodiments, the first layer may be aluminium oxide. In preferred embodiments, the first layer is from 20 to 40 nm thick, while the second layer is 100 to 150 nm thick, and the third layer is 10 to 35 nm thick. The thickness of the various layers can be measured with a variety of techniques, including, but not limited to, scanning electron microscopy, ion beam backscattering, X-ray scattering, transmission electron microscopy, and ellipsometry.The conformal layer may comprise a parylene, a siloxane, or an epoxy. It may be a thin protective parylene coating in between the insulating dielectric and the hydrophobic coating. Typically, parylene is used as a dielectric layer on simple devices. In this invention, the rationale for deposition of parylene is not to improve insulation / dielectric properties such as reduction in pinholes, but rather to act as a conformal layer between the dielectric and hydrophobic layers. The inventors find that parylene, as opposed to other similar insulating coatings of the same thickness such as PDMS (polydimethylsiloxane), prevent contact angle hysteresis caused by high conductivity solutions or solutions deviating from neutral pH for extended hours. The conformal layer may be between 10 nm and 100 pm thick. The conformal layer may be between 100 nm and 200 nm thick.Disclosed is a method for splitting an aqueous liquid volume into multiple droplets, comprising:providing an electrokinetic device, including:a first substrate having a matrix of electrodes, wherein each of the matrix electrodes is coupled to a thin film transistor, and wherein the matrix electrodes are overcoated with a functional coating comprising:one or more dielectric layer(s) comprising silicon nitride, hafnium oxide or aluminium oxide in contact with the matrix electrodes,a conformal layer comprising parylene in contact with the dielectric layer, and a hydrophobic layer in contact with the conformal layer;a second substrate comprising a top electrode;a spacer disposed between the first substrate and the second substrate and defining an electrokinetic workspace; anda voltage source operatively coupled to the matrix electrodes;providing a first aqueous volume covering at least first, second and third matrix electrodes; andproviding a series of electrical pulses to the first and second electrodes and a second weaker series of electrical pulses to the third electrodes such that the aqueous volume is split into droplets which cover the first and second electrodes and fail to cover the third electrode.The second substrate may also comprise a second hydrophobic layer disposed on the second substrate. The first and second substrates may be disposed so that the hydrophobic layer and the second hydrophobic layer face each other, thereby defining the electrokinetic workspace between the hydrophobic layers.The present invention can be used to contact adjacent aqueous droplets by disposing a second aqueous droplet on a third matrix electrode and providing a differential electrical potential between the third matrix electrode and the second matrix electrode with the voltage source.The invention further provides an assay, nucleic acid synthesis, nucleic acid assembly, nucleic acid amplification, nucleic acid manipulation, next-generation sequencing library preparation, protein synthesis, or cellular manipulation comprising repeating the method steps described above.The insulator / dielectric may be made of SiC>2, silicon oxynitride, SislSk, hafnium oxide, yttrium oxide, lanthanum oxide, titanium dioxide, aluminium oxide, tantalum oxide, hafnium silicate, zirconium oxide, zirconium silicate, barium titanate, lead zirconate titanate, strontium titanate, barium strontium titanate, parylene siloxane, epoxy or a mixture thereof. The insulator / dielectric layer has a thickness of 10-10,000 nm.The hydrophobic coat may comprise a fluoropolymer such as, for example, Teflon, CYTOP or PTFE. The hydrophobic coating layer may be made of an amorphous fluoropolymer or siloxane or organic silane. The hydrophobic layer has a thickness of 1-1,000 nm.A second substrate is positioned opposite the array of individually controllable elements and the second substrate and the individually controllable elements are separated by a spacer which defines an electrokinetic workspace.In order to promote adhesion between the different layer gaseous precursors are often used. This can be used when the layers are deposited using a spin coating or a dip coating.The EWoD-based devices shown and described below are active matrix thin film transistor devices containing a thin film dielectric coating with a Teflon hydrophobic top coat. These devices are based on devices described in the E Ink Corp patent filing on “Digital microfluidic devices including dual substrate with thin-film transistors and capacitive sensing”, US patent application no 2019 / 0111433, incorporated herein by reference.Described herein are electrokinetic devices, including:a first substrate having a matrix of electrodes, wherein each of the matrix electrodes is coupled to a thin film transistor, and wherein the matrix electrodes are overcoated with a functional coating comprising:a dielectric layer in contact with the matrix electrodes,a conformal layer in contact with the dielectric layer, anda hydrophobic layer in contact with the conformal layer;a second substrate comprising a top electrode;a spacer disposed between the first substrate and the second substrate and defining an electrokinetic workspace; anda voltage source operatively coupled to the matrix electrodes.The electrokinetic devices as described may be used with other elements, such as for example devices for heating and cooling the device or reagent cartridges for the introduction of reagents as needed. The workspace is defined between the two opposing hydrophobic surfaces separated by the spacer.The devices can be used for any biochemical assay process involving high solute (ionic) strength solutions where the high concentration of ions would otherwise degrade and prevent use of prior art devices. The devices are particularly advantageous for processes involvingthe synthesis of biomolecules such as for example nucleic acid synthesis, for example using template independent strand extensions, or cell-free protein expression using a population of different nucleic acid templates.FIGURESFigure 1 shows a schematic representation of a droplet being split laterally into two equal sized droplets. The splitting pattern splits rectangles along their shortest axis by moving the ends laterally (up and down rather than apart). White colour represents the pixel electrodes which are turned ON and black represents the OFF electrodes.Figure 2 shows a schematic representation of a droplet being split laterally into four equal sized droplets. The splitting pattern splits rectangles along their shortest axis by moving the ends laterally (up and down rather than apart). White colour represents the pixel electrodes which are turned ON and black represents the OFF electrodes.Figure 3 shows electrode actuation pattern for lateral split method (white shows electrodes ON, black is OFF) of 14x14 pixel droplet into two 20x5 pixel droplets. The square droplet (a) is resized to the rectangular shape (b) which is followed by the lateral (sliding) motion of two halves toward the perpendicular direction of the resized droplet (c-d). It is observed as a controlled split of the initial droplet into two even halves. The reliability of the method relies in maximized radiuses of curvature for both droplets (R1 and R2). It minimizes the Laplace pressure from both opposite corners which is especially essential if two droplets are not identical. It reduces the flow of the aqueous phase from the smaller droplet (smaller radius of curvature and higher Laplace pressure) toward the larger droplet (larger radius of curvature and smaller Laplace pressure). This can occur for example if the device experiences lack of applied voltage to the pixels, for example due to reloading images etc. White colour represents the pixel electrodes which are turned ON and black represents the OFF electrodes.Figure 4 shows splitting of 4x4 pixel droplets into two 3x3 pixel droplets using sliding splitting method:a: 4x4 pixel droplets before splittingb: droplets are resized to 6x3 pixels to initiate splittingc: sliding splitting into two 3x2 pixels dropletsd: an additional area is actuated to finalize splittinge: resized and rearranged 3x3 pixels dropletsWhite colour represents the pixel electrodes which are turned ON and black represents the OFF electrodes.Figure 5 shows a split of an array of 192 7x7 pixel droplets to 3845x5 pixel droplets using sliding split:a: 7x7 pixels droplets before splittingb: droplets are resized to 16x3 pixels to initiate splittingc-d: sliding splitting into two 8x3 pixel dropletse: resized and rearranged 5x5 pixel droplets.and their associated actuation patterns.White colour represents the pixel electrodes which are turned ON and black represents the OFF electrodes.Figure 6 shows a split from 14x14 pixel droplets to four 7x7 pixel droplets:la, 2a: 14x14 pixel droplets before splittinglb, 2b: droplets are resized to 40x5 pixels to initiate splitting1c-d, 2c-d: sliding splitting into two 20x5 pixel droplets1 e-f, 2e-f: another sliding splitting into 10x5 pixel droplets1g, 2g: resized and rearranged 7x7 pixel dropletsand their associated actuation patterns (3a-g).Top and middle panel show images of 14 x 14 aqueous reagent undergoing splitting at room temperature and 37 °C and the bottom panel shows actuation pattern. White colour represents the pixel electrodes which are turned ON and black represents the OFF electrodes.Figure 7 shows high throughput 7x7 pixel droplets dispensed using sliding split method: 1a-c: formation of 14x42 pixels daughter reservoirs to dispense twelve 7x7 pixels droplets 1d-e: split of daughter reservoirs into three 14x14 pixel droplets1 f-h: following split into 7x14 pixel droplets1 i-j: last split into 7x7 pixel dropletsand their associated actuation patterns (2a-h).White colour represents the pixel electrodes which are turned ON and black represents the OFF electrodes.Figure 8 shows static images from a video of aqueous droplet containing magnetic particles being split on an EWoD device. The splitting pattern splits a rectangle along its shortest axisby moving the ends and central parts laterally (up and down rather than apart). The rectangle can be split directly into four droplets (squares) using lateral movements.Figure 9 shows the spacing requirement of a split array. The repeated splitting allows generation of a large number of droplets in a space similar to the starting space of the initial droplet.Figure 10 depicts an array of individually controllable elements forming an electrode array 202. Figure 10 is a diagrammatic view of an exemplary driving system 200 for controlling droplet operation by an AM-EWoD propulsion electrode array 202. The AM-EWoD driving system 200 may be in the form of an integrated circuit adhered to a support plate. The elements of the EWoD device are arranged in the form of a matrix having a plurality of data lines and a plurality of gate lines. Each element of the matrix contains a TFT for controlling the electrode potential of a corresponding electrode, and each TFT is connected to one of the gate lines and one of the data lines. The electrode of the element is indicated as a capacitor Cp. The storage capacitor Cs is arranged in parallel with Cp and is not separately shown in Figure 10. The controller shown comprises a microcontroller 204 including control logic and switching logic. It receives input data relating to droplet operations to be performed from the input data lines 22. The microcontroller has an output for each data line of the EWoD matrix, providing a data signal. A data signal line 206 connects each output to a data line of the matrix. The microcontroller also has an output for each gate line of the matrix, providing a gate line selection signal. A gate signal line 208 connects each output to a gate line of the matrix. A data line driver 210 and a gate line driver 212 is arranged in each data and gate signal line, respectively. The figure shows the signals lines only for those data lines and gate lines shown in the figure. The gate line drivers may be integrated in a single integrated circuit. Similarly, the data line drivers may be integrated in a single integrated circuit. The integrated circuit may include the complete gate driver assembly together with the microcontroller. The integrated circuit may be integrated on a support plate of the AM-EWoD device. The integrated circuit may include the entire AM-EWoD device driving system. The data line drivers provide the signal levels corresponding to a droplet operation. The gate line drivers provide the signals for selecting the gate line of which the electrodes are to be actuated. As illustrated in Figure 10, traditional AM-EWoD cells use line-at-a-time addressing, in which one gate line n is high while all the others are low. The signals on all of the data lines are then transferred to all of the pixels in row n. At the end of the line time gate line n signal goes low and the next gate line n+1 goes high, so that data for the next line is transferred to the TFT pixels in row n+1. This continues with all of the gate lines being scanned sequentially so the whole matrix is driven. This is the same method that is used in almost all AM-LCDs, such asmobile phone screens, laptop screens and LC-TVs, whereby TFTs control the voltage maintained across the liquid crystal layer, and in AM-EPDs (electrophoretic displays).Figure 11 shows a histogram of droplet volume after splitting (measured as fluorescence intensity). The bimodal distribution arises from overactuation of the aqueous phase and the aspect ratio of the elongated droplet. The 8x8 droplet is resized to 12x6 and then to split droplets of 6x6.Figure 12 shows a histogram of droplet volume after splitting (measured as fluorescence intensity) using optimised aspect ratio of 16x4. Using an elongated volume to split means a bimodal distribution is not seen and the droplets have a low coefficient of variation (CV).Figure 13 shows a method for accurately forming rectangular volumes without overloading. An actuated meniscus is used to control the volume of the rectangles prior to splitting. The script generates a smooth transition from an initial step with the daughter reservoir partially protruding from the mother reservoir through to full detachment while keeping the full actuated area constant at all times by resizing the mother reservoir iteratively. The figure shows snapshots during the process of forming rectangular volumes. The middle of the figure describes an ideal case where the meniscus between mother and daughter reservoir is continuous and the bottom sketch shows the stepwise approximation used in the algorithm. Once the daughter reservoir is formed, it is stepwise shifted forward one pixel column at a time. The mother reservoir is resized to match, with a single row at its back (height f) resized to adjust the total volume. As the daughter reservoir moves forward, different conditions are encountered depending on whether the near end of the daughter reservoir is located inside the mother reservoir proper or within the meniscus. This process is complete once the daughter reservoir has travelled far enough forward to sit at the end of the meniscus. In the next steps, the meniscus switches shape from undulating to fully concave. Then, a bridge (here of width I = 4 px) is formed and its width is gradually reduced until the daughter reservoir is detached. The volumes taken up by each fluid area are shown as calculations.Figure 14 shows results with and without meniscus actuation. Without meniscus actuation (top), several of the droplets dispensed are overfilled. Using meniscus actuation to control the volume of the initial rectangle, the split volumes are accurate and not overfilled (bottom).Figure 15 shows the movement of an aqueous droplet from an EWoD region with a weaker actuation pattern (1,0,0, 0,-1) to a stronger actuation pattern (1,-1). Top image shows whenthe actuation duty cycle is identical and image below shows when differential electrowetting is used. 1, -1 and 0 corresponds to 15V, -15V and 0V respectively.Figure 16 shows an actuation pattern for improved splitting. The central part of the elongated volume is actuated with a weaker pattern (1,0,0, 0,-1) (lighter shading pixel areas) than the ends of the liquid volume (1,-1) (heavier shading). Splitting using this pattern gives improved uniformity over simply turning off the central regions. 1, -1 and 0 corresponds to 15V, -15V and 0V respectively.Figure 17 shows differential electrowetting for splitting.Figure 18 shows a zoomed in view of an actuation pattern for reservoir with a marker; where the marker is actuated with a lower duty cycle actuation. Grey is (1,0,0, 0,-1), white is (1,-1) and black is no actuation. 1, -1 and 0 corresponds to 15V, -15V and 0V respectively.Figure 19 shows four different types of sliding split actuation pattern using differential electrowetting where light gray is actuated with a weaker pattern (1,0,0, 0,-1), heavier shading with a stronger pattern (1,-1), and black is electrode OFF. Plots show advantage of using differential electrowetting for various split patterns used. BL is baseline sliding split (no differential electrowetting) and A, B, C, D use differential electrowetting with actuation patterns as shown in the top image. R1, R2 correspond to two dataset with relaxation at the end of the actuation cycle and C1 and C2 is without relaxation.Figure 20 shows movement of droplets using differential electrowetting, only the leading edge is actuated with stronger pattern (1,-1) compared to the rest of the aqueous phase which is actuated with a weaker pattern (1,0,0, 0,-1). This is beneficial to reduce the fouling and to keep the contents mixed with continuous movement.Figure 21 shows a sequence of images generating a single pixel droplet. The neck has a differential actuation at the center to hold preferentially a small volume of liquid and controlling the shape of the long neck into 2 distinct necks with a bulge that will create the single pixel droplet. When the 2 necks snap, the bulk of the liquid leaves with the two ends but the differential bulge at the center creates a central droplet of controlled size. The generation of single pixel droplets is aided by the use of differential wetting in the elongated neck. The larger ends allow for generation of enough force to break off a droplet and the differential control of the bulge in the neck allows for breaking off the central drop of volume much smaller than would be possible by simple elongation of the 2 ends. The starting drop is 3x13 pixelsand the ends are not smaller than 3x3 pixels to exert enough pull to split off such a small droplet. Without the extra differential driving of the center region of the neck, the continued pulling of the 2 ends would normally split the neck approximately in the middle and generate 2 droplets with about 20 pixels each. Controlling the differential voltage at the center of the neck creates a capability to generate a third droplet of controlled and much smaller size along with the 2 endsFigure 22 shows a traditional microfluidic device including a common top electrode.Figure 23 is a schematic diagram of a TFT architecture for a plurality of propulsion electrodes of an EWoD device.Figure 24 is a schematic diagram of showing the pixels of an array showing as conventional actuation pattern where the pixels are either on (positively or negatively polarized relatively to common top electrode) or off underneath the actuation area of the droplet. Adjacent electrodes have the same charge (or zero charge). The actuated pixels are shown as paler shading, the darker shading represents no actuation. The paler shading has the same pattern, showing common driving waveforms across the whole droplet.Figure 25 is a schematic diagram of a pattern of three patterns the invention where the pixels are actuated in rows or columns to generate a circuit through the droplet and thereby cause movement. The actuated pixels are shown as paler shading, the darker shading represents no actuation. The paler shading has varying patterns between adjacent pixels, showing the use of different driving waveforms under the droplet.Figure 26 is a circuit drawing of a conventional actuation using a common top plate electrode.Figure 27 is a circuit diagram of the invention showing an adjacent electrode circuit. Different electrodes under the droplet are differently charged.Figure 28 illustrates droplet dispensing using actuation patterns of the invention. Figure 28 shows the start and Figure 29 the end of a series of droplet handling patterns. The baseline patterns shows poor dispensing and a failure to split.Figure 29 shows split droplet and dispensed droplets using the actuation patterns of the invention.Figure 30 shows construction of a device with a coated plastic top plate.Figure 31 shows droplet generation on a device with a coated plastic top plate.Figure 32 is a flow chart of a method for implementing a droplet operation on a microfluidic device of the present application.Figure 33 shows images comparing top plate corrosion with and without top plate driving. R1 shows adjacent electrode driving and little top plate corrosion. When the top plate electrode is used as part of the driving circuit, the aqueous reagents cause corrosion of the top plate (R2).Figure 34 shows an open device having no top plate. The device contain the TFT arrays and a layer of base fluid. Droplets can be dispensed directly into the base fluid.Figure 35 shows dispensing of droplets from the reagents of Figure 34 in a device with no top plate. The droplets can be dispensed from reservoirs using alternating charging of the pixel electrodes.Applications of the inventionThe invention can be used in a myriad of different applications. In particular the invention can be used to move cells, nucleic acids, nucleic acid templates, proteins, initiation oligonucleotide sequences for nucleic acid synthesis, beads, magnetic beads, cells immobilised on magnetic beads, or biopolymers immobilised on magnetic beads.In these applications the steps of disposing an aqueous droplet having an ionic strength on a first matrix electrode and providing a differential electrical potential may be repeated many times. They may be repeated over 1000 times or over 10,000 times, sometimes over a 24 hour period.Nucleic acid syntheses applicationsThe present method can be used in the synthesis of nucleic acids, such as phosphoramidite-based nucleic acid synthesis, templated or non-templated enzymatic nucleic acid synthesis, or more specifically, terminal deoxynucleotidyl transferase (TdT) mediated addition of 3'-O-reversibly terminated nucleoside 5'-triphosphates to the 3'-end of 5'-immobilized nucleic acids. During enzymatic nucleic acid synthesis, the following steps are taken on the instrument:1. Addition solution containing TdT, optionally pyrophosphatase (PPiase), 3'-O- reversibly terminated dNTPs, and required buffer (including salts and necessary reaction components such as metal divalents) is brought to a reaction zone containing an immobilized nucleic acid, where the nucleic acid is immobilized on a surface such as through magnetic beads via a covalent linkage to the 5’ terminus of the nucleic acid. The initial immobilized nucleic acid may be known as an initiator oligonucleotide and comprises N nucleotides, for example 3-100 nucleotides, preferably 10-80 nucleotides, and more preferably 20-65 nucleotides. Initiator oligonucleotides may contain a cleavage site, such as a restriction site or a non-canonical DNA base such as II or 8-oxoG. Addition solution may optionally contain a phosphate sensor, such as E. coli phosphate-binding protein conjugated to MDCC fluorophore, to assess the quality of nucleic acid synthesis as a fluorescent output. dNTPs can be combined in ratios to make DNA libraries, such as NNK syntheses.2. Wash solution, either in bulk or in discrete droplets, is applied to reaction zones to wash away the addition solution. Wash solution typically has a high solute concentration (>1 M NaCI).3. Deprotection solution, either in bulk or in discrete droplets, is applied to reaction zones to deprotect the 3'-O-reversible terminator added to the immobilized nucleic acids in the immobilized nucleic acid zone in step I. Deprotection solution typically has a high solute concentration.4. Wash solution, either in bulk or in discrete droplets, is applied to reaction zones to wash away the deprotection solution.5. Steps l-l V are repeated until desired sequences are synthesized, for example steps l-l V are repeated 10, 50, 100, 200 or 1000 times.The present method can be used in the preparation of oligonucleotide sequences, either via synthesis or assembly. The device allows synthesis and movement of defined sequences. Using the present method the initiation sequences can be modified at a specific location above an electrode and the extended oligonucleotides prepared. The initiation sequences at different locations can be exposed to different nucleotides, thereby synthesising different sequences in different regions of the electrokinetic device.After synthesis of a defined population of different sequences in different regions of the electrokinetic device, the sequences can be further assembled in longer contiguous sequences by joining two or more synthesised strands together.Described herein is a method for preparing a contiguous oligonucleotide sequence of at least 2n bases in length comprising taking the electrokinetic device as described herein having a plurality of immobilised initiation oligonucleotide sequences, one or more of which contains a cleavage site, using the initiation oligonucleotide sequences to synthesise a plurality of immobilised oligonucleotide sequences of at least n bases in length, using cycles of extension of reversibly blocked nucleotide monomers, selectively cleaving at least two of the immobilised oligonucleotide sequences of least n bases in length into a reaction solution whilst leaving one or more of the immobilised oligonucleotide sequences attached, hybridizing at least two of the cleaved oligonucleotides to each other, to form a splint, and hybridizing one end of the splint to one of the immobilized oligonucleotide sequences and joining at least one of the cleaved oligonucleotides to the immobilised oligonucleotide sequences, thereby preparing a contiguous oligonucleotide sequence of at least 2n bases in length.The steps of synthesis and assembly may involve high solute concentrations where the ionic strength would degrade the devices without the protecting conformal layer.The method of moving aqueous droplets may also be used to help facilitate cell-free expression of peptides or proteins. In particular, droplets containing a nucleic acid template and a cell-free system having components for protein expression in an oil-filled environment can be moved using a method of the invention in the described electrokinetic device.The present invention can be used to automate the movements of droplets in a cartridge. For example, droplets intended for analysis can be moved according to the present invention. The present invention could be incorporated into a cartridge used for local clinician diagnostics. For example it could be used in conjunction with nucleic acid amplification testing (NAAT) to determine nucleic acid targets in, for example, genetic testing for indications such as cancer biomarkers, pathogen testing for example detecting bacteria in a blood sample or virus detection, such as a coronavirus, e.g. SARS-CoV-2 for the diagnosis of COVID-19.The device may be thermocycled to enable nucleic acid amplification, or the device may be held at a desired temperature for isothermal amplification. Having different sequences synthesised in different regions of the device allows multiplex amplification using different primers in different regions of the device.Furthermore the invention can be used in conjunction with next generation sequencing in which DNA is synthesised by the addition of nucleotides and large numbers of samples aresequenced in parallel. The present invention can be used to accurately locate the individual samples used in next generation sequencing.The invention can be used to automate library preparation for next generation sequencing. For example the steps of ligation of sequencing adaptors can be carried out on the device. Amplification of a selective subset of sequences from a sample can then have adaptors attached to enable sequencing of the amplified population.Protein Expression ApplicationsThe method of moving aqueous droplets may also be used to help facilitate cell-free expression of peptides or proteins. In particular, droplets containing a nucleic acid template and a cell-free system having components for protein expression in an oil-filled environment can be moved using a method of the invention in the described electrokinetic device.Proteins are biological macromolecules that maintain the structural and functional integrity of the cell, and many diseases are associated with protein malfunction. Protein purification is a fundamental step for analysing individual proteins and protein complexes and identifying interactions with other proteins, DNA or RNA. A variety of protein purification strategies exist to address desired scale, throughput and downstream applications. However, protein production can be challenging for many reasons. One major challenge is finding a suitable expression system, for example sourced from mammalian, bacterial, fungal, or plant cells. This can take months of work.Cell-free protein synthesis (CFPS), also known as coupled or uncoupled in-vitro transcription and translation, is the production of peptides or proteins using biological machinery in a cell-free system, that is, without the use of living cells. The CFPS environment is not constrained within a cell wall or limited by conditions necessary to maintain cell viability, and enables the rapid production of any desired protein from a nucleic acid template, usually plasmid DNA or RNA from an in-vitro transcription. CFPS has been known for decades, and many commercial systems are available. Cell-free protein synthesis encompasses systems based on crude lysate (Cold Spring Harb Perspect Biol. 2016 Dec; 8(12): a023853) and systems based on reconstituted, purified molecular reagents, such as the PURE system for protein production (Methods Mol Biol. 2014; 1118: 275-284). CFPS requires significant concentrations of biomacromolecules, including DNA, RNA, proteins, polysaccharides, molecular crowding agents, and more (Febs Letters 2013, 2, 58, 261-268).Split detectors such as split green fluorescent protein (GFP) systems are known for use in protein expression. A protein of interest having a GFP subcomponent can be detected by complementing with a detector species having the remainder of the GFP. Cabantous and Waldo describe such a system (In-vivo and in-vitro protein solubility assays using split GFP (NATURE METHODS | VOL.3 NO.10 | OCTOBER 2006 | 845). The system described relies on expression in cells, from which the proteins of interest are lysed and then exposed to the detector in order to measure the level of expression.WO2022 / 038353 describes cell-free expression of a protein having a GFP tag in the presence of a GFP detector species to measure signal as expression progresses.Many proteins are expressed containing endogenous solubility factors. However factors such as for example maltose binding protein (MBP), Small Ubiquitin-like Modifier (SUMO), Glutathione S-transferase (GST) or thioredoxin (TRX) have a substantial size and may have undesirable effects on the proteins of interest to which they are attached. Additionally expression of large solubility elements uses the reagent resources during expression. Such sequences also take time to fold before acting at solubility enhancers, hence synthesising proteins of interest with solubility enhancing sequences attached thereto is far from ideal.To date protein purification and analysis typically requires complex analysis techniques involving electrophoresis or requiring purified proteins. The inventors herein have developed simplified protein analysis and purification methods allowing multiple ways of characterising expressed proteins in their crude form.One of the current challenges for cell-free protein synthesis is to increase the soluble yield of the expressed and purified proteins and to avoid aggregation or insolubility.Disclosed herein is a method for the real-time monitoring of in-vitro protein synthesis comprising1. In-vitro transcription and translation of a protein of interest fused to a peptide tag; and2. monitoring the presence of the peptide tag using a further polypeptide which in the presence of the peptide tag produces a detectable signal.Disclosed herein is a method for the monitoring of cell-free protein synthesis in a droplet on a digital microfluidic device comprisinga. cell-free transcription and translation of a protein of interest fused to a peptide tag; andb. monitoring the presence of the peptide tag using a further polypeptide which in the presence of the peptide tag produces a detectable signal.The use of the terms “in-vitro” and “cell-free” may be used interchangeably herein.The detectable signal may be for example fluorescence or luminescence. The detectable signal may also be caused by the binding of a ligand to the complemented oligopeptide, peptide, or polypeptide tag fused to the protein of interest.The detectable signal may also be caused by the binding of the polypeptide to the protein of interest fused to a His-tag.Any in-vitro transcription and translation may be used, for example extract-based systems derived from rabbit reticulocyte lysate, human lysate, Chinese Hamster Ovary lysate, a wheat germ, HEK293 lysate, E. coli lysate, yeast lysate.Alternatively the in-vitro transcription and translation may be assembled from purified components, for example a system of purified recombinant elements (PURE).The in-vitro transcription and translation may be coupled or uncoupled.The peptide tag may be one component of a fluorescent protein and the further polypeptide a complementary portion of the fluorescent protein. The fluorescent protein could include sfGFP, GFP, ccGFP, eGFP, deGFP, frGFP, eYFP, eBFP, eCFP, Citrine, Venus, Cerulean, Dronpa, DsRED, mKate, mCherry, mRFP, FAST, SmURFP, miRFP670nano. For example the peptide tag may be GFPn and the further polypeptide GFP1-10. The peptide tag may be one component of sfCherry. The peptide tag may be sfCherryn and the further polypeptide sfCherryi-10. The peptide tag may be CFASTn or CFAST10 and the further polypeptide NFAST in the presence of a hydroxybenzylidene rhodanine analog.The peptide tag may also be one component of a protein that forms a detectable substrate, such as a luminescent or colorigenic substrate. The protein could include beta-galactosidase, beta-lactamase, or luciferase.The protein may be fused to multiple tags. For example the protein may be fused to multiple GFPn peptide tags and the synthesis occurs in the presence of multiple GFP1-10 polypeptides. For example the protein may be fused to multiple sfCherryn peptide tags and the synthesis occurs in the presence of multiple sfCherryi-10 polypeptides. The protein of interest may be fused to one or more sfCherryn peptide tags and one or more GFPn peptide tags and the synthesis occurs in the presence of one or more GFP1-10 polypeptides and one or more sfCherryi-10 polypeptides.Any protein of interest may be synthesised. The protein may be an enzyme, for example a terminal deoxynucleotidyl transferase (TdT) enzyme or a truncated version thereof or the homologous amino acid sequence of a terminal deoxynucleotidyl transferase (TdT) enzyme in other species or the homologous amino acid sequence of Polp, Poip, PolA, and PolQ of any species or the homologous amino acid sequence of X family polymerases of any species.Protein expression typically requires an ample supply of oxygen. The most convenient and high yielding way to power CFPS is via oxidative phosphorylation where O2 serves as the final electron acceptor; however, there are other ways that involve replenishing with energy molecules not involved in oxidative phosphorylation. In a confined microfluidic or digital microfluidic system of droplets, insufficient oxygen is available to enable efficient protein synthesis.Described herein are improved methods allowing for the cell-free expression of peptides or proteins in a digital microfluidic device. Included is a method for the cell-free expression of peptides or proteins in a microfluidic device wherein the method comprises one or more droplets containing a nucleic acid template (i.e. , DNA or RNA) and a cell-free system having components for protein expression in an oil-filled environment, and moving said droplets using electrowetting. The components for the cell-free protein synthesis droplet can be premixed prior to introduction to or mixed on the digital microfluidic device.The droplet can be repeatedly moved for at least a period of 30 minutes whilst the protein is expressed. The droplet can be repeatedly moved for at least a period of two hours whilst the protein is expressed. The droplet can be repeatedly moved for at least a period of twelve hours whilst the protein is expressed. The act of moving the droplet allows oxygen to be supplied to the droplet and dispersed throughout the droplet. The act of moving improves the level of protein expression over a droplet which remains static.The droplet can be moved using any means of electrowetting. The droplet can be moved using electrowetting-on-dielectric (EWoD). The electrical signal on the EWoD or optical EWoD device can be delivered through segmented electrodes, active-matrix thin-film transistors, or digital micromirrors.The filler fluid in the device can be any water immiscible liquid. The filler fluid can be mineral oil, silicone oil such as dodecamethylpentasiloxane (DM PS), an alkyl-based solvent such as decane or dodecane, or a fluorinated oil. The filler fluid can be oxygenated prior to or during the expression process.A source of supplemental oxygen can be supplied to the droplets. For example droplets or gas bubbles containing gaseous or dissolved oxygen can be merged with the droplets during the protein expression. Additionally, a source of supplemental oxygen can be found by oxygenating the oil that is used as the filler medium. It is well-known in the art that oils such as hexadecane, HFE-7500, and others can be oxygenated to support the oxygen requirements of cell growth, especially E. coliceW growth (RSC Adv., 2017, 7, 40990-40995). Oxygenation can be achieved by aerating the oil with pure oxygen or atmospheric air.The droplets can be formed before entering the microfluidic device and flowed into the device. Alternatively the droplets can be merged on the device. Included is a method comprising merging a first droplet containing a nucleic acid template such as a plasmid with a second droplet containing a cell-free extract having the components for protein expression to form a combined droplet capable of cell-free protein synthesis.The droplets can be split on the device either before or after expression. Included herein is a method further comprising splitting the aqueous droplet into multiple droplets. If desired the split droplets can be screened with further additives. Included is a method wherein one or more of the split droplets are merged with additive droplets for screening.The cell-free expression of peptides or proteins can use a cell lysate having the reagents to enable protein expression. Common components of a cell-free reaction include an energy source, a supply of amino acids, cofactors such as magnesium, and the relevant enzymes. A cell extract is obtained by lysing the cell of interest and removing the cell walls, DNA genome, and other debris by centrifugation. The remains are the cell machinery including ribosomes, aminoacyl-tRNA synthetases, translation initiation and elongation factors, nucleases, etc. Once a suitable nucleic acid template is added, the nucleic acid template can be expressed as a peptide or protein using the cell derived expression machinery.Any particular nucleic acid template can be expressed using the system described herein. Three types of nucleic acid templates used in CFPS include plasmids, linear expression templates (LETs), and mRNA. Plasmids are circular templates, which can be produced either in cells or synthetically. LETs can be made via PCR. While LETs are easier and faster to make, plasmid yields are usually higher in CFPS. mRNA can be produced through in-vitro transcription systems. The methods use a single nucleic acid template per droplet. The methods can use multiple droplets having a different nucleic acid template per droplet.An energy source is an important part of a cell-free reaction. Usually, a separate mixture containing the needed energy source, along with a supply of amino acids, is added to the extract for the reaction. Common sources are phosphoenolpyruvate, acetyl phosphate, and creatine phosphate. The energy source can be replenished during the expression process by adding further reagents to the droplet during the process.The cell-free extract having the components for protein expression includes everything required for protein expression apart from the nucleic acid template. Thus the term includes all the relevant ribosomes, enzymes, initiation factors, nucleotide monomers, amino acid monomers, metal ions and energy sources. Once the nucleic acid template is added, protein expression is initiated without further reagents being required.Thus the cell-lysate can be supplemented with additional reagents prior to the template being added. The cell-free extract having the components for protein expression would typically be produced as a bulk reagent or ‘master mix’ which can be formulated into many identical droplets prior to the distinct template being separately added to separate droplets. Common cell extracts in use today are made from E. coli (ECE), rabbit reticulocytes (RRL), wheatgerm (WGE), insect cells (ICE) and Yeast Kluyveromyces (the D2P system). All of these extracts are commercially available.Rather than originating from a cell extract, the cell-free system can be assembled from the required reagents. Systems based on reconstituted, purified molecular reagents are commercially available, for example the PURE system for protein production, and can be used as supplied. The PURE system is composed of all the enzymes that are involved in transcription and translation, as well as highly purified 70S ribosomes. The protein synthesis reaction of the PURE system lacks proteases and ribonucleases, which are often present as undesired molecules in cell extracts.Once the CFPS reagents have been enclosed in the droplets, additional reagents can be supplied by merging the original droplet with a second droplet. The second droplet can carry any desired additional reagents, including for example oxygen or ‘power’ sources, or test reagents to which it is desired to expose to the expressed protein.The droplets can be aqueous droplets. The droplets can contain an oil immiscible organic solvent such as for example DMSO. The droplets can be a mixture of water and solvent, providing the droplets do not dissolve into the bulk filler liquid.The droplets containing the cell-free extract having the components for protein expression will therefore typically be in the oil filled environment before the nucleic acid templates are added to the droplets. The templates can be added by merging droplets on the microfluidic device. Alternatively, the templates can be added to the droplets outside the device and then flowed into the device for the expression process. For example the expression process can be initiated on the device by increasing the temperature. The expression system typically operates optimally at temperatures above standard room temperatures, for example at or above 29 °C.The expression process typically takes many hours. Thus the process should be left for at least 30 minutes or 1 hour, typically at least 2 hours. Expression can be left for at least 12 hours. During the process of expression the droplets should be moved within the device. The moving improves the process by mixing the reagents and ensuring sufficient oxygen is available within the droplet. The moving can be continuous, or can be repeated with intervening periods of non-movement.Thus the aqueous droplet can be repeatedly moved for at least a period of 30 minutes or one hour whilst the protein is expressed. The aqueous droplet can be repeatedly moved for at least a period of two hours whilst the protein is expressed. The aqueous droplet can be repeatedly moved for at least a period of twelve hours whilst the protein is expressed. The act of moving the droplet allows mixing within the droplet, and allows oxygen or other reagents to be supplied to the droplet. The act of moving improves the level of protein expression over a droplet which remains static.The filler fluid in the device can be any water immiscible, non-ionic or hydrophobic liquid. The oil can be mineral oil, silicone oil such as dodecamethylpentasiloxane (DMPS), an alkyl-based solvent such as decane or dodecane, or a fluorinated oil.A source of supplemental oxygen can be supplied to the droplets. For example droplets or gas bubbles containing gaseous or dissolved oxygen can be merged with the aqueous droplets during the protein expression. Alternatively the source of oxygen can be a molecular source which releases oxygen. Alternatively the droplets can be moved to an air / liquid boundary to enable increased diffusion of oxygen from a gaseous environment. Alternatively the oil can be oxygenated.The droplet can be formed before entering the microfluidic device and flowed into the device. Alternatively the droplets can be merged on the device. Included is a method comprising merging a first droplet containing a nucleic acid template such as a plasmid with a second droplet containing a cell-free system having the components for protein expression to form the droplet.The droplets can be split on the device either before, during or after expression. Included herein is a method further comprising splitting the droplet into multiple droplets. If desired the split droplets can be screened with further additives. Included is a method wherein one of more of the split droplets are merged with additive droplets for screening.Through an affinity tag, such as a FLAG-tag, HIS-tag, GST-tag, MBP-tag, STREP-tag, or other form of affinity tag, CFPS-expressed proteins can be immobilized to a solid-support affinity resin and fresh batches of CFPS reagent can be delivered over the said resin. Thus, renewed reagents can be used to carry out protein synthesis, closely mimicking industrial methods of continuous flow (CF) and continuous exchange (CE) CFPS. By mimicking CF-and CE-CFPS, users can scale up their CFPS production methods.Droplets can also contain additives to reduce the effects of biofouling on digital microfluidic surfaces. Specifically, droplets containing CFPS components can also contain additives such as surfactants or detergents to reduce the effects of biofouling on the hydrophobic or superhydrophobic surface of a digital microfluidic device (Langmuir 2011 , 27, 13, 8586-8594). Such droplets may use antifouling additives such as TWEEN 20, Triton X-100, and / or Pluronic F127. Specifically, droplets containing CFPS components may contain TWEEN 20 at 0.1% v / v, Triton X-100 at 0.1% v / v, and / or Pluronic F127 at 0.08% w / v.Rather than adding surfactants to the aqueous sample, it is instead possible to add surfactant, such as a sorbitan ester such as Span85 (e.g. Sorbitan trioleate, Sigma Aldrich, SKU 8401240025), to the filler liquid. This has the advantages of enabling CFPS reactions to proceed on-DMF without dilution or adulteration. Additionally, it simplifies the samplepreparation procedure for setting up the reactions, increasing the ease of use and the consistency of results. Using 1% w / w Span85 in dodecane allows for dilution-free CFPS reactions on-DMF, as well as dilution-free detection of the expressed non-fluorescent proteins. Other surfactants besides Span85, and oils other than dodecane could be used. A range of concentrations of Span85 could be used. Surfactants could be nonionic, anionic, cationic, amphoteric or a mixture thereof. Oils could be mineral oils or synthetic oils, including silicone oils, petroleum oils, and perfluorinated oils. Surfactants can have a detrimental effect on (1) the CFPS reactions and (2) the efficiency of the detection system (if the detection system involves complementation of a tag and detector). For example, by performing the CFPS reaction on-DMF with oil-surfactant mix, the detection of the expressed protein can also proceed without dilution and without adding aqueous surfactant. It has been shown that surfactants reduce the efficiency of some detection systems, including but not limited to the Split GFP (e.g. GFP11 / GFP1-10) system, so removing surfactants from the reagent mix and instead adding them to the oil can be beneficial.The peptide tag can be attached to the C or N terminus of the protein. The peptide tag may be one component of a green fluorescent protein (GFP). For example the peptide tag may be GFPn and the further polypeptide GFP1-10. The peptide tag may be one component of sfCherry. The peptide tag may be sfCherryn and the further polypeptide sfCherryi-10.The POI may be expressed with a binding sequence which binds to a detector moiety. In which case a further droplet containing a detector moiety may be added to the droplet containing the POI to create an detectable signal in the combined droplet. The binding sequences may contain four or more amino acids. The binding sequences may contain 4-30 amino acids.The detector moiety may be a protein. The detector moiety may comprise a component of a fluorescent protein such as for example sfGFP or ccGFP.The expressed protein may contain a sequence acting as a solubility enhancer, for example selected from:The detection tag may be one component of a fluorescent protein and the detector reagent a complementary portion of the fluorescent protein. The fluorescent protein could include sfGFP, ccGFP, GFP, eGFP, deGFP, frGFP, eYFP, eBFP, eCFP, Citrine, Venus, Cerulean, Dronpa, DsRED, mKate, mCherry, mRFP, FAST, SmllRFP, miRFP670nano. For example the tag may be GFPn and the detector GFP1-10. The tag may be one component of sfCherry. The tag may be sfCherryn and the detector sfCherryi-10. The tag may be CFASTn or CFAST10 and the detector NFAST in the presence of a hydroxybenzylidene rhodanine analog.The tag may be ccGFPn and the detector ccGFPi- .The expression may be performed using cell-free protein synthesis reagents derived from whole cell extracts. The expression may be performed using cell-free protein synthesis reagents derived from reconstituted systems comprising assembled components for transcription and translation in a system of purified recombinant elements (PURE).Disclosed is a method for expressing proteins in droplets on a digital microfluidic device having a two-dimensional array of planar microelectrodes wherein the proteins have a ccGFPn peptide amino sequence tag, wherein a portion of the droplets contain ccGFPi-during the expression process and a further portion have ccGFPi- added after expression and comparing the level of ccGFPi-n signal from the droplets.The binding moiety for purification may contain four or more amino acids. The binding sequences may contain 4-30 amino acids. The binding moiety may be selected from:Alfa-tag (SRLEEELRRRLTE) (SEQ ID NO: 1)Avi-tag (GLNDIFEAQKIEWHE) (SEQ ID NO: 2)C-tag (EPEA) (SEQ ID NO: 3)Calmodulin-tag (KRRWKKNFIAVSAANRFKKISSSGAL) (SEQ ID NO: 4)Dogtag (DIPATYEFTDGKHYITNEPIPPK) (SEQ ID NO: 5)E-tag (GAPVPYPDPLEPR) (SEQ ID NO: 6)FLAG (DYKDDDDK) (SEQ ID NO: 7)G4T (EELLSKNYHLENEVARLKK) (SEQ ID NO: 8)HA (YPYDVPDYA) (SEQ ID NO: 9)His (HHHHHH) (SEQ ID NO: 10)Isopeptag (TDKDMTITFTNKKDAE) (SEQ ID NO: 11)lanthanide binding tag (LBT) (FIDTNNDGWIEGDELLLEEG) (SEQ ID NO: 12)Myc (EQKLISEEDL) (SEQ ID NO: 13)NE-Tag (TKENPRSNQEESYDDNES) (SEQ ID NO: 14)Poly Glutamate-tag (EEEEEEE) (SEQ ID NO: 15)Poly Arginine-tag (RRRRRRR) (SEQ ID NO: 16)Rho1D4-tag (TETSQVAPA) (SEQ ID NO: 17)SBP-tag (MDEKTTGWRGGHVVEGLAGELEQLRARLEHHPQGQREP) (SEQ ID NO: 18) Sdytag (DPIVMIDNDKPIT) (SEQ ID NO: 19)SH3 (STVPVAPPRRRRG) (SEQ ID NO: 20)SNAC (GSHHW) (SEQ ID NO: 21)Snooptag (KLGDIEFIKVNK) (SEQ ID NO: 22)Softag 1 (SLAELLNAGLGGS) (SEQ ID NO: 23)Softag 3 (TQDPSRVG) (SEQ ID NO: 24)Spot-tag (PDRVRAVSHWSS) (SEQ ID NO: 25)Spytag (AHIVMVDAYKPTK) (SEQ ID NO: 26)S-tag (KETAAAKFERQHMDS) (SEQ ID NO: 27)Strep-tag (AWAHPQPGG) (SEQ ID NO: 28) (AWRHPQFGG) (SEQ ID NO: 29)Strep-tag II (WSHPQFEK) (SEQ ID NO: 30)T7tag (MASMTGGQQMG) (SEQ ID NO: 31)TC-tag (EVHTNQDPLD) (SEQ ID NO: 32)Ty-tag (CCPGCC) (SEQ ID NO: 33)VSV-tag (YTDIEMNRLGK) (SEQ ID NO: 34)Xpress-tag (DLYDDDDK) (SEQ ID NO: 35)The method may be performed on different sequences in parallel. The method may use at least 8 different nucleic acid templates, which may be screened against at least 4 different expression reagents on the same device. The device may be capable of handling many droplets in parallel, for example the device may separately manipulate at least 192 droplets. The expression comparison and purification screen can identify the optimal conditions for expression and purification of the desired protein in its most soluble and stable form.The POI may be expressed with a binding sequence which binds to a detector moiety. In which case a further droplet containing a detector moiety may be added to the droplet containing the POI to create an detectable signal in the combined droplet.The computer readable program instructions may be stored on a non-transitory, tangible computer readable medium. The computer readable storage medium may include one or more of an electronic storage device, a magnetic storage device, an optical storage device, an electromagnetic storage device, a semiconductor storage device, a portable computer disk, a hard disk, a random access memory (RAM), a read-only memory (ROM), an erasable programmable read-only memory (EPROM or Flash memory), a static random access memory (SRAM), a portable compact disc read-only memory (CD-ROM), a digital versatile disk (DVD), a memory stick, a floppy disk.As will be appreciated by one of skill in the art, the invention described herein may be embodied in whole or in part as a method, a data processing system, or a computer program product including computer readable instructions. Accordingly, the invention may take the form of an entirely hardware embodiment or an embodiment combining software, hardware and any other suitable approach or apparatus.Exemplary embodiments of the invention may be implemented as a circuit board which may include a CPU, a bus, RAM, flash memory, one or more ports for operation of connected I / O apparatus such as printers, display, keypads, sensors and cameras, ROM, a communications sub-system such as a modem, and communications media.Experimental DetailsAdhesion promotionAdding 0.5% v / v Silane A-174 to a 1 :1 ratio of isopropanol / water and stirring for 30 seconds formed solution 1. Solution 1 was left to stand for at least 2 hours to fully react and was used within 24 hours. Substrates were immersed in the Solution 1 for 30 minutes, while ensuring the flex strips of the TFT arrays were kept dry. Substrates were removed and air dried for 15 minutes and then cleaned in isopropanol for 15-30 seconds with agitation using tweezers. Substrates were dried with an air gun and stored in a Teflon box for Parylene C coating within 30 hours.Parylene CoatingPrepared substrates (silanised and non-silanised) were arranged face up on a rotating stage alongside a clean glass slide within the deposition chamber of a thoroughly clean SCS Labcoter 2 and the chamber was sealed. 50 mg of Parylene C dimer was weighed into a disposable aluminium boat and loaded into the sublimation chamber. The system was sealed and pumped down to 50 milliTorr before liquid nitrogen was added to the cold trap. The system continued to evacuate throughout the deposition process. The sublimation chamber was heated to 175°C and the heater cycled to maintain a target pressure of 0.1 Torr. The sublimation chamber was connected to the deposition chamber by a pyrolysis zone which was heated to 690°C at a target pressure of 0.5 Torr. The deposition zone remained at ambient temperature, circa 25°C, and around 50 milliTorr. The system was maintained at temperature and pressure for two hours. The system was allowed to return gradually to ambient temperature over 30-40 minutes before the stage and vacuum pump were turned off and the system vented. The samples were removed from the deposition chamber and the coating thickness verified as circa 100 nm by profilometry.Split MethodsInfluence of the aspect ratio on the splitting success and daughter droplet properties (accuracy and CV).Droplets containing 50 iM fluorescein were dispensed into an electrowetting device. Droplets of size 12x6 pixels were split into two 6x6 droplets using a lateral motion. The intensity of the post split droplets is plotted in Figure 11. The post-split droplet sizes show a clear bimodal distribution, confirming that the split is systematically unequal. The overall CV of the droplet intensity of 128 droplets after the split is 32.18%.The splitting is improved is the droplets are further elongated before splitting. Elongation of the aspect ratio (from 12 x 6 to 16 x 4) for split helps in providing uniform split ratio by improving the radius of curvature. Figure 12 shows the droplet intensity after splitting, and indicates the bimodal distribution is removed. The resulting CV across the array is 5.02% when the drops are 8x4 and 4.94% when resized to 6x6. Thus having the split droplets elongated prior to splitting makes splitting of the droplets more even as it increases tolerance against starting volume variations and compensates for reagent that is not actually actuated as it is part of the meniscus during the split.Differential ElectrowettinqFigure 15 shows how EWOD regions with different duty cycles can be used to move a droplet from an EWoD region with a lower duty cycle (lower voltage, lower EW) to a region with a higher duty cycle (higher voltage, higher EW). To obtain the result shown in the figure, twodroplets of 14 x14 pixel electrodes were moved next to each other using electrowetting forces and resized to 22 x 22 pixels electrodes to initiate flow in a preferred direction. (Top L) Actuation pattern for droplets energized with the same duty cycle post resizing (white is 1 ,-1 actuation and black is no actuation). (Top R) Fluorescent aqueous phase shows no preferential movement. (Bottom L) Actuation pattern for droplets energized with the different duty cycle (grey is 1,0,0, 0,-1, white is 1,-1 and black is no actuation) (Bottom R) Fluorescent aqueous phase moving from region of low EWto high EW force. The arrows in the bottom left image show the direction of reagent movement due to differential electrowetting. 1, -1 and 0 corresponds to 15V, -15V and 0V respectively.Symmetric droplet splitting with unknown reagent volumeStretching a droplet in opposing directions with a lower EW force in the middle reduces the ‘snapping of’ of aqueous phase which otherwise negatively affects repeatability and split success. The presence of lower duty cycle patterns guides the aqueous phase to the higher duty cycle pattern i.e. stronger EW regions. Figure 16 shows an actuation pattern for improved symmetric splitting. The central part of the elongated volume is actuated with a weaker pattern (1,0,0, 0,-1) (heavier shading pixel areas) than the ends of the liquid volume (1,-1) (white shading). Splitting using these patterns gives improved uniformity over simply turning off the central regions. 1 , -1 and 0 corresponds to 15 V, -15 V and 0 V respectively.Figure 17 shows the application of differential electrowetting for splitting. The top row illustrates the splitting process, where white is 1,-1, heavy shading is 1,0,0, 0,-1 and black is no actuation. The arrows show the direction of reagent movement due to differential electrowetting. Middle row shows droplets in the left column which were intentionally prepared to contain a reagent volume that is smaller than the actuated area. This constellation, which can result as a consequence of intentional or unintentional earlier droplet operations, can be challenging to address with conventional means and frequently results in split failures or very uneven droplet sizes. Bottom row shows droplet in the left column which are correctly sized. Using differential electrowetting, for correctly sized droplets, the splitting pattern yields 100% success and low CV (right peak in the histogram of Figure 17). Crucially, even for underfilled droplets, the splitting pattern yields 100% success and a relatively uniform splitting ratio.Reagent loading- reducing false positives with marker-based filling indicatorsShaped markers are employed to indicate whether a given reservoir or loaded reagent volume, contains sufficient reagent for the downstream droplet operations. In someinstances, this can result in a false positive, where the marker is fully or partially present, but the reservoir volume is missing reagent in its main body.Using actuation patterns where the marker is actuated with a lower duty cycle than the rest of the body of the reservoir promotes flow of the aqueous phase into the body from the marker first, avoiding false positives of the kind described above. The markers will be filled only if the reservoirs are correctly sized. Correctly formed reservoirs are essential for downstream droplet operations.Figure 18 shows a zoomed in view of an actuation pattern for a reservoir with a marker, where the marker is actuated with a lower duty cycle actuation (heavy shading is 1,0,0, 0,-1, white is 1,-1 and black is no actuation). The arrow shows the direction of reagent movement due to differential electrowetting. Images show red dye with 0.05% F127 in a basefluid of siloxane oil. The middle image shows how an incorrectly formed reservoir is taken as correctly formed (false positive) due to the presence of a marker which is using the same duty cycle as the reservoir body. The last image shows that the use of differential electrowetting removes false positives as the incorrectly filled reservoir (with higher duty cycle) pulls the aqueous phase from lower duty cycle marker as shown in schematic.Where used herein “and / or” is to be taken as specific disclosure of each of the two specified features or components with or without the other. For example “A and / or B” is to be taken as specific disclosure of each of (i) A, (ii) B and (iii) A and B, just as if each is set out individually herein.Unless context dictates otherwise, the descriptions and definitions of the features set out above are not limited to any particular aspect or embodiment of the invention and apply equally to all aspects and embodiments which are described.It will further be appreciated by those skilled in the art that although the invention has been described by way of example with reference to several embodiments. It is not limited to the disclosed embodiments and that alternative embodiments could be constructed without departing from the scope of the invention as defined in the appended claims.Embodiments disclosed herein include:A digital microfluidic device for manipulating aqueous droplets, comprising:(a) a bottom plate array comprising:a thin-film transistor matrix, wherein each transistor of the matrix is operably connected to a gate line, a data line, and a separate propulsion electrode;a plurality of gate lines, wherein each gate line is operably connected to a gate driver; a plurality of data lines, wherein each data line is operably connected to a data driver; a first dielectric layer covering the bottom electrode array; anda hydrophobic upper layer;(b) a controller operably connected to the gate driver and the data driver; and(c) a processing unit operably connected to the controller and programmed to perform a microfluidic driving method, the method comprising:receiving input instructions in the processing unit, the input instructions relating to a droplet operation to be performed by the digital microfluidic system;outputting gate line and data line selecting instructions from the processing unit to the controller;outputting gate line signals from the controller to the gate driver, to drive a first gate line and an adjacent second gate line, andoutputting data line signals from the controller to the data driver, to drive a first data line and an adjacent second data line to simultaneously provide different signals to adjacent electrodes under a single droplet.A digital microfluidic device wherein the device has a top plate opposing the bottom plate.A digital microfluidic device wherein the top plate does not contact the aqueous droplets.A digital microfluidic device wherein the top plate is not in an electrical connection with the bottom plate.A digital microfluidic device wherein the top plate has no electrodes or dielectric coatings.A digital microfluidic device wherein the top plate has electrodes and / or dielectric coatings and is disconnected via a switch.A digital microfluidic device wherein the top plate is a hydrophobic polymer or glass or polymer coated with a hydrophobic layer.A digital microfluidic device wherein the propulsion voltages take the form of providing voltages to the electrodes in parallel stripes or in a checkerboard pattern.A digital microfluidic device wherein stripes are perpendicular to the direction of droplet propulsion.A digital microfluidic device wherein the width of the electrodes of the electrode array is smaller than the gap defining the microfluidic region.A digital microfluidic device wherein each electrode is smaller than 80 .m.A digital microfluidic device wherein the adjacent electrodes are charged with the opposite polarity in the same frame.A digital microfluidic device wherein the adjacent electrodes are charged with +15 V and -15 V in the same frame.A digital microfluidic device wherein the device has a hydrophobic base fluid layer on top of the bottom electrode array containing the aqueous droplets and the device has no top plate such that the hydrophobic base fluid layer is open to atmospheric gas exchange.A digital microfluidic device wherein the electrokinetic device has square electrodes.A digital microfluidic device wherein two adjacent electrodes are supplied with different signals selected from the pulse sequences: 1,-1; 1,0,-1; 1,0, 0,-1; 1 ,0,0, 0,-1 ;1 ,0,-1 ,-1 ; 1 ,-1 ,0,-1; 1,1, 0,0,0, -1,-1; or 1, -1,0, 1,-1.

Claims

1. Claims1. A method for manipulating an aqueous liquid volume on a digital microfluidic device having an array of electrodes, the method comprising simultaneously supplying different electronic pulse sequences to different electrodes underneath the area covered by a single aqueous liquid volume, wherein the different electronic pulse sequences comprise either supplying stronger and weaker voltage pulses or sequences to adjacent electrodes or supplying positive and negative sequences of pulses to adjacent electrodes.

2. The method according to claim 1, wherein the aqueous liquid volume is split into smaller droplets.

3. The method according to claim 2, wherein the aqueous liquid volume is split into two or more smaller droplets by creating a neck of fluid between two parts of the aqueous liquid volume, wherein the electrodes under the neck region are actuated with a weaker pulse sequence than the electrodes under the two resulting droplets.

4. The method according to any one preceding claim, wherein the method comprises splitting the aqueous liquid volume into multiple evenly sized smaller droplets by moving the aqueous liquid volume in opposing lateral directions, wherein the lateral movement is at 90 degrees to the longer axis such that the ends of the aqueous liquid volume move perpendicular to the longer axis.

5. The method according to any one preceding claim, wherein the aqueous liquid volume is rectangular.

6. The method according to any one preceding claim, wherein the smaller droplets are square.

7. The method according to claim 5, wherein each rectangle makes two, three or four smaller droplets.

8. The method according to any one preceding claim, wherein multiple droplets are split simultaneously.

9. The method according to any one preceding claim, wherein the aqueous liquid volume is elongated and split into evenly sized smaller droplets, and the split droplets are then elongated and further split into smaller droplets.

10. The method according to claim 9, wherein each first droplet is split at least twice in order to make at least 4 smaller evenly sized droplets from each of the first droplets.

11. The method according to any one preceding claim wherein the final droplets are less than 250 nL in volume.

12. The method according to any one preceding claim wherein the final droplets occupy 4x4 pixels or fewer of the electrokinetic device per droplet.6813. The method according to any one preceding claim wherein the aqueous liquid volume occupies at least 10x10 pixels of the electrokinetic device per droplet.

14. The method according to any one of claims 1 to 13 for performing droplet based cell- free expression of peptides or proteins, wherein the droplets contain a cell-free system having components for protein expression.

15. The method according to claim 14, wherein the droplets contain a nucleic acid template.

16. The method according to any one preceding claim, wherein the electrokinetic device has square electrodes.

17. The method according to any one preceding claim, wherein the weaker sequence is selected from the pulse sequences:1 ,0,-1 ; 1,0, 0,-1; 1,0,0, 0,-1; 1,0, -1,-1; 1,-1, 0,-1; 1,1, 0,0,0, -1,-1; or 1,-1, 0, 1,-1.

18. The method according to any one preceding claim, wherein the stronger sequence is a sequence 1,-1 without rest pulses.

19. The method according to any one preceding claim, wherein the weaker sequence uses a voltage of less than 15 V and the stronger sequences uses a voltage of 15 V or greater.

20. The method according to any one preceding claim, wherein a first electrode receives +15 V and a second electrode receives -15 V.

21. The method according to any one preceding claim for splitting an aqueous liquid volume into multiple droplets, comprising:providing an electrokinetic device, including:(a) a first substrate having a matrix of electrodes, wherein each of the matrix electrodes is coupled to a thin film transistor, and wherein the matrix electrodes are overcoated with a functional coating comprising:one or more dielectric layer(s) comprising silicon nitride, hafnium oxide or aluminium oxide in contact with the matrix electrodes,a conformal layer comprising parylene in contact with the dielectric layer, and a hydrophobic layer in contact with the conformal layer;(b) a controller operatively coupled to the matrix of electrodes, wherein the controller is configured to simultaneously provide different propulsion voltages to adjacent electrodes of the electrode matrix; and(c) a hydrophobic base fluid layer on top of the bottom electrode array containing the aqueous droplets;wherein the device manipulates the aqueous droplets under application of propulsion voltages by providing different propulsion voltages to adjacent electrodes of the electrode matrix.6922. The method according to claim 21, wherein the method splits a first aqueous volume covering at least first, second and third matrix electrodes by providing a series of electrical pulses to the first and second electrodes and a second weaker series of electrical pulses to the third electrodes such that the aqueous volume is split into droplets which cover the first and second electrodes and fail to cover the third electrode.

23. The method according to any one preceding claim comprising using a droplet with an elongated neck and controlling the center of the elongated neck to create 2 necks and a central bulge, wherein the central bulge forms a droplet which is smaller than could be generated with elongation of a single neck.

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