Patent Publication Number: US-10330919-B2

Title: AM-EWOD device and control methods with intermittent actuation patterns

Description:
TECHNICAL FIELD 
     The present invention relates to droplet microfluidic devices, and more specifically to Active Matrix Electro-wetting-On-Dielectric (AM-EWOD) devices and control methods for actuating device elements. 
     BACKGROUND ART 
     Electrowetting on dielectric (EWOD) is a well-known technique for manipulating droplets of fluid by the application of an electric field. Active Matrix EWOD (AM-EWOD) refers to implementation of EWOD in an active matrix array incorporating transistors, for example by using thin film transistors (TFTs). It is thus a candidate technology for digital microfluidics for lab-on-a-chip technology. An introduction to the basic principles of the technology can be found in “Digital microfluidics: is a true lab-on-a-chip possible?”, R. B. Fair, Microfluid Nanofluid (2007) 3:245-281). 
       FIG. 1  shows a part of a conventional EWOD device in cross section. The device includes a lower substrate  10 , the uppermost layer of which is formed from a conductive material which is patterned so that a plurality of array element electrodes  12  (e.g.,  12 A and  12 B in  FIG. 1 ) are realized. The electrode of a given array element may be termed the element electrode  12 . A liquid droplet  14 , including a polar material (which is commonly also aqueous and/or ionic), is constrained in a plane between the lower substrate  10  and a top substrate  16 . A suitable gap between the two substrates may be realized by means of a spacer  18 , and a non-polar surround fluid  20  (e.g. oil) may be used to occupy the volume not occupied by the liquid droplet  14 . An insulator layer  22  disposed upon the lower substrate  10  separates the conductive element electrodes  12 A,  12 B from a first hydrophobic coating  24  upon which the liquid droplet  14  sits with a contact angle  26  represented by θ. The hydrophobic coating is formed from a hydrophobic material (commonly, but not necessarily, a fluoropolymer). 
     On the top substrate  16  is a second hydrophobic coating  28  with which the liquid droplet  14  may come into contact. Interposed between the top substrate  16  and the second hydrophobic coating  28  is a reference electrode  30 . 
     The contact angle θ is defined as shown in  FIG. 1 , and is determined by the balancing of the surface tension components between the solid-to liquid (γ SL ), the liquid-to non-polar surrounding fluid (γ LG ) and the solid to non-polar surrounding fluid (γ SG ) interfaces, and in the case where no voltages are applied satisfies Young&#39;s law, the equation being given by: 
     
       
         
           
             
               
                 
                   
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     In operation, voltages termed the EW drive voltages, (e.g. V T , V 0  and V 00  in  FIG. 1 ) may be externally applied to different electrodes (e.g. reference electrode  30 , element electrodes  12 ,  12 A and  12 B, respectively). The resulting electrical forces that are set up effectively control the hydrophobicity of the hydrophobic coating  24 . By arranging for different EW drive voltages (e.g. V 0  and V 00 ) to be applied to different element electrodes (e.g.  12 A and  12 B), the liquid droplet  14  may be moved in the lateral plane between the two substrates  10  and  16 . 
     Example configurations and operation of EWOD devices are described in the following. U.S. Pat. No. 6,911,132 (Pamula et al., issued Jun. 28, 2005) discloses a two dimensional EWOD array to control the position and movement of droplets in two dimensions. U.S. Pat. No. 6,565,727 (Shenderov, issued May 20, 2003) further discloses methods for other droplet operations including the splitting and merging of droplets, and the mixing together of droplets of different materials. U.S. Pat. No. 7,163,612 (Sterling et al., issued Jan. 16, 2007) describes how TFT based thin film electronics may be used to control the addressing of voltage pulses to an EWOD array by using circuit arrangements very similar to those employed in AM display technologies. 
     The approach of U.S. Pat. No. 7,163,612 may be termed “Active Matrix Electrowetting on Dielectric” (AM-EWOD). There are several advantages in using TFT based thin film electronics to control an EWOD array, namely:
         Electronic driver circuits can be integrated onto the lower substrate  10 .   TFT-based thin film electronics are well suited to the AM-EWOD application. They are cheap to produce so that relatively large substrate areas can be produced at relatively low cost.   TFTs fabricated in standard processes can be designed to operate at much higher voltages than transistors fabricated in standard CMOS processes. This is significant since many EWOD technologies require electro-wetting voltages in excess of 20V to be applied.       

     To perform various droplet operations in an AM-EWOD device, it can be desirable to be able to sense a droplet property, such as droplet size or location on the array of elements. US application 2010/0194408 (Sturmer et al., published Aug. 5, 2010) describes a method, circuit and apparatus for detecting capacitance on a droplet actuator, inter alia, for determining the presence, partial presence or absence of a droplet at an electrode. U.S. Pat. No. 8,653,832 (Hadwen et al., issued Feb. 18, 2014) describes how an impedance (capacitance) sensing function can be incorporated into the array element circuit of each array element of an AM-EWOD device. The impedance sensor circuit may be used for determining the presence and size of liquid droplets present at each electrode in the array. 
     Upon adequate sensing, droplet operations may then be performed, such as for example holding a droplet position, moving a droplet across the device, splitting a droplet into multiple droplets, mixing different droplets, and others. These various operations may be performed by actuating a suitable pattern of elements on the AM-EWOD device. For example, U.S. Pat. No. 7,569,129 (Pamula et al., issued Aug. 4, 2009) describes the use of element actuation to hold a droplet isolated from other droplets. Sturmer referenced above describes the use of capacitance detection as real time feedback to determine whether a droplet operation has been successful. WO2008055256A3 (Kim et al., published May 8, 2008) describes the use of capacitance detection as real time feedback to control the volume of a droplet being dispensed or split from a reservoir. Other actuation methods for performing various droplet operations are known. 
     Problem to be Solved by the Invention 
     The inventors have found that the electric fields generated from excessive or prolonged actuation of the EWOD or AM-EWOD elements can be damaging to both the subject droplet and to components of the device itself. The protocols performed on EWOD platforms may use reagents which are delicate and may be adversely affected by actuation. Damage to reagents and other functional chemicals contained within a droplet can result in undesirable bubbles forming within the droplet (for example due to the release of gas dissolved in the droplet or the surrounding oil). In addition, excessive or prolonged actuation may reduce the lifetime of the EWOD device as the electric fields involved can have deleterious effects on components of the device. For example, the insulator layers and the hydrophobic coatings have been found in particular to be susceptible to damage from the electric fields that result from prolonged actuation of the EWOD elements. 
     The present invention solves this problem through enhanced control of the actuation patterns of the EWOD elements. In particular, the control system and related control methods of the present invention operate to minimize the time over which EWOD elements are actuated while still effectively performing requisite droplet operations. By minimizing actuation time of the EWOD elements, which minimizes exposure to the generated electric fields, the propensity to damage the subject droplets or device components is reduced. 
     SUMMARY OF INVENTION 
     The present invention pertains to enhanced control systems and methods for the actuation of array elements in an EWOD device, and AM-EWOD devices in particular. The control system implements a method of driving the array elements by which intermittent actuation of pertinent array elements is employed to maintain a droplet in a desired state. By employing intermittent actuation patterns, the control system minimizes actuation time of the EWOD elements, which in turn minimizes exposure to the generated electric fields and the resultant damage to the subject droplets or device components. 
     In exemplary embodiments, the control system operates to apply suitable actuation voltages to pertinent array elements at a predetermined time, rate, and duration in accordance with a specified or preset duty cycle, regardless of the actual real time properties of the droplet. In other exemplary embodiments, the EWOD or AM-EWOD device incorporates one or more sensors, such as for example sensor circuitry within each array element circuit, which provides information and feedback regarding a droplet state. In embodiments employing sensor circuitry or other sensors, the control system operates to apply suitable actuation voltages only when an intervention is necessary to maintain the droplet in a desired state (e.g., to maintain droplet position and stop a droplet drifting out of position, maintain a particular droplet shape or aspect ratio, maintain a particular droplet size, prevent droplet collision with a second object or droplet, or the like). 
     The present invention provides for an enhance microfluidic system including an electro-wetting on dielectric (EWOD) device and a control system, and a related control method. The EWOD device includes an element array configured to receive one or more fluid droplets, the element array comprising a plurality of individual array elements. The control system is configured to control actuation voltages applied to the element array to perform manipulation operations as to the fluid droplets. In exemplary embodiments, the control system is configured to apply a sequence of actuation voltages to a portion of the array elements associated with a droplet to maintain the droplet in a desired droplet state corresponding to a predetermined droplet property. The sequence of actuation voltages includes an actuation-on period in which the portion of the array elements associated with the droplet is actuated and an actuation-off period in which the portion of the array elements associated with the droplet is not actuated, and the actuation-off period is non-zero. 
     In exemplary embodiments, the control system may be configured to apply a sequence of actuation voltages comprising a predetermined duty cycle including a predetermined time, rate and duration of actuation voltages to the portion of the array elements associated with the droplet. In exemplary embodiments, the system further may include a sensor for sensing a droplet state. With sensor based control, the control system may be configured to: receive droplet state information from the sensor; determine whether a droplet is in a state that deviates from the desired droplet state in accordance with predetermined criteria based on the droplet state information; and apply actuation voltages to the portion of the array elements associated with the droplet when the control system determines that the droplet state satisfies the predetermined criteria to return the droplet to the desired droplet state. 
     These and further features of the present invention will be apparent with reference to the following description and attached drawings. In the description and drawings, particular embodiments of the invention have been disclosed in detail as being indicative of some of the ways in which the principles of the invention may be employed, but it is understood that the invention is not limited correspondingly in scope. Rather, the invention includes all changes, modifications and equivalents coming within the spirit and terms of the claims appended hereto. Features that are described and/or illustrated with respect to one embodiment may be used in the same way or in a similar way in one or more other embodiments and/or in combination with or instead of the features of the other embodiments. 
     Advantageous Effects of the Invention 
     The control system and related control methods of the present invention operate to provide intermittent actuation of the array elements to minimize the time over which EWOD or AM-EWOD elements are actuated while still effectively performing requisite droplet operations (e.g. move, merge, split, dispense and hold). By minimizing actuation time of the EWOD or AM-EWOD elements, which minimizes exposure to the generated electric fields, the propensity to damage the subject droplets or device components is reduced. Intermittent actuation thus advantageously limits the duty cycle or time period over which the array elements and associated droplets are actuated. This improves the device reliability and/or prevents damage to chemically or biologically fragile reagents within the droplet. 
    
    
     
       BRIEF DESCRIPTION OF DRAWINGS 
         FIG. 1  is a drawing depicting a conventional EWOD device in cross-section. 
         FIG. 2  is a drawing depicting an exemplary EWOD based microfluidic system according to embodiments of the present invention. 
         FIG. 3  is a drawing depicting an exemplary AM-EWOD device in schematic perspective in accordance with embodiments of the present invention. 
         FIG. 4  is a drawing depicting a cross section through some of the array elements of the exemplary AM-EWOD device of  FIG. 3 . 
         FIG. 5A  is a drawing depicting a circuit representation of the electrical load presented at the element electrode when a liquid droplet is present. 
         FIG. 5B  is a drawing depicting a circuit representation of the electrical load presented at the element electrode when no liquid droplet is present. 
         FIG. 6  is a drawing depicting an exemplary arrangement of thin film electronics in the exemplary AM-EWOD device of  FIG. 3  in accordance with embodiments of the present invention. 
         FIG. 7  is a drawing depicting an exemplary arrangement of the array element circuit in accordance with embodiments of the present invention. 
         FIG. 8  is a drawing depicting a sequence of electrode actuation comprising a duty cycle for maintaining droplet position on an array of elements in an EWOD device. 
         FIG. 9  is a drawing depicting a sequence of electrode actuation comprising a duty cycle for maintaining droplet shape on an array of elements in an EWOD device. 
         FIG. 10  is a drawing depicting a sequence of electrode actuation comprising a duty cycle for maintaining droplet size on an array of elements in an EWOD device. 
         FIG. 11  is a drawing depicting a sequence of electrode actuation comprising a sensor-based intervention for maintaining droplet position on an array of elements in an EWOD device. 
         FIG. 12  is a drawing depicting a sequence of electrode actuation comprising a sensor-based intervention for maintaining droplet shape on an array of elements in an EWOD device. 
         FIG. 13  is a drawing depicting a sequence of electrode actuation comprising a sensor-based intervention for maintaining droplet size on an array of elements in an EWOD device. 
         FIG. 14  is a drawing depicting a sequence of electrode actuation comprising a sensor-based intervention for maintaining droplet position on an array of elements in an EWOD device relative to a second object in the EWOD device. 
         FIG. 15A  and  FIG. 15B  are drawings depicting a sequence of electrode actuation comprising a sensor-based intervention for maintaining droplet position of a first droplet relative to a second moving droplet on an array of elements in an EWOD device. 
     
    
    
     DESCRIPTION OF EMBODIMENTS 
     Embodiments of the present invention will now be described with reference to the drawings, wherein like reference numerals are used to refer to like elements throughout. It will be understood that the figures are not necessarily to scale. 
       FIG. 2  is a drawing depicting an exemplary EWOD based microfluidic system according to embodiments of the present invention. In the example of  FIG. 2 , the measurement system includes a reader  32  and a cartridge  34 . The cartridge  34  may contain a microfluidic device, such as an EWOD or AM-EWOD device  36 , as well as (not shown) fluid input ports into the device and an electrical connection as are conventional. The fluid input ports may perform the function of inputting fluid into the AM-EWOD device  36  and generating droplets within the device, for example by dispensing from input reservoirs as controlled by electro-wetting. As further detailed below, the microfluidic device includes an electrode array configured to receive the inputted fluid droplets. 
     The microfluidic system further may include a control system configured to control actuation voltages applied to the electrode array of the microfluidic device to perform manipulation operations to the fluid droplets. For example, the reader  32  may contain such a control system configured as control electronics  38  and a storage device  40  that may store any application software any data associated with the system. The control electronics  38  may include suitable circuitry and/or processing devices that are configured to carry out various control operations relating to control of the AM-EWOD device  36 , such as a CPU, microcontroller or microprocessor. 
     Among their functions, to implement the features of the present invention, the control electronics may comprise a part of the overall control system that may execute program code embodied as a control application within the storage device  40 . It will be apparent to a person having ordinary skill in the art of computer programming, and specifically in application programming for electronic control devices, how to program the control system to operate and carry out logical functions associated with the stored control application. Accordingly, details as to specific programming code have been left out for the sake of brevity. The storage device  40  may be configured as a non-transitory computer readable medium, such as random access memory (RAM), a read-only memory (ROM), an erasable programmable read-only memory (EPROM or Flash memory), or any other suitable medium. Also, while the code may be executed by control electronics  38  in accordance with an exemplary embodiment, such control system functionality could also be carried out via dedicated hardware, firmware, software, or combinations thereof, without departing from the scope of the invention. 
     The control system may be configured to perform some or all of the following functions:
         Define the appropriate timing signals to manipulate liquid droplets on the AM-EWOD device  36 .   Interpret input data representative of sensor information measured by a sensor or sensor circuitry associated with the AM-EWOD device  36 , including computing the locations, sizes, centroids and perimeters of liquid droplets on the AM-EWOD device  36 .   Use calculated sensor data to define the appropriate timing signals to manipulate liquid droplets on the AM-EWOD device  36 , i.e. acting in a feedback mode.   Provide for implementation of a graphical user interface (GUI) whereby the user may program commands such as droplet operations (e.g. move a droplet), assay operations (e.g. perform an assay), and the GUI may report the results of such operations to the user.       

     In the example of  FIG. 2 , an external sensor module  35  is provided for sensing droplet properties. For example, optical sensors as are known in the art may be employed as external sensors for sensing droplet properties. Suitable optical sensors include camera devices, light sensors, charged coupled devices (CCDs) and image similar image sensors, and the like. As further detailed below, a sensor alternatively may be configured as internal sensor circuitry incorporated as part of the drive circuitry in each array element. Such sensor circuitry may sense droplet properties by the detection of an electrical property at the array element, such as impedance or capacitance. 
     The control system, such as via the control electronics  38 , may supply and control the actuation voltages applied to the electrode array of the microfluidics device  36 , such as required voltage and timing signals to perform droplet manipulation operations and sense liquid droplets on the AM-EWOD device  36 . The control electronics further may execute the application software to generate and output control voltages for droplet sensing and performing sensing operations. The reader  32  and cartridge  34  may be electrically connected together while in use, for example by a cable of connecting wires  42 , although various other methods (e.g. wireless connection) of providing electrical communication may be used as are known to those of ordinary skill in the art. 
       FIG. 3  is a drawing depicting additional details of the exemplary AM-EWOD device  36  in schematic perspective in accordance with embodiments of the present invention. The AM-EWOD device  36  has a lower substrate  44  with thin film electronics  46  disposed upon the lower substrate  44 . The thin film electronics  46  are arranged to drive array element electrodes  48 . A plurality of array element electrodes  48  are arranged in an electrode or element array  50 , having X by Y array elements where X and Y may be any integer. A liquid droplet  52  which may include any polar liquid and which typically may be aqueous, is enclosed between the lower substrate  44  and a top substrate  54  separated by a spacer  56 , although it will be appreciated that multiple liquid droplets  52  can be present. 
       FIG. 4  is a drawing depicting a cross section through some of the array elements of the exemplary AM-EWOD  36  device of  FIG. 3 . In the portion of the AM-EWOD device depicted in  FIG. 4 , the device includes a pair of the array element electrodes  48 A and  48 B that are shown in cross section that may be utilized in the electrode or element array  50  of the AM-EWOD device  36  of  FIG. 3 . The device configuration is similar to the conventional configuration shown in  FIG. 1 , with the AM-EWOD device  36  further incorporating the thin-film electronics  46  disposed on the lower substrate  44 , which is separated from the upper substrate  54  by the spacer  56 . The uppermost layer of the lower substrate  44  (which may be considered a part of the thin film electronics layer  46 ) is patterned so that a plurality of the array element electrodes  48  (e.g. specific examples of array element electrodes are  48 A and  48 B in  FIG. 4 ) are realized. The term element electrode  48  may be taken in what follows to refer both to the physical electrode structure  48  associated with a particular array element, and also to the node of an electrical circuit directly connected to this physical structure. A reference electrode  58  is shown in  FIG. 4  disposed upon the top substrate  54 , but the reference electrode alternatively may be disposed upon the lower substrate  44  to realize an in-plane reference electrode geometry. The term reference electrode  58  may also be taken in what follows to refer to both or either of the physical electrode structure and also to the node of an electrical circuit directly connected to this physical structure. 
     Also similarly to the conventional structure of  FIG. 1 , in the AM-EWOD device  36 , a non-polar fluid  60  (e.g. oil) may be used to occupy the volume not occupied by the liquid droplet  52 . An insulator layer  62  may be disposed upon the lower substrate  44  that separates the conductive element electrodes  48 A and  48 B from a first hydrophobic coating  64  upon which the liquid droplet  52  sits with a contact angle  66  represented by θ. The hydrophobic coating is formed from a hydrophobic material (commonly, but not necessarily, a fluoropolymer). On the top substrate  54  is a second hydrophobic coating  68  with which the liquid droplet  52  may come into contact. The reference electrode  58  is interposed between the top substrate  54  and the second hydrophobic coating  68 . 
       FIG. 5A  shows a circuit representation of the electrical load  70 A between the element electrode  48  and the reference electrode  58  in the case where a liquid droplet  52  is present. The liquid droplet  52  can usually be modeled as a resistor and capacitor in parallel. Typically, the resistance of the droplet will be relatively low (e.g. if the droplet contains ions) and the capacitance of the droplet will be relatively high (e.g. because the relative permittivity of polar liquids is relatively high, e.g. ˜80 if the liquid droplet is aqueous). In many situations the droplet resistance is relatively small, such that at the frequencies of interest for electro-wetting, the liquid droplet  52  may function effectively as an electrical short circuit. The hydrophobic coatings  64  and  68  have electrical characteristics that may be modelled as capacitors, and the insulator  62  may also be modelled as a capacitor. The overall impedance between the element electrode  48  and the reference electrode  58  may be approximated by a capacitor whose value is typically dominated by the contribution of the insulator  62  and hydrophobic coatings  64  and  68  contributions, and which for typical layer thicknesses and materials may be on the order of a pico-Farad in value. 
       FIG. 5B  shows a circuit representation of the electrical load  70 B between the element electrode  48  and the reference electrode  58  in the case where no liquid droplet is present. In this case the liquid droplet components are replaced by a capacitor representing the capacitance of the non-polar fluid  60  which occupies the space between the top and lower substrates. In this case the overall impedance between the element electrode  48  and the reference electrode  58  may be approximated by a capacitor whose value is dominated by the capacitance of the non-polar fluid and which is typically small, of the order of femto-Farads. 
     For the purposes of driving and sensing the array elements, the electrical load  70 A/ 70 B overall functions in effect as a capacitor, whose value depends on whether a liquid droplet  52  is present or not at a given element electrode  48 . In the case where a droplet is present, the capacitance is relatively high (typically of order pico-Farads), whereas if there is no liquid droplet present the capacitance is low (typically of order femto-Farads). If a droplet partially covers a given electrode  48  then the capacitance may approximately represent the extent of coverage of the element electrode  48  by the liquid droplet  52 . 
       FIG. 6  is a drawing depicting an exemplary arrangement of thin film electronics  46  in the exemplary AM-EWOD device  36  of  FIG. 3  in accordance with embodiments of the present invention. The thin film electronics  46  is located upon the lower substrate  44 . Each array element  51  of the array of elements  50  contains an array element circuit  72  for controlling the electrode potential of a corresponding element electrode  48 . Integrated row driver  74  and column driver  76  circuits are also implemented in thin film electronics  46  to supply control signals to the array element circuit  72 . The array element circuit  72  may also contain a sensing capability for detecting the presence or absence of a liquid droplet in the location of the array element. Integrated sensor row addressing  78  and column detection circuits  80  may further be implemented in thin film electronics for the addressing and readout of the sensor circuitry in each array element. 
     A serial interface  82  may also be provided to process a serial input data stream and facilitate the programming of the required voltages to the element electrodes  48  in the array  50 . A voltage supply interface  84  provides the corresponding supply voltages, top substrate drive voltages, and other requisite voltage inputs as further described herein. A number of connecting wires  86  between the lower substrate  44  and external control electronics, power supplies and any other components can be made relatively few, even for large array sizes. Optionally, the serial data input may be partially parallelized. For example, if two data input lines are used the first may supply data for columns 1 to X/2, and the second for columns (1+X/2) to M with minor modifications to the column driver circuits  76 . In this way the rate at which data can be programmed to the array is increased, which is a standard technique used in Liquid Crystal Display driving circuitry. 
     Generally, an exemplary AM-EWOD device  36  that includes thin film electronics  46  may be configured as follows. The AM-EWOD device  36  includes the reference electrode  58  mentioned above (which, optionally, could be an in-plane reference electrode) and a plurality of individual array elements  51  on the array of elements  50 , each array element  51  including an array element electrode  48  and array element circuitry  72 . Relatedly, the AM-EWOD device  36  may be configured to perform a method of actuating the array elements to manipulate liquid droplets on the array by controlling an electro-wetting voltage to be applied to a plurality of array elements. The applied voltages may be provided by operation of the control system described as to  FIG. 2 , including the control electronics  38  and applications and data stored on the storage device  40 . The electro-wetting voltage at each array element  51  is defined by a potential difference between the array element electrode  48  and the reference electrode  58 . The method of controlling the electro-wetting voltage at a given array element typically includes the steps of supplying a voltage to the array element electrode  48 , and supplying a voltage to the reference electrode  58 , by operation of the control system. 
       FIG. 7  is a drawing depicting an exemplary arrangement of the array element circuit  72  present in each array element  51 , in accordance with embodiments of the present invention. The array element circuit  72  may contain an actuation circuit  88 , having inputs ENABLE, DATA and ACTUATE, and an output which is connected to an element electrode  48 . The array element circuit  72  also may contain a droplet sensor circuit  90 , which may be in electrical communication with the element electrode  48 . Typically, the read-out of the droplet sensor circuit  90  may be controlled by one or more addressing lines (e.g. RW) that may be common to elements in the same row of the array, and may also have one or more outputs, e.g. OUT, which may be common to all elements in the same column of the array. 
     The array element circuit  72  may typically perform the functions of:
         (i) Selectively actuating the element electrode  48  by supplying a voltage to the array element electrode. Accordingly, any liquid droplet present at the array element  51  may be actuated or de-actuated by the electro-wetting effect.   (ii) Sensing the presence or absence of a liquid droplet at the location of the array element  51 . The means of sensing may be capacitive, optical, thermal or some other means. Capacitive sensing may be employed conveniently and effectively using an impedance sensor circuit as part of the array element circuitry.       

     Exemplary configurations of array element circuits  72  including impedance sensor circuitry are known in the art, and for example are described in detail in U.S. Pat. No. 8,653,832 referenced in the background art section, and commonly assigned UK application GB1500261.1, both of which are incorporated here by reference. These patent documents include descriptions of how the droplet may be actuated (by means of electro-wetting) and how the droplet may be sensed by capacitive or impedance sensing means. Typically, capacitive and impedance sensing may be analogue and may be performed simultaneously, or near simultaneously, at every element in the array. By processing the returned information from such a sensor (for example in the application software in the storage device  40  of the reader  32 ), the control system described above can determine in real-time, or almost real-time the position, size, centroid and perimeter of each liquid droplet present in the array of elements  50 . As referenced in connection with  FIG. 2 , an alternative to sensor circuitry is to provide an external sensor (e.g., sensor  35 ), such as an optical sensor that can be used to sense droplet properties. 
     The present invention pertains to an enhanced control system and control methods for the actuation of array elements in an EWOD device, including more specifically an AM-EWOD device. The control system implements a method of driving the array elements by which intermittent actuation of pertinent array elements is employed to maintain a droplet in a desired state. By employing an intermittent actuation, the control system minimizes actuation time of the AM-EWOD elements, which in turn minimizes exposure to the generated electric fields and the resultant damage to the subject droplets or device components. 
     Certain terms as used in the specification are defined as follows. The “state” of a droplet may refer to any property of the droplet such as size, shape, centroid position, aspect ratio, edge position, location on the device array, proximity to other objects on the device array, and the like. A “desired droplet state” may refer to a droplet state (i.e., any droplet property as above), that otherwise may be measured relative to a reference item, such as for example various components of the device such as the array elements, input or output ports and other physical structures in the device, other droplets in the device, and the like. A desired state typically may be thought of as a preferred droplet state as determined with reference to particular circumstances. A “sensor measurement” may refer to any means of measuring the droplet properties such as impedance measurements performed by an impedance sensor circuit in the array element circuitry, or optical measurements performed by an external optical sensor device (e.g., camera, light sensor, CCD and the like) as are known in the art. “Array elements associated with a droplet” are a subset of the array elements on an EWOD device that when actuated can control or affect a droplet state or otherwise manipulate a droplet. 
     Intermittent actuation is intended to provide actuation of array elements associated with a droplet for a sufficient amount of time to maintain the state of a droplet in a desired droplet state, while minimizing actuation time to substantially reduce the propensity to damage droplet constituents or device components. 
     Damage to the device may occur due to the exposure of the insulator  62  and hydrophobic coatings ( 64  and  68 ) to a high electric field, a situation that occurs when a droplet is actuated. In particular, the hydrophobic coatings are commonly formed from an electret material and thus have a tendency to trap charge, within them or at their interfaces. Trapped charge may have the effect of screening the applied electric field and reducing the actuation force applied to the droplet. Additionally, the high electric field may result in polarization of the insulator/hydrophobic coatings, which may also reduce the actuation strength. Additionally, the high electric field may occasionally result in defect paths forming through the insulator/hydrophobic coatings resulting in current flow, electrolysis and failure of the device. 
     Damage to the liquid due to exposure to the electric field may occur if the liquid contains components that may be denatured by the electric field, e.g. proteins, enzymes, cells or nucleic acids. The extent of the damage may be a function of the accumulated exposure time to the electric field. 
     When actuation voltages are turned off, small non-uniformities that may be present in droplet shape and thickness can result in migration or movement of the droplet. The result can be deviations of the droplet state away from a desired droplet state. For example, droplet size, shape, centroid position, edge position, location on the device array, and the like may change overtime in an undesirable manner that can undermine the purpose and use of the droplet, and perhaps undesirably result in the droplet coming in contact with or otherwise interfering with other droplets in close proximity. When such deviations occur, intermittent actuation can be performed to return the droplet from a current droplet state to a desired droplet state. The following are non-limiting example reasons why a droplet may deviate over time:
         1. The spacing between the top substrate  54  and bottom substrate  44  (e.g. as controlled by the spacer  56 ) may be non-uniform over the lateral extent of the droplet. A non-uniform spacing may result in a difference in the Laplace pressure (the internal droplet pressure, determined in part by the constraining dimensions) over the lateral extent of the droplet. This will result in a lateral force, causing the droplet to move towards the part of the device where the spacing between the substrates is greater.   2. If either of the hydrophobic coatings  64  and  68  may be slightly non-uniform so as to have a differing hydrophobicity over the lateral extent of the droplet, the droplet may move or change shape of its own accord to cover the area of lower hydrophobicity.   3. If the profile of the droplet in plan-view is non-circular, the droplet may be observed to relax over time into a more circular shape.   4. If the whole device is operated on a non-horizontal surface, the droplet may drift over time due to the effects of gravity.       

     In general, in accordance with principles of the present invention, a sequence of actuation of array elements associated with a droplet in an EWOD device is performed wherein a ratio of the actuation-on period to actuation-off period is less than infinite, i.e., the actuation-off period is non-zero meaning that the actuation voltages are not applied for at least a portion of the sequence of actuation. In this regard, the inventors have observed that droplet changes in state tend to occur very gradually over time (and sometimes not at all if the device and/or droplet is highly uniform). Accordingly, an actuation-off time period in which actuation voltages are not applied can be significantly greater than an actuation-on period in which the actuation voltages are applied to the array elements associated with a droplet. The figures described below illustrate different sequences of actuation of array elements associated with a droplet in accordance with various control methods in accordance with the principles of the present invention. 
     The present invention provides for an enhance microfluidic system including an electro-wetting on dielectric (EWOD) device and a control system, and a related control method. The EWOD device includes an element array configured to receive one or more fluid droplets, the element array comprising a plurality of individual array elements. The control system is configured to control actuation voltages applied to the element array to perform manipulation operations as to the fluid droplets. In exemplary embodiments, the control system is configured to apply a sequence of actuation voltages to a portion of the array elements associated with a droplet to maintain the droplet in a desired droplet state corresponding to a predetermined droplet property. The sequence of actuation voltages includes an actuation-on period in which the portion of the array elements associated with the droplet is actuated and an actuation-off period in which the portion of the array elements associated with the droplet is not actuated, and the actuation-off period is non-zero. In exemplary embodiments, the control system may be configured to apply a sequence of actuation voltages comprising a predetermined duty cycle, and/or the actuation voltages may be applied in accordance with a sensor based intervention. 
       FIGS. 8-15  are drawings depicting sequences of electrode actuation of array elements associated with a droplet in an EWOD device, the sequences being performed for maintaining or establishing a desired state of a droplet in the EWOD device. Generally, such figures depict an array of elements  100  that is comparable to the array of elements  50  described above. The additional details of the EWOD device are omitted from these figures for convenience of illustration, but an EWOD device employing the array of elements  100  may be configured as described above with respect to  FIGS. 2-7 . Accordingly, the array of elements  100  may include individual array elements  102  each comparable to the array element  51 , and each for example thus including electrodes  48  and  58 , and the corresponding array element circuitry  72  which may include a droplet sensor circuit  90 , such as for example an impedance or capacitive sensing circuit. As defined above, array elements associated with a droplet refers to a portion of the array elements on an EWOD device that when actuated can control of affect a droplet state or otherwise manipulate a droplet. In the examples of  FIGS. 8-15 , array elements  102   a  are designated as being associated with a droplet  104 , and remaining array elements  102   b  are in regions of the array in which the array elements are not associated with the droplet  104 , i.e., the array elements  102   b  are not involved in controlling or affecting the droplet state. 
     In exemplary embodiments, the control system operates to apply suitable actuation voltages to perform a sequence of electrode actuation of array elements associated with a droplet at a predetermined time, rate, and duration in accordance with a specified or preset duty cycle, regardless of the actual real time properties constituting the state of the droplet. In general, a predetermined duty cycle may be configured whereby a ratio of the actuation-on period to actuation-off period is less than infinite, i.e., again, the actuation-off period is non-zero meaning that the actuation voltages are not applied for at least a portion of the duty cycle. As referenced above, droplet changes in state may occur very gradually over time which permits the actuation-off period to be significantly greater than the actuation-on period. Based on such observation, a suitable duty cycle may be characterized by approximately 10% actuation-on/90% actuation-off, i.e., a ratio of actuation-on period to actuation-off period is less than or equal to 1:10. Under such parameters, an example of a suitable duty cycle may be to apply actuation voltages to electrodes of array elements associated with a droplet for 0.5 seconds once every 5.0 seconds. Other suitable duty cycles may be employed as may be appropriate to particular circumstances or applications. 
     In other circumstances, droplet changes in state may occur more rapidly over time, which requires that the actuation-off period not be as significantly greater than the actuation-on period. For example, during loading of the droplet into the EWOD device, its size changes relatively rapidly and in order to maintain a desirable droplet state (such as, but not limited to, a desirable droplet centroid position or droplet aspect ratio) a suitable duty cycle may be characterized by a ratio of actuation-on period to actuation-off period greater than or equal to 1:2. Under such parameters, an example of a suitable duty cycle may be to apply actuation voltages to electrodes of array elements associated with a droplet of 0.5 seconds every 1.5 seconds. Again, any suitable duty cycles may be employed as may be appropriate to particular circumstances or applications. 
       FIGS. 8-10  are drawings depicting sequences of electrode actuation comprising a predetermined duty cycle for maintaining a desired state of a droplet on an associated array of elements in an EWOD device. In the example of  FIG. 8 , a sequence of electrode actuation comprising a predetermined duty cycle is applied for maintaining droplet position on the array of elements  100 . The steps of the sequence of actuation in  FIG. 8  respectively are labeled A, B, C, and D. Further in the example of  FIG. 8 , there are nine array elements  102   a  associated with the droplet  104 , which can be actuated to maintain a current position of the droplet  104  on the array  100 . It will be appreciated that the number and position of elements associated with the droplet can be varied as suitable for any particular circumstances. The remaining elements  102   b  are in regions of the array that are not associated with the droplet  104 , i.e., the droplets  102   b  are not involved in maintaining the droplet current position and thus remain unactuated during the entire actuation sequence A-D. 
     The sequence of actuation of  FIG. 8  illustrates a duty cycle of alternating actuation-off periods, sequence steps A and C, with actuation-on periods, sequence steps B and D. As shown in  FIG. 8 , during the actuation-off periods, the droplet  104  is located at a particular position on the array  100 . To ensure that the droplet  104  maintains this position, periodically the control system applies actuation voltages to the portion of array elements  102   a  associated with the droplet. When actuated, the electrical field draws the droplet to the array element electrodes. As shown in sequence steps B and D, the boundary of droplet  104  is drawn by the electric field to be commensurate with the actuated array element boundaries. When the actuation voltages are removed, the droplet  104  resumes its unactuated state as shown, for example, from the progression of sequence step B to step C. 
     By applying intermittent actuation in accordance with the predetermined duty cycle, the droplet  104  generally maintains its desired state and does not move substantially from the initial position of sequence step A. Again, because droplet changes in state occur very gradually over time, the actuation-off period may be significantly greater than the actuation-on period (e.g., ratio actuation-on period to actuation-off period is no greater than 1:10). 
       FIG. 9  is a variation in which a sequence of electrode actuation comprises a predetermined duty cycle for maintaining a desired shape or aspect ratio of a droplet. The steps of the sequence of actuation in  FIG. 9  similarly are respectively labeled A, B, C, and D. Further in the example of  FIG. 9 , there are 32 array elements  102   a  associated with the droplet  104  due to the elongated or ovular shape of the droplet  104  in the desired state shown in sequence step A. The array elements  102   a  can be actuated to maintain such shape and aspect ratio of the droplet  104  on the array  100 . The sequence of actuation of  FIG. 9 , therefore, illustrates a duty cycle of alternating actuation-off periods, sequence steps A and C, with actuation-on periods, sequence steps B and D. During the actuation-off periods, the droplet  104  maintains the desired ovular and elongated shape on the array  100 . To ensure that the droplet  104  maintains this shape, periodically the control system applies actuation voltages to the portion of array elements  102   a  associated with the droplet. When actuated, the electrical field draws the droplet to the array element electrodes. As shown in sequence steps B and D, the boundary of droplet  104  is drawn by the electric field to be commensurate with the actuated array element boundaries. When the actuation voltages are removed, the droplet  104  resumes its unactuated state as shown, for example, from the progression of sequence step B to step C. A comparable duty cycle may be employed in the sequence of  FIG. 9  as in  FIG. 8 . 
       FIG. 10  is a variation in which a sequence of electrode actuation comprises a predetermined duty cycle for maintaining a desired size of a droplet. The steps of the sequence of actuation in  FIG. 10  similarly are respectively labeled A-F. Further in the example of  FIG. 10 , there are nine array elements  102   a  associated with the droplet  104  that may be actuated to maintain the desired size of the droplet  104  shown in sequence step A. The array elements  102   a  can be actuated to maintain such size of the droplet  104  on the array  100 . The sequence of actuation of  FIG. 10 , therefore, illustrates a duty cycle of alternating actuation-off periods, sequence steps A-B and D-E, with actuation-on periods, sequence steps C and F. A comparable duty cycle may be employed in the sequence of  FIG. 10  as in  FIGS. 8-9 . 
     The example of  FIG. 10  illustrates the potential for migration of droplet material to change the state of the droplet from the desired state during the actuation-off period. In this particular example of droplet size, during the actuation-off period spanning steps A-B, the droplet has spread out and the size has changed in the sense that the droplet covers more of the array  100  in step B than in the desired state of step A. When actuated, the electrical field draws the droplet to the actuated array element electrodes. As shown in sequence step C, the boundary of droplet  104  is drawn by the electric field to be commensurate with the actuated array element boundaries. When the actuation voltage is removed, the droplet  104  resumes its unactuated state as shown, for example, from the progression of sequence steps C to D. In this example, there actually is no droplet migration during the actuation-off period spanning sequence steps D-E. However, because actuation occurs in accordance with a predetermined or preset duty cycle, the electrodes  102   a  are actuated at sequence step F even though the droplet state has not changed, insofar as the predetermined duty cycle is independent of the real-time state of the droplet. Migration can also change the state of the droplet from the desired state with respect to location ( FIG. 8 ) or shape ( FIG. 9 ), or other droplet properties, with actuation in accordance with the duty cycle then returning the droplet to the state corresponding to the desired state of the droplet. 
     In exemplary embodiments, the control system may store and execute any number of predetermined or preset duty cycles as executable program code as part of the control application. For example, the control application may include executable program code for any number of duty cycles for different operational modes of the device, which may be stored in the storage device  40  and executed by processor devices of the control electronics  38  (see  FIG. 2 ). Initiation of a particular duty cycle may be initiated by user selection through interface devices of the control electronics, or otherwise selected automatically as part of the control application. In addition, the control application may be executed to apply different duty cycles to different portions of the array of elements. 
     In other exemplary embodiments, the AM-EWOD device incorporates one or more sensors, such as for example sensor circuitry  90  within each array element, or external sensors  35 , that provide information and feedback regarding a droplet state. In embodiments employing sensor circuitry or other sensors, the control system operates to apply suitable actuation voltages only when an intervention is necessary to maintain the droplet in a desired state based on data gathered by the sensors (e.g., to maintain droplet position and stop a droplet drifting out of position, maintain a particular droplet shape, maintain a particular droplet size, or the like). For example, the control system may determine when a droplet state has deviated from a desired state by a predetermined amount or other predetermined criteria, and apply actuation voltages to array elements associated with the droplet to return the droplet to the desired state. 
     In exemplary embodiments of the invention, therefore, the microfluidic system further may include a sensor for sensing a droplet state. The control system may be configured to: receive droplet state information from the sensor; determine whether a droplet is in a state that deviates from the desired droplet state in accordance with predetermined criteria based on the droplet state information; and apply actuation voltages to the portion of the array elements associated with the droplet when the control system determines that the droplet state satisfies the predetermined criteria to return the droplet to the desired droplet state. 
       FIGS. 11-15  are drawings depicting a sequence of electrode actuation comprising a sensor-based intervention for maintaining a desired state of a droplet on an associated array of elements in an EWOD device. In the example of  FIG. 11 , a sequence of electrode actuation comprising a sensor-based intervention is applied for maintaining droplet position on the array of elements  100 . The steps of the sequence of actuation in  FIG. 11  respectively are labeled A-E. Further in the example of  FIG. 11 , there are nine array elements  102   a  associated with the droplet  104 , which can be actuated to maintain a current position of the droplet  104  on the array  100 . Again, the number and position of elements associated with the droplet can be varied as suitable for any particular circumstances. The remaining droplets  102   b  are in regions of the array that are not associated with the droplet  104 , i.e., the droplets  102   b  are not involved in maintaining the droplet current position and thus remain unactuated during the entire actuation sequence A-F. 
     As shown in  FIG. 11 , sequence step A is an actuation-off period in which the droplet  104  is located at a particular position on the array  100 . In sequence step B, the droplet has drifted from the desired state position of sequence step A. The droplet position may be tracked by the sensor until the droplet has deviated from the desired position by a predetermined amount or other predetermined criteria. In the example of  FIG. 11 , the droplet position drifts further until at sequence step C, the droplet  104  has deviated from the desired position by a predetermined amount. Accordingly, at sequence step C the control system applies actuation voltages to the portion of array elements  102   a  associated with the droplet. When actuated, the electrical field draws the droplet to the array element electrodes, and as shown in sequence step D, the droplet  104  is drawn by the electric field to be commensurate with the actuated array element boundaries. The result is the droplet  104  is drawn back to the position of the desired state, and when the actuation voltages are removed as shown in sequence step E, the droplet  104  resumes its unactuated state located back at the desired position commensurate with sequence step A. 
     By applying intermittent actuation in accordance with the sensor information, the droplet  104  generally maintains its desired state and any deviations from the initial position of sequence step A beyond predetermined criteria are eliminated. Again, because droplet changes in state occur very gradually over time, the actuation-off period may be significantly greater than the actuation-on period. In addition, by using sensor-based actuation, the actuation-off period is minimized insofar as actuation voltages are applied only as needed to return the droplet to the desired state (the desired position in the example of  FIG. 11 ). Comparable actuation control may be applied to properties associated with any suitable desired state, as described with respect to the examples of the additional figures below. 
       FIG. 12  is a variation in which a sequence of electrode actuation comprises a sensor-based intervention for maintaining a desired shape or aspect ratio of a droplet. The steps of the sequence of actuation in  FIG. 12  similarly are respectively labeled A-E. Sequence step A is an actuation-off period in which the droplet  104  has a particular shape and aspect ratio on the array  100 . Further in the example of  FIG. 12 , there are 32 array elements  102   a  associated with the droplet  104  due to the elongated or ovular shape of the droplet  104  in the desired state shown in sequence step A. In sequence step B, the droplet has deviated from the desired state shape of sequence step A, flattening out to become more circular. The droplet shape and aspect ratio may be tracked by the sensor until the droplet has deviated from the desired shape of sequence step A by a predetermined amount or other predetermined criteria. Accordingly, at sequence step C the control system applies actuation voltages to the portion of array elements  102   a  associated with the droplet. When actuated, the electrical field draws the droplet to the array element electrodes, and as shown in sequence step D, the droplet  104  is drawn by the electric field to be commensurate with the actuated array element boundaries. The result is the droplet  104  is drawn back to the shape and aspect ratio of the desired state, and when the actuation voltages are removed as shown in sequence step E, the droplet  104  resumes its unactuated state having the desired shape and aspect ratio commensurate with sequence step A. 
       FIG. 13  shows a variation in which a sequence of electrode actuation comprises a sensor-based intervention for maintaining a desired size of a droplet. The steps of the sequence of actuation in  FIG. 13  similarly are respectively labeled A-E. Sequence step A is an actuation-off period in which the droplet  104  has a particular size on the array  100 . In sequence step B, the droplet has deviated from the desired state size of sequence step A, widening out into a larger circle. The droplet size may be tracked by the sensor until the droplet has deviated from the desired size by a predetermined amount or other predetermined criteria. In the example of  FIG. 13 , the droplet size deviates further until at sequence step C, the droplet  104  has deviated from the desired size by a predetermined amount. Accordingly, at sequence step C the control system applies actuation voltages to the portion of array elements  102   a  associated with the droplet. When actuated, the electric field draws the droplet to the array element electrodes, and as shown in sequence step D, the droplet  104  is drawn by the electric field to be commensurate with the actuated array element boundaries. The result is the droplet  104  is drawn back to the size of the desired state, and when the actuation voltages are removed as shown in sequence step E, the droplet  104  resumes its unactuated state with the desired size commensurate with sequence step A. 
       FIG. 14  shows another variation in which a sequence of electrode actuation comprises a sensor-based intervention for maintaining a desired position of a droplet, similar to  FIG. 11 . The steps of the sequence of actuation in  FIG. 14  similarly are respectively labeled A-E. In this particular example, the droplet  104  also is positioned in proximity to a second object whose position is known and with which a collision is non-desirable. In the specific example of  FIG. 14 , the second object is a second droplet  106 , whose position also may be sensed by any suitable sensor (e.g., impedance sensor circuit or suitable external sensor). The second object, however, may be an object other than another droplet, such as for example a physical barrier within the device, like a device wall or spacer. 
     As shown in the example of  FIG. 14 , sequence step A is an actuation-off period in which the first droplet  104  is located at a particular position on the array  100  with suitable spacing apart from the second object (e.g., second droplet)  106 . In sequence step B, the first droplet  104  has drifted from the desired state position of sequence step A. The droplet position may be tracked by the sensor until the first droplet  104  has deviated from the desired position by a predetermined amount or other predetermined criteria. In this example, the predetermined criteria may be a threshold proximity to the second object (e.g., second droplet  106 ), which can present a potential for collision. In the example of  FIG. 14 , the first droplet position drifts further until at sequence step C, the first droplet  104  has deviated from the desired position in accordance with the predetermined criteria being in close proximity to the second droplet  106 . Accordingly, at sequence step C the control system applies actuation voltages to the portion of array elements  102   a  associated with the first droplet  104 . When actuated, the electrical field draws the first droplet to the array element electrodes, and as shown in sequence step D, the first droplet  104  is drawn by the electric field to be commensurate with the actuated array element boundaries. The result is the first droplet  104  is drawn back to the position of the desired state, and when the actuation voltages are removed as shown in sequence step E, the first droplet  104  resumes its unactuated state located back at the desired position suitably spaced apart from the second droplet  106  commensurate with sequence step A. 
       FIGS. 15A and 15B  show another variation in which a sequence of electrode actuation comprises a sensor-based intervention for maintaining a desired position of a droplet, similar to  FIG. 14  except the second droplet  106  is being moved across the array  100  in a direction indicated by the arrows in the sub-figures. As referenced above, the position of the second droplet  106  also may be sensed by any suitable sensor (e.g., impedance sensor circuit or suitable external sensor), and the movement of the second droplet  106  may be achieved by a sequential actuation of another portion of the array elements  102   c  associated with moving the second droplet  106  as is known in the art. A collision of the two droplets is undesirable, and the steps of the sequence of actuation in  FIGS. 15A and 15B  to avoid such a collision is shown similarly in sequence steps A-E. 
     As shown in the example of  FIGS. 15A and 15B , sequence step A is an actuation-off period in which the first droplet  104  is located at a particular position on the array  100  without suitable spacing apart from a path of the moving second droplet  106  indicated by the arrow. At such position, there is a potential for collision with the moving second droplet  106 . As in previous embodiments, the droplet position may be tracked by the sensor to determine if the droplet position is different from a desired position by a predetermined amount or other predetermined criteria. In this example, the predetermined criteria may be a threshold proximity to the path of the moving second droplet  106 , which can present a potential for collision. In this example, in view of the path of the moving second droplet  106 , the droplet state of sequence step A can be considered a non-desirable state even though such position previously may have been a desirable state. Accordingly, at sequence step B the control system applies actuation voltages to the portion of array elements  102   a  associated with the first droplet  104 . When actuated, the electrical field draws the first droplet to the array element electrodes, and as shown in sequence step C, the first droplet  104  is drawn by the electric field to be commensurate with the actuated array element boundaries. The result is the first droplet  104  is drawn to the position of a desired state suitably spaced apart from the path of the moving droplet  106 . This actuation may be maintained as shown in sequence step D as the second droplet  106  passes the first droplet  104 , thereby precluding any collision. When the actuation voltages are removed as shown in sequence step E, the first droplet  104  resumes its unactuated state located now at the desired position suitably spaced apart from the second droplet  106 . Alternatively, after the second droplet  106  has passed the first droplet  104 , the control system may apply actuation voltages to a portion of array elements as appropriate to return the droplet to the initial position of sequence step A. 
     The control system and related control methods of the present invention, therefore, operate to provide intermittent actuation of the array elements to minimize the time over which EWOD or AM-EWOD elements are actuated while still effectively performing requisite droplet operations. By minimizing actuation time of the array elements, which minimizes exposure to the generated electric fields, the propensity to damage the subject droplets or device components is reduced. Intermittent actuation thus advantageously limits the time period over which the array elements and associated droplet are actuated. This improves the device reliability and/or prevents damage to chemically or biologically fragile reagents within the droplet. 
     Because the sensor-based intervention is targeted to the droplet state, more optimized actuation time periods are achieved, and therefore sensor-based intervention is preferred in EWOD systems that employ sensors. The presence of sensors, however, is not beneficial or feasible in all EWOD applications or technologies. For example, sensors typically are not employed in passive EWOD devices, and in some EWOD devices the pixel size may be too small to incorporate effective sensor circuitry or other sensors. For such EWOD devices that do not employ sensors, intermittent actuation by a preset or predetermined duty cycle is advantageous. 
     As aspect of the invention, therefore, is an enhanced microfluidic system including an electro-wetting on dielectric (EWOD) device comprising an element array configured to receive one or more fluid droplets, the element array comprising a plurality of individual array elements, and a control system configured to control actuation voltages applied to the element array to perform manipulation operations as to the fluid droplets. In exemplary embodiments, the control system is configured to apply a sequence of actuation voltages to a portion of the array elements associated with a droplet to maintain the droplet in a desired droplet state corresponding to a predetermined droplet property. The sequence of actuation voltages includes an actuation-on period in which the portion of the array elements associated with the droplet is actuated and an actuation-off period in which the portion of the array elements associated with the droplet is not actuated, and the actuation-off period is non-zero. The microfluidic system may include one or more of the following features, either individually or in combination. 
     In an exemplary embodiment of the microfluidic system, the control system is configured to apply a sequence of actuation voltages comprising a predetermined duty cycle including a predetermined time, rate and duration of actuation voltages to the portion of the array elements associated with the droplet. 
     In an exemplary embodiment of the microfluidic system, the actuation-off period of the duty cycle is greater than the actuation-on period of the duty cycle. 
     In an exemplary embodiment of the microfluidic system, a ratio of the actuation-on period of the duty cycle to the actuation-off period of the duty cycle is less than or equal to 1:10. 
     In an exemplary embodiment of the microfluidic system, the system further includes a sensor for sensing a droplet state, and the control system is configured to: receive droplet state information from the sensor; determine whether a droplet is in a state that deviates from the desired droplet state in accordance with predetermined criteria based on the droplet state information; and apply actuation voltages to the portion of the array elements associated with the droplet when the control system determines that the droplet state satisfies the predetermined criteria to return the droplet to the desired droplet state. 
     In an exemplary embodiment of the microfluidic system, the sensor comprises sensor circuitry incorporated into one or more array elements. 
     In an exemplary embodiment of the microfluidic system, the predetermined droplet property of the desired droplet state is based on at least one of droplet position on the element array, droplet shape or aspect ratio, droplet size, proximity of the droplet to a second object or second droplet on the element array, and proximity of the droplet to a path of a second droplet moving along the array. 
     Another aspect of the invention is a related control method for controlling actuation voltages applied to array elements of an element array on an electro-wetting on dielectric (EWOD) device. In exemplary embodiments, the control method includes the steps of: receiving one or more fluid droplets on the element array; and applying a sequence of actuation voltages to a portion of the array elements associated with a droplet to maintain the droplet in a desired droplet state corresponding to a predetermined droplet property; wherein the sequence of actuation voltages includes an actuation-on period in which the portion of the array elements associated with the droplet is actuated and an actuation-off period in which the portion of the array elements associated with the droplet is not actuated, and the actuation-off period is non-zero. The microfluidic system may include one or more of the following features, either individually or in combination. 
     In an exemplary embodiment of the control method, applying a sequence of actuation voltages comprises applying actuation voltages in accordance with a predetermined duty cycle including a predetermined time, rate and duration of actuation voltages to the portion of the array elements associated with the droplet. 
     In an exemplary embodiment of the control method, the actuation-off period of the duty cycle is greater than the actuation-on period of the duty cycle. 
     In an exemplary embodiment of the control method, a ratio of the actuation-on period of the duty cycle to the actuation-off period of the duty cycle is less than or equal to 1:10. 
     In an exemplary embodiment of the control method, the duty cycle comprises applying actuation voltages to the array elements associated with a droplet for 0.5 seconds once every 5.0 seconds. 
     In an exemplary embodiment of the control method, the control method further includes: sensing a droplet state with a sensor; determining whether the sensed droplet state is a state that deviates from the desired droplet state in accordance with predetermined criteria; and applying actuation voltages to the portion of the array elements associated with the droplet when it is determined that the droplet state satisfies the predetermined criteria to return the droplet state to the desired droplet state. 
     In an exemplary embodiment of the control method, sensing a droplet state with the sensor comprises sensing a droplet position on the element array, the control method further comprising: determining whether the sensed droplet state is a state in which the droplet position deviates from a desired droplet state position in accordance with the predetermined criteria; and applying actuation voltages to the portion of the array elements associated with the droplet when it is determined that the droplet state satisfies the predetermined criteria to return the droplet state to the desired droplet state position. 
     In an exemplary embodiment of the control method, the predetermined criteria includes whether the droplet is at a position within a preset proximity to a second object on the element array. 
     In an exemplary embodiment of the control method, the predetermined criteria includes whether the droplet is at a position within a preset proximity to a path of a moving second droplet on the element array. 
     In an exemplary embodiment of the control method, sensing a droplet state with the sensor comprises sensing a droplet shape or aspect ratio, the control method further comprising: determining whether the sensed droplet state is a state in which the droplet shape or aspect ratio deviates from a desired droplet state shape or aspect ratio in accordance with predetermined criteria; and applying actuation voltages to the portion of the array elements associated with the droplet when it is determined that the droplet state satisfies the predetermined criteria to return the droplet state to the desired droplet state shape or aspect ratio. 
     In an exemplary embodiment of the control method, sensing a droplet state with the sensor comprises sensing a droplet size, the control method further comprising: determining whether the sensed droplet state is a state in which the droplet size deviates from a desired droplet state size in accordance with predetermined criteria; and applying actuation voltages to the portion of the array elements associated with the droplet when it is determined that the droplet state satisfies the predetermined criteria to return the droplet state to the desired droplet state size. 
     Another aspect of the invention is a non-transitory computer-readable medium storing program code which is executed by a processing device for controlling actuation voltages applied to array elements of an element array of an electro-wetting on dielectric (EWOD) device for performing droplet manipulations on droplets on the element array. The program code is executable by the processing device to perform the steps of the control method. 
     Although the invention has been shown and described with respect to a certain embodiment or embodiments, equivalent alterations and modifications may occur to others skilled in the art upon the reading and understanding of this specification and the annexed drawings. In particular regard to the various functions performed by the above described elements (components, assemblies, devices, compositions, etc.), the terms (including a reference to a “means”) used to describe such elements are intended to correspond, unless otherwise indicated, to any element which performs the specified function of the described element (i.e., that is functionally equivalent), even though not structurally equivalent to the disclosed structure which performs the function in the herein exemplary embodiment or embodiments of the invention. In addition, while a particular feature of the invention may have been described above with respect to only one or more of several embodiments, such feature may be combined with one or more other features of the other embodiments, as may be desired and advantageous for any given or particular application. 
     INDUSTRIAL APPLICABILITY 
     The described embodiments could be used to provide an enhance AM-EWOD device. The AM-EWOD device could form a part of a lab-on-a-chip system. Such devices could be used in manipulating, reacting and sensing chemical, biochemical or physiological materials. Applications include healthcare diagnostic testing, material testing, chemical or biochemical material synthesis, proteomics, tools for research in life sciences and forensic science. 
     REFERENCE SIGNS LIST 
     
         
         
           
               10 —lower substrate 
               12 / 12 A. 12 B—element electrodes 
               14 —liquid droplet 
               16 —top substrate 
               18 —spacer 
               20 —non-polar surround fluid 
               22 —insulator layer 
               24 —first hydrophobic coating 
               26 —contact angle 
               28 —second hydrophobic coating 
               30 —reference electrode 
               32 —reader 
               34 —cartridge 
               35 —external sensor module 
               36 —EWOD or AM-EWOD device 
               38 —control electronics 
               40 —storage device 
               42 —cable of connecting wires 
               44 —lower substrate 
               46 —thin film electronics 
               48 / 48 A/ 48 B—element electrodes 
               50 —electrode element array 
               51 —array element 
               52 —liquid droplet 
               54 —top substrate 
               56 —spacer 
               58 —reference electrode 
               60 —non-polar fluid 
               62 —insulator layer 
               64 —first hydrophobic coating 
               66 —contact angle 
               68 —second hydrophobic coating 
               70 A—electrical load with droplet present 
               70 B—electrical load without droplet present 
               72 —array element circuit 
               74 —row driver 
               76 —column driver 
               78 —sensor row addressing circuits 
               80 —column detection circuits 
               82 —serial interface 
               84 —voltage supply interface 
               86 —number of connecting wires 
               88 —actuation circuit 
               90 —sensor circuit 
               100 —array of elements 
               102 —individual array elements 
               102   a —array elements associated with the droplet 
               102   b —array elements not associated with the droplet 
               102   c —array elements associated with a second droplet 
               104 —droplet 
               106 —second droplet