Patent Publication Number: US-10315911-B2

Title: Spacer for side loaded EWOD device

Description:
TECHNICAL FIELD 
     The present invention relates to droplet microfluidic devices, and more specifically to Active Matrix Electro-wetting-On-Dielectric (AM-EWOD) devices, including input structures for enhanced loading of fluid into such devices. 
     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 or channel between the two substrates may be realized by means of a spacer  18 , and a nonpolar surround fluid  20  (e.g. oil) may be used to occupy the volume not occupied by the liquid droplet  14 . The function of the oil is to reduce the surface tension at the surfaces of the polar droplets, and to increase the electro-wetting force, which ultimately leads to the ability to create small droplets and to move them quickly. It is usually beneficial, therefore, for the oil to be present within the channel of the device before any polar fluids are introduced therein. 
     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: 
     
       
         
           
             
               
                 
                   
                     cos 
                     ⁢ 
                     
                         
                     
                     ⁢ 
                     θ 
                   
                   = 
                   
                     
                       
                         γ 
                         SG 
                       
                       - 
                       
                         γ 
                         SL 
                       
                     
                     
                       γ 
                       LG 
                     
                   
                 
               
               
                 
                   ( 
                   
                     equation 
                     ⁢ 
                     
                         
                     
                     ⁢ 
                     1 
                   
                   ) 
                 
               
             
           
         
       
     
     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.       

       FIG. 2  is a drawing depicting additional details of an exemplary AM-EWOD device  36  in schematic perspective, which may incorporate the layered structures in  FIG. 1 . The AM-EWOD device  36  has a lower substrate  44  with thin film electronics  46  disposed upon the lower substrate  44 , and a reference electrode (comparable to reference electrode  30  above) is incorporated into an upper substrate  54 . The electrode configuration may be reversed, with the thin film electronics being incorporated into the upper substrate and the reference electrode being incorporated into the lower substrate. 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 the upper substrate  54  separated by a spacer  56 , although it will be appreciated that multiple liquid droplets  52  can be present. 
     As described above with respect to the representative EWOD structure, the EWOD channel or gap defined by the two substrates initially is filled with the nonpolar fluid (oil). The liquid droplets  14 / 52  including a polar material, i.e., the droplets to be manipulated by operation of the EWOD device, must be inputted from an external “reservoir” of fluid into the EWOD channel or gap. The external reservoir may for example be a pipette, or may be a structure incorporated into the plastic housing of the device. As the fluid from the reservoir for the droplets is inputted, oil gets displaced and is removed from the EWOD channel. 
     Different mechanisms have been devised for the inputting or loading of fluid into such devices. For example, U.S. Pat. No. 8,686,344 (Sudarsan et al., issued Apr. 1, 2014) describes a method of fluid loading utilizing patterning of the hydrophobic layers disposed upon the device surfaces. WO 2015/023747 (Yi et al., published Feb. 19, 2016) and US 2016/0016170 (Lay et al., published Jan. 21, 2016) both describe an EWOD cartridge assembly including upper and lower EWOD substrates and a plastic part which functions as a pipette guide. 
     Achieving entry of the polar fluid into the EWOD channel is non-trivial because the internal surfaces of the EWOD device are hydrophobic. Additionally, the direction of travel of the fluids once in the EWOD channel must be controlled, for example such that different fluids input through different and adjacent ports do not accidentally combine together or mix. 
     A conventional method of achieving controlled fluid entry is to create an upper substrate with apertures (holes) drilled or otherwise incorporated into the upper substrate. Apertures in the upper substrate provide for convenient fluid input, but of course require the thin film electronics to be on the bottom substrate. The apertures define fluid input ports and a fluid path from the exterior of the EWOD device into the EWOD channel directly above the electrowetting array. Employing apertures in the upper substrate, however, may be difficult and expensive to manufacture, particularly because the preferred material of the upper and lower substrates is glass. Glass is commonly the preferred material because glass is compatible with common LCD manufacturing technologies. It furthermore is advantageous to make the EWOD device with the same materials for the upper and lower substrates to achieve a high precision in the EWOD channel gap, and to minimize deleterious effects of working with different materials of different thermal expansion coefficients for usages in which the device is to be heated. In normal usage, the number of distinct polar fluid entry points is determined by the number of apertures within the upper substrate. As the number of apertures in the upper substrate is increased, both the cost of production and the fragility of the upper substrate goes up. 
     An alternative approach is to use a side loading configuration that does not require apertures formed in the upper substrate (e.g., the upper substrate is simply rectangular). Fluids that are to enter the EWOD channel are inputted through a side of the EWOD channel between the two substrates, rather than through apertures in the upper substrate. By using an upper substrate with no apertures, the cost and mechanical strength of the upper substrate is completely independent of the number of polar fluid entry points that are required, potentially enabling a higher density of fluid entry points than can be incorporated into the EWOD device when the upper substrate has apertures. It has been difficult, however, to achieve precise control of polar fluid input with current side loading configurations. 
     GB 2542372 (Walton et al., published Mar. 22, 2017) is another design by the current inventors. Such disclosure describes a method of fluid loading and discloses a spacer design for side-loading of fluid into the EWOD channel. This simple side loading, however, does not incorporate any particular components for ensuring that polar fluid input from outside of the EWOD device is specifically able to enter the EWOD channel. 
     SUMMARY OF INVENTION 
     The manner and efficiency of the input of the polar fluid for the liquid droplets can affect the overall performance of the EWOD device. The present invention relates to EWOD and AM-EWOD microfluidic devices, and to controlling the input and output of fluids to and from the hydrophobic EWOD channel defined between the substrate assemblies of such devices. The present disclosure describes an enhanced spacer configuration that permits side loading, and with added structures for ensuring fluid input from outside the EWOD device is able to enter the EWOD channel in a well-controlled manner. 
     The inventors have recognized that prior side loading designs, while providing advantages over apertures in the upper glass substrate, can encounter deficiencies with respect to how to control the input of the fluids in the case where an incomplete oil filling loading method (as described in GB 2542372) is used. This problem is illustrated, for example, in  FIGS. 3 and 4  showing an example of a side loading EWOD device  60  that is insufficient for controlling the position of the air-oil interface when an incomplete oil filling method is to be used. Shown schematically from a top view are an upper glass substrate  62  atop a lower substrate  64 , and separated by a spacer  66 . An active area of the device  67  ( FIG. 3 ) may be defined as the region containing electrowetting electrodes, atop which liquid droplets may be manipulated. In operation, the droplets are typically manipulated within a non-polar liquid (e.g. oil) as the surrounding medium. It is not necessary, however, that to manipulate droplets the entirety of the active area be filled with oil. Rather, the oil only needs to be present at the boundaries of the liquid droplets. This is exploited in the filling mechanism of the device whereby the channel (optionally including some of the channel that is also the active area) is incompletely filled with oil prior to the polar fluid loading. This incomplete filling by the oil is able to assist in the filling of the polar fluid. Thus, one of the problems being solved by the invention is how to incompletely fill the oil while also ensuring that the oil goes to the correct parts of the channel to assist with the filling of the polar fluid. 
     In typical operation, the oil is initially loaded into the device. After the initial loading of oil  68 , an air bubble  70  will position itself to be along the edges of the upper substrate where the spacer  66  is absent as shown in  FIG. 4 . If subsequently fluid loading is performed along the edges of the EWOD device where the spacer is absent, proper fluid loading can fail along the edge of the device which has the air bubble. 
     As an example of successful versus failed side loading,  FIG. 5  shows a possible scenario that can occur if the polar fluid is loaded vertically (e.g. via a pipette dock) along the edge that does have the oil present at the air boundary.  FIG. 5  depicts a side view of the EWOD device  60  comparable as in  FIGS. 3 and 4 . A pipette  72  (which may be part of a pipette dock, not shown) may be employed to load the polar fluid  74  into the oil  68 . The lower left portion of  FIG. 5  shows a successful loading by which the polar fluid  74  comes into contact with the nonpolar fluid (oil)  68 , and the polar fluid can be drawn into the EWOD channel using the electrowetting force. However, alternatively a failed loading of the polar fluid  74  can occur in which the polar fluid  74  does not make contact with the oil  68 . An unsuccessful fluid loading is shown in the lower right portion of  FIG. 5 . In such case, the polar fluid may load in the direction away from the upper substrate and EWOD channel and become stranded spaced away from the EWOD channel. If this happens, no contact is made with the oil at the edge of the EWOD channel, and loading of the polar fluid cannot be achieved using the electrowetting force. Previous side loading designs are not configured with appropriate structures to ensure that proper side loading occurs to avoid the unsuccessful loading shown in  FIG. 5 . 
     The device of the present disclosure describes structures and related methods of side loading of fluid whereby the above deficiencies associated with prior side loading designs are overcome, and with an EWOD device structure that is simple and compatible with a low-cost assembly process. The described designs include fluid input ports incorporated into a spacer component, configured to facilitate loading of both the polar and nonpolar fluids. The side loading of the present invention has advantageous characteristics. When the EWOD device is initially, and incompletely, filled with the nonpolar filler fluid (oil), the nonpolar filler fluid is presented at positions at which the polar fluid enters via the fluid input ports to ensure contact of the polar fluid with the nonpolar oil. This is significant because contact of the polar fluid with the nonpolar filler fluid provides an enhanced condition for successful loading of the polar fluid into the EWOD channel. When the polar fluid is loaded in a direction that is substantially orthogonal to the EWOD substrates, the polar fluid always enters the EWOD channel without being stranded apart from the EWOD channel as can occur in prior configurations. 
     The described EWOD device configurations address several significant issues associated with proper loading of fluid into an EWOD device. Such issues include, for example: spacing the EWOD substrates apart uniformly and by the appropriate gap to form the EWOD channel; ensuring that when the device is partially filled with oil that oil will be present at the points at which the polar fluid subsequently is introduced; and controlling the entry of the polar fluid into the EWOD channel of the EWOD device. 
     An enhanced EWOD device configuration facilitates side loading and resolves the above problems and issues associated with prior designs. In exemplary embodiments, an EWOD device includes a spacer (typically made of plastic). The spacer extends into the EWOD channel between the upper and lower substrates, having at least a spacer part located at least at one edge of the EWOD channel, and the spacer part defines at least one fluid port including an air gap defining an input (or output) from the EWOD channel to the EWOD device exterior. 
     In other exemplary embodiments, the spacer part may include or perform the functions of one or more of defining the cell gap spacing of the EWOD channel between the upper and lower substrates; defining geometries of fluid input ports optimized for inputting (or outputting) of nonpolar (e.g. oil) or polar (e.g. aqueous) fluid input into the EWOD channel; and defining an interface to the outside of the EWOD device for the input and output of fluids from the device, for example pipette guides. The design of the spacer for a given EWOD device may be optimized specific to a design of the active area of the EWOD device; a position of the oil filling port of the EWOD device; points at which polar fluid is subsequently to be loaded in the EWOD channel; a number and volume of separate polar reagents to be loaded into the EWOD channel; and a number and volume of separate polar reagents to be unloaded/extracted from the EWOD channel. 
     An aspect of the invention, therefore, is an EWOD device that has an enhanced spacer configuration, by which the spacer defines the fluid input ports and is configured in a manner that ensures fluid input from the outside of the device is able to enter the EWOD channel. In exemplary embodiments, the EWOD device includes a first substrate assembly and a second substrate assembly, and a spacer that spaces apart the first substrate assembly from the second substrate assembly to define a channel between the first and second substrate assemblies. The spacer defines a plurality of fluid input ports that are in fluid communication with the channel, and the spacer is configured for directing fluid from the fluid input ports into the channel. The spacer has a combed spacer configuration to define the plurality of fluid input ports, the combed spacer configuration including alternating teeth that extend into the channel from a base region, and the teeth isolate adjacent fluid input ports from each other. The spacer may contact only a portion of the first and second substrate assemblies to form a spacerless region within the EWOD device, and the spacer includes regions that are in contact with both the first and second substrate assemblies and extend into the channel to define a cell-gap of the channel. 
     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. 
    
    
     
       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 AM-EWOD device in schematic perspective. 
         FIG. 3  is a drawing depicting an exemplary side loading EWOD device. 
         FIG. 4  is a drawing depicting the exemplary side loading EWOD device of  FIG. 3  with incomplete oil filling. 
         FIG. 5  is a drawing depicting an exemplary side loading EWOD device illustrating successful and unsuccessful side fluid loading. 
         FIG. 6 , including the additional views of  FIG. 6( a ) ,  FIG. 6( b ) ,  FIG. 6( c ) , and  FIG. 6( d ) , are drawings depicting a plan view and side cross section views along different sections of a first exemplary EWOD device in accordance with embodiments of the present invention. 
         FIG. 7  is a drawing depicting a close-up plan view of a portion of the exemplary EWOD device of  FIG. 6 . 
         FIG. 8  is a drawing depicting the plan view of the exemplary EWOD device of  FIG. 6  with incomplete oil filling. 
         FIG. 9  is a drawing depicting side views of the exemplary EWOD device of  FIG. 6  illustrating successful and recoverable side loading. 
         FIG. 10  is a drawing depicting a side cross section view and a plan view of another exemplary EWOD device in accordance with embodiments of the present invention. 
         FIGS. 11( a ), 11( b ), and 11( c )  are drawings depicting side cross-section views of variations of an exemplary EWOD device in accordance with embodiments of the present invention. 
         FIG. 12  is a drawing depicting a side cross-section view of another exemplary EWOD device in accordance with embodiments of the present invention. 
         FIGS. 13( a ), 13( b ), 13( c ), and 13( d )  are drawings depicting side cross-section views of another an exemplary EWOD device in accordance with embodiments of the present invention, with the figures showing a progression of side fluid loading. 
         FIG. 14  is a drawing depicting a plan view of an EWOD device using an oil shell method of fluid loading. 
         FIG. 15  is a drawing depicting a plan view of the exemplary EWOD device of  FIG. 6 , as shown using an oil shell method of fluid loading. 
     
    
    
     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. 
     Generally, an aspect of the invention is an EWOD device that has an enhanced spacer configuration, by which the spacer defines the fluid input ports and is configured in a manner that ensures fluid input from the outside of the device is able to enter the EWOD channel. In exemplary embodiments, the EWOD device includes a first substrate assembly and a second substrate assembly, and a spacer that spaces apart the first substrate assembly from the second substrate assembly to define a channel between the first and second substrate assemblies. The spacer defines a plurality of fluid input ports that are in fluid communication with the channel, and the spacer is configured for directing fluid from the fluid input ports into the channel. The spacer has a combed spacer configuration to define the plurality of fluid input ports, the combed spacer configuration including alternating teeth that extend into the channel from a base region, and the teeth isolate adjacent fluid input ports from each other. The spacer may contact only a portion of the first and second substrate assemblies to form a spacerless region within the EWOD device, and the spacer includes regions that are in contact with both the first and second substrate assemblies and extend into the channel to define a cell-gap of the channel 
       FIG. 6  is a drawing depicting a top view of an exemplary EWOD device  80  in accordance with embodiments of the present invention.  FIG. 6  further shows respectively different cross-sections (a)-(d) of the EWOD device  80  along the lines A-A′, B-B′, C-C′ and D-D′. 
     The EWOD device  80  includes a first or upper substrate assembly  82  and a second or lower substrate assembly  84  separated by a spacer  86 . For simplicity of illustration of pertinent features, the individual layers of the EWOD device components are omitted. Accordingly, the first and second substrate assemblies may include the associated substrate layers, insulating layers, electrode layers, and related structures that form the EWOD device as are known in the art. Typically, the second (lower) substrate assembly  84  constitutes the TFT substrate that would include the thin film electronics, with the first (upper) substrate assembly  82  incorporating the reference electrode. This, however, may be reversed with the first (upper) substrate assembly  82  constituting the TFT substrate and the second (lower) substrate assembly  84  incorporating the reference electrode. 
     The spacer is configured to have a desired width or thickness so as to correctly position the substrates relative to each other to define an EWOD channel  88 . Example materials for the spacer can include suitable rigid sheet plastics, such as for example polycarbonate, PET, polystyrene, polyester, polyimides (e.g. Kapton, Cirlex), or Mylar. As seen in the cross-section views, the spacer  86  may rest on one of the substrates, such as the lower substrate  84 , and generally may not contact the other substrate, e.g., upper substrate  82 , at all surfaces of the spacer. The spacer, however, has at least regions  90  (see cross sections (b) and (d)) that extend into the EWOD channel  88  and thus are in contact with both substrates so as to properly space the substrates apart to form the EWOD channel of a desired width. The spacer thereby addresses the issue of spacing the EWOD substrates apart uniformly and with the proper spacing to form a suitable cell-gap of the EWOD channel  88 . 
     The spacer  86  is configured for side loading of fluids into the EWOD channel. Accordingly, there is a spacerless region  92  of the EWOD device at which the perimeter of the EWOD device has no spacer portion. The spacerless region  92  is therefore open to air and has no spacer.  FIG. 6  shows the device in plan view including an active area  93  as may be filled at least partially with the non-polar fluid or oil. The oil tends to pin to the spacer by capillary action, and thus there is no oil that fills the spacerless region  92 . Accordingly, the open spacerless region  92  permits a side loading of polar fluid where the spacer is absent in a well-controlled position and manner. 
     The spacer  86  is configured as a “combed” spacer including alternating teeth  96  that extend from a base region  97  to define a plurality of fluid input ports  98 .  FIG. 7  is a drawing depicting a close-up view of a portion of the EWOD device to better illustrate the sub-components of the spacer that form the fluid input ports  98 . The teeth  96  extend from the base region  97  into the EWOD channel, and thus provide overlapping regions of the spacer with the oil  94 . In the example of  FIG. 6 , there are twenty-two fluid input ports  98  along the opposing longitudinal sides of the spacer, although any suitable number and location of fluid input ports may be formed. By forming fluid input ports using the combed nature of the spacer  86 , the configuration of the EWOD device  80  obviates the need to form fluid input ports by drilling or otherwise providing holes in the either of the glass substrates  82  or  84 . Additional details regarding the configuration and functions of the combed spacer and associated fluid input ports are detailed below. 
     As described above, in certain circumstances it may be desirable to fill the EWOD channel incompletely with oil, which permits the formation of an air bubble comparably as formed in  FIG. 4 .  FIG. 8  is a drawing depicting the EWOD device  80  when the EWOD channel is incompletely filled with oil. When the EWOD channel is incompletely filled with oil, the configuration of EWOD device  80  forms an air bubble  100 , the air bubble being formed in a controlled position in the spacerless region  92  to permit in turn well-controlled fluid loading by venting air when oil is displaced by the input of polar fluid. 
     When incompletely filled with oil, the oil still will tend to pin to those areas of the spacer that overlap with the EWOD channel of the device, and therefore the air bubble  100  will tend to reside at the spacerless region  92  where there is no spacer in the EWOD channel, as shown in  FIG. 8 . This is useful because it allows polar fluids subsequently to be loaded along the edges of the upper substrate where the combed spacer forms the fluid inputs  98  where polar fluid may be injected. Such configuration performs the function of ensuring that when the EWOD channel is only partially or incompletely filled with oil, oil will be present at points at which the polar fluid subsequently may be inputted into the EWOD channel. When the polar fluid is inputted, oil gets displaced and by virtue of such displacement, air may vent out from the air bubble  100 . 
     As referenced above, the spacer teeth  96  form overlapping regions where the spacer overlaps the oil, and in between the overlapping regions the teeth define the fluid input ports  98 . The fluid input ports  98  thus are openings in the spacer that allow polar fluid to enter the device in contact with the oil. Under circumstances in which the polar fluid is loaded at an angle, the polar fluid can easily be injected into the EWOD channel of the device in a well-controlled manner. The combed nature of the spacer also operates such that the polar fluid will not come into contact with parts of the spacer other than the overlapping regions for a controlled fluid input. In other words, the configuration of the teeth  96  and base region  97  confines inputted polar fluid within a particular fluid input port  98  to prevent contact and mixing of different polar fluids. 
       FIG. 9  is drawing depicting scenarios of fluid input in the EWOD device  80  from a side view, similarly as depicted in  FIG. 5 , but illustrating how the spacer  86  ensures successful fluid input. The lower left side of  FIG. 9  shows ordinary successful fluid loading as would occur when pipetted polar fluid  102  contacts the oil upon input, similarly as the successful loading depicted by the left portion of  FIG. 5 . Electrowetting forces can be employed to draw the polar fluid into the EWOD channel. 
     In addition, the configuration of the EWOD device  80  with the spacer  86  further avoids the problem associated with  FIG. 5  of the polar fluid potentially being stranded away from the EWOD channel. Rather, the design of the fluid input ports  98  can be tailored to ensure that that any backward flowing fluid will be caught by the spacer and directed into the EWOD channel, referred to herein as “recoverable loading”. The lower right portions of  FIG. 9  illustrate recoverable loading and how the spacer configuration overcomes the problem associated with unsuccessful loading of prior designs (e.g.,  FIG. 5 ). As  FIG. 9  shows, provided that sufficient polar fluid  102  is loaded, the teeth and base region of spacer  86  that define the fluid input port will prevent any backward flowing fluid from moving too far from the EWOD channel of the device, as shown in the first step of recoverable loading. Eventually, the fluid issuing from the pipette will touch the oil and enter the EWOD channel of the device before the pipette is retracted as shown in the second step of recoverable loading. What is required is to control the polar fluid so that the polar fluid cannot be loaded into the device without coming into contact with the oil within the EWOD channel. So long as contact with the oil is made, and there exists in the EWOD channel of the device an air bubble (e.g., air bubble  100 ) that is open to the exterior of the EWOD device so that air can vent from oil displacement by the polar fluid, then the polar fluid will enter the EWOD channel under control of the electrowetting voltage. In this manner, the EWOD device  80  addresses the issue of how to control entry of the polar fluid into the EWOD channel of the device. 
     The optimum design for the dimensions of the spacer may depend on the dimensions and location of the pipette or other object that is injecting the polar fluid, the angle of fluid injection, the contact angle of the polar fluid with the lower EWOD substrate, and the nature of the polar fluid and its affinity for the spacer material. Typically, there should be one fluid input for  98  in the spacer for each reagent to be injected, and that the smaller the volume to be injected, the closer the back of the spacer should be to the end of the polar fluid injector. In addition, it is typically advantageous to avoid sharp corners in the spacer, for example, by using curved edges or rounded edges where the fluid input ports are defined, as shown for example in the close-up view of  FIG. 7 . Typically also, independently of the design of the opening part of the spacer, there will be a minimum loadable volume which is determined by the proximity of the polar fluid injector to the edge of the upper EWOD substrate. 
     A principal advantage of the described configuration is that by defining fluid input ports with the spacer, the requirement for forming holes in the upper or lower substrate is removed. This advantage is particularly appreciable in the case of AM-EWOD devices in which the upper and lower substrates are both preferentially made of glass. This advantage is also particularly appreciable if there is a requirement for the device to have a large number of fluid input ports. Such devices are difficult and expensive to manufacture if the upper substrate has numerous holes, since the holes typically have to be created (e.g. by drilling) one at a time. Numerous holes in the substrate also renders the substrate more fragile This EWOD device  80 , enabling side-loading, thus greatly reduces the manufacturing cost of the device while enhancing durability. A further advantage is that the arrangement described facilitates ease of assembly, utilizing the dual function of the spacer to function as both the definer of the EWOD channel gap and the fluid input ports. 
     The described EWOD device configuration also addresses the significant issues associated with proper loading of fluid into an EWOD device referenced above, including for example: spacing the EWOD substrates apart uniformly and by the appropriate gap to form the EWOD channel; ensuring that when the device is partially filled with oil that oil will be present at the points at which the polar fluid subsequently is introduced; and controlling the entry of the polar fluid into the EWOD channel of the EWOD device. These issues are addressed in a cost-effective manner that is easy to implement without the deficiencies of prior designs. 
     The spacer  86  also can be configured to enhance fluid extraction to remove processed fluids to permit entry of new polar fluid for droplet manipulations. Referring back to  FIG. 6 ,  FIG. 6  illustrates a representative droplet  120  that has been moved to an exit port  122  formed in the spacer  86 . In particular, the exit port  122  may be configured as an extension of the spacer that forms a fluid passage into the EWOD channel. Although the exit port  122  is shown as extending from a spacer region that lacks fluid input ports, any suitable location may be employed. In addition, more than one exit port may be provided. 
     For successful droplet extraction, the port  122  should have an opening that has a diameter or width dimension that is no larger than a diameter of the smallest droplet  120  that may be subject to extraction. When the droplet  120  is moved to the exit port as shown in  FIG. 6 , the droplet initially blocks the exit port, essentially precluding additional oil from entering the exit port. The polar fluid of the droplet  120  may then be extracted through the exit port by electrowetting or other suitable extraction mechanism. Although a slight amount of the oil that already is within the exit port  122  may be extracted, such amount would be small and negligible, with mainly the polar fluid droplet being subject to extraction. 
     Such extraction process can also be combined with an adaptive electrode pattern that is applied to the polar droplet being extracted to keep the polar droplet in the correct position within the EWOD channel. Such electrode patterning is described, for example, in Applicant&#39;s patent application EP 16194633, which is incorporated here by reference. In this manner, the polar droplet  120  never moves away from the opening in the spacer exit port  122  through which the extraction draw electrowetting force is being applied, which results in a smooth and continuous extraction of the droplet with very little excess oil being drawn out at the same time. 
     Subsequent figures illustrate structural variations to the above. For ease of illustration, like structures are identified with like reference numerals as in the previous figures and generally are comparably configured. Additional explanation is provided as to the structural differences among the various embodiments. Relatedly, the various embodiments of the EWOD device generally will operate as described above, with certain modifications based on the structural variations as detailed below. 
       FIG. 10  is a drawing depicting a side view and a plan view (the plan view being a partial and close-up view) of an exemplary EWOD device  80   a  in accordance with another embodiment of the present invention. In this embodiment, first or upper substrate  82  is the active substrate including thin film drive electronics  104 , and the second or lower substrate  84  incorporates the reference electrode. Oil  94  being located with the EWOD channel  88  is shown. This arrangement may be advantageous if the application requires the droplets to be interrogated optically, for example to read out the results of an assay. Depending on the preferred geometry, it may be advantageous to perform optical interrogation of the EWOD device from below, in which case it is advantageous to have the EWOD drive electronics (which are partially absorbing and can be auto-fluorescent) on the upper substrate. 
     Typically in an AM-EWOD array, it is inconvenient to arrange for the droplet manipulation area of the substrate i.e., the active area, where the surface properties are controllable by electrowetting, to extend right to the edges of the lower substrate. This is due to the need to allow space for row and column driver circuits, and also because there is necessarily some tolerance in the precision to which the glass edge can be defined by the cutting process used to dice individual devices from large mother-glass sources upon which the substrates are typically fabricated. 
     Accordingly, the EWOD device  80   a  may include an active area  106  to which electrowetting voltages may be applied, and an inactive border region  108  that is not under active EWOD control. Given this arrangement, the fluid input ports must therefore be sized to receive a minimum volume of polar fluid that must be loaded (for each step of fluid input) to ensure that the fluid input at least partially overlaps the active area  106  of the device. This ensures that the entirety of the fluid input volume can then further be attracted onto the active area  106  by the electrowetting force and then subsequently manipulated in a controlled manner by the EWOD electrodes. To achieve such a configuration, as shown in the plan view, the teeth  96  are configured to extend beyond the inactive region  108  and into the active area  106 , which ensures that the fluid input ports  98  are contiguous with the active area. In addition, as referenced above and as shown in  FIG. 10  (see also  FIG. 7 ), a portion of a back of the spacer opposite from of the channel that defines the fluid input ports  98  is rounded in shape. 
     If, for example, a width of the border region is denoted “w” and the cell-gap dimension of the EWOD channel between the two substrates is denoted “d”, and with a back portion of the spacer being rounded as shown in  FIG. 10 , then the fluid input ports must be sized to receive a minimum volume of input fluid that is at least the volume of a disk of radius w/2 and height d, i.e. πdw 2 /4. For example, if the border region is 2 mm wide, and the cell-gap dimension is 250 um, then the minimum polar fluid that can be loaded (and still be useful on the EWOD array) is at least 0.8 ul. Applications which require a volume less than 0.8 ul to be loaded would not be suited to this particular geometry, because the polar fluid may not reach the active area. In applications in which relatively larger volumes of fluid are to be loaded, this geometry has certain advantages, in that the geometry may allow the storage of a certain amount of polar fluid volume within the inactive border region of the EWOD channel of the device, so that if large volumes of polar fluid are required to perform an assay on the EWOD array, the polar fluid can be stored after fluid loading without taking up valuable space on the active area of the device. 
     For the configuration of  FIG. 10 , as referenced above it is advantageous to extend the overlap regions of the spacer teeth  96  into the EWOD channel of the device to an extent that the spacer teeth  96  overlap with the active area  106  of the device, again as shown in the plan view of  FIG. 10 . This further retains different polar fluids isolated from each other in the inactive region  108  of the device in separated fluid input ports  98 , so that unwanted mixing of different fluids is prevented. 
       FIGS. 11( a ), 11( b ), and 11( c )  are drawings depicting side views of different exemplary alternative variations of an EWOD device in accordance with embodiments of the present invention. In these embodiments (in contrast to the previous embodiment), the second or lower substrate  84  is the active substrate including the thin film drive electronics  104 , and the first or upper substrate  82  incorporates the reference electrode. 
     These embodiments demonstrate different variations on how to configure the inactive border region. In the example EWOD device  80   b  of  FIG. 11( a ) , there is a symmetric inactive border region  108  within the EWOD channel where the upper substrate having the counter electrode extends beyond the edges of the active area  106  of the device, at least on some opposing edges. Because the size of the inactive border region  108  of the EWOD channel is determined by the size and shape chosen for the counter electrode (i.e., not by the design of the drive electronics for the active EWOD array), there is a greater degree of control over the size and shape of the border region. Accordingly, for example in another EWOD device configuration, an asymmetric border region  112  can be configured to have different sizes on differing edges of the device to suit the needs of the fluid volumes to be loaded in a particular application, as shown for example in an exemplary EWOD device  80   c  shown in  FIG. 11( b ) . 
     In addition, as referenced above in connection with the previous embodiment, one advantage of having the inactive border region is the potential for storage of larger volumes of polar fluid before the polar fluid is brought onto the active area of the device for droplet operations to be performed. A further advantage of this particular embodiment would be the ease of creating even further volume storage by varying the profile of the upper substrate  82 , as shown for example in another exemplary EWOD device  80   d  shown  FIG. 11( c ) . In such example, the upper substrate  82  has a ridge  114  against which the nonpolar fluid will tend to pin by capillary action to create an additional storage capacity. The ridge  114  is more advantageously formed in the upper substrate  82  because the physical profile of the counter electrode EWOD substrate is more easily varied than the physical profile of the active EWOD substrate. 
       FIG. 12  is a drawing depicting a side view of another exemplary alternative variation of an EWOD device  80   e  in accordance with embodiments of the present invention. In this embodiment, again the second or lower substrate  84  is the active substrate including the thin film drive electronics  104 , and the first or upper substrate  82  incorporates the reference electrode. The upper substrate  82  is arranged so that along the edges where polar fluid is to be loaded, the edge of the upper substrate is co-incident with the active area  106 . In other words, there is no inactive border region along edges of the EWOD device where polar fluid is to be inputted. This arrangement has an advantage in that with no inactive region of the in the EWOD channel where the polar fluid is to be inputted, in principle there is no minimum loadable polar reagent volume as the inputted fluid will always be in contact with the active area. Relatedly, there is no part of the active area of the that is outside the EWOD channel, and so no part of the active area is wasted. As in the previous embodiment, the border region (or lack thereof) can be chosen to be different along different edges of the device, such as in the embodiment of  FIG. 11( b ) , and also may be combined with the variable profile upper substrate to minimize the area taken up by larger volumes of fluid stored on the array, as shown in  FIG. 11( c ) . 
       FIG. 13  is a drawing depicting a side view of another exemplary alternative variation of an EWOD device  80   f  in accordance with embodiments of the present invention. In this embodiment, again the second or lower substrate  84  is the active substrate including the thin film drive electronics  104 , and the first or upper substrate  82  incorporates the reference electrode. In this example, the upper substrate  82  arranged so that at least part of the active area  106  of the lower EWOD substrate extends beyond the edges of the upper substrate. Such a configuration may be advantageous when very small volumes of fluid are to be loaded into the device, which otherwise may render it more difficult for the inputted fluid to contact the oil or active area. Accordingly, in this embodiment, there always is a portion of the active area EWOD electrodes directly below the fluid injection point, which enables better control of the input of polar fluid at the point where the polar fluid has not yet come into contact with the oil and therefore not yet entered the EWOD channel of the device. 
     To facilitate the loading of very small volumes of polar fluid, electrode patterns may be synched with the loading of the polar fluid, as shown for example in the progressive series of views (a)-(d) of  FIG. 13 . In the first view (a), a small volume of polar fluid  102  is located within the injector  116 , such as a pipetting device. In the second view (b), initially the small volume of polar fluid  102  exits the injector  116  and does not move laterally due to the small volume. Accordingly, an EWOD electrode portion  118  of the active area  106  is activated in an applied electrode pattern as the polar fluid touches the lower substrate, in readiness to steer the polar fluid  102  towards the EWOD channel before the injector  116  is retracted. As shown in the third view (c), the applied electrode pattern provides an electrowetting force on the polar fluid, which biases a direction of fluid flow towards the EWOD channel of the device (and the oil within), therefore aiding successful loading of small volumes of polar fluid. As shown in the fourth view (d), once the small volume of polar fluid  102  is loaded into the EWOD channel, the injector can be removed from the device. By waiting until the polar fluid has been moved into the EWOD channel before retracting the pipetting device, undesirable backflow of polar fluid upon retraction is avoided, which can occur in conventional configurations. 
     If the EWOD device includes sensor feedback, this process can be done in an automatic fashion via the use of a software function which automatically detects fluid as soon as it touches the EWOD substrate, and can adapt the applied EWOD electrode pattern in an automated fashion. If the fluid input is being done robotically, this software function can be linked to the pumps so that the injector is only retracted once the polar fluid has safely been moved away from the edge of the EWOD channel. If the fluid input is being done manually (e.g. by pipette) then a signal (audio or visual) can be given to the user when the polar fluid has been moved into the EWOD channel and it is safe to remove the pipette. As in the previous embodiments, the EWOD device  80   f  can be designed so that this feature is present along only some of the edges of the device, if this suits the fluid loading requirements of the assay to be performed for a given application. 
       FIGS. 14 and 15  are drawings depicting another exemplary method of employing the EWOD device  80  to input polar fluid into the EWOD channel. In previous embodiments, a feature of the spacer  86  is that the teeth  96  form overlapping regions where the spacer is inside the EWOD channel of the device, and the oil pins oil along the edges of the spacer where fluid inputs are positioned to load the polar fluid. In this manner, the spacer configuration ensures that the polar fluid contacts the oil within the EWOD channel of the device before the object delivering the polar fluid into the EWOD device is retracted. 
       FIG. 14  depicts an alternative fluid input method referred to herein as an “oil shell” method of fluid input. The oil shell method achieves a comparable effect of contacting the polar fluid and the oil, which negates the need for the oil to be specifically present at the relevant boundary of the upper EWOD substrate and spacer.  FIG. 14  depicts a non-specific EWOD device  125  for illustration purposes of the oil shell method. As shown in  FIG. 14 , in the oil shell method an input droplet  130  is formed including a polar fluid droplet  132  encased within an oil shell  134 . If the fluid is to be delivered by pipette, for example, then the input droplet  130  can be formed by dipping the pipette tip into oil after loading the polar fluid into the pipette tip. Alternatively, a double draw can be performed with the pipette, so that two fluids are drawn into the pipette tip including both a controlled quantity of polar fluid simultaneously with a suitable quantity of oil. The two fluids can then be injected into the EWOD channel without the need for oil to be present specifically at the boundary of the input port. The presence of the oil around the polar fluid permits input of the droplet despite the hydrophobicity of the substrate surfaces. Provided the input droplet  130  can be moved by electrowetting forces to the main body of oil or other desired location, proper fluid input is achieved. 
       FIG. 15  is a drawing depicting the oil shell method as may be employed in connection with the EWOD device  80  shown in  FIG. 6 . It will be appreciated that the oil shell method comparably may be employed using the EWOD device of any of the described embodiments. As shown in  FIG. 15 , an oil shell input droplet  130  may be inputted at one of the input ports  98  that is spaced apart from the main body of oil  94 . Electrowetting voltages may then be employed to move the input droplet  130  to the main body of oil. The oil shell method may be used in conjunction with the input methods of the other embodiments to load extra polar fluid into any remaining air bubble  100  present in the EWOD device, e.g. such as when insufficient oil  94  has been loaded at the start of a reaction protocol, as shown in  FIG. 15 . 
     An aspect of the invention is an EWOD device that has an enhanced spacer configuration, by which the spacer defines the fluid input ports and is configured in a manner that ensures fluid input from the outside of the device is able to enter the EWOD channel. In exemplary embodiments, the EWOD device includes 1 a first substrate assembly and a second substrate assembly, and a spacer that spaces apart the first substrate assembly from the second substrate assembly to define a channel between the first and second substrate assemblies. The spacer defines a plurality of fluid input ports that are in fluid communication with the channel, and the spacer is configured for directing fluid from the fluid input ports into the channel. The EWOD device may include one or more of the following features, either individually or in combination. 
     In an exemplary embodiment of the EWOD device, the spacer has a combed spacer configuration to define the plurality of fluid input ports, the combed spacer configuration including alternating teeth that extend into the channel from a base region. 
     In an exemplary embodiment of the EWOD device, externally from the channel the teeth isolate adjacent fluid input ports from each other. 
     In an exemplary embodiment of the EWOD device, the spacer contacts only a portion of the first and second substrate assemblies to form a spacerless region within the EWOD device. 
     In an exemplary embodiment of the EWOD device, the spacer includes regions that are in contact with both the first and second substrate assemblies and extend into the channel so as to define a uniform cell-gap of the channel. 
     In an exemplary embodiment of the EWOD device, the EWOD device further includes an exit port configured as an extension of the spacer that forms a fluid passage into the channel. 
     In an exemplary embodiment of the EWOD device, a portion of the spacer opposite of the channel that defines the fluid input ports is rounded in shape. 
     In an exemplary embodiment of the EWOD device, one of the first substrate assembly or the second substrate assembly includes thin film electronics for applying an electrowetting voltage into the channel, the thin film electronics defining an active area within the channel; and the substrate assembly including the thin film electronics defines an inactive border region adjacent to the active area in which the electrowetting voltage is not applicable. 
     In an exemplary embodiment of the EWOD device, the spacer has a combed spacer configuration to define the plurality of fluid input ports, the combed spacer configuration including alternating teeth that extend into the channel from a base region beyond the inactive border region and into the active area. 
     In an exemplary embodiment of the EWOD device, if a width of the inactive border region is denoted “w” and a cell-gap dimension of the channel between the first and second substrate assemblies is denoted “d”, then the fluid input ports are sized to receive a minimum volume of input fluid of at least a volume of a disk of radius w/2 and height d. 
     In an exemplary embodiment of the EWOD device, the inactive border region is symmetric on at least some opposing edges of the substrate assembly including the thin film electronics. 
     In an exemplary embodiment of the EWOD device, the inactive border region is asymmetric to have different sizes on differing edges of the substrate assembly including the thin film electronics. 
     In an exemplary embodiment of the EWOD device, the substrate assembly including the thin film electronics has a ridge adjacent to the inactive border region. 
     In an exemplary embodiment of the EWOD device, the first substrate assembly is an upper substrate assembly and includes the thin film electronics. 
     In an exemplary embodiment of the EWOD device, the second substrate assembly is a lower substrate assembly and includes the thin film electronics. 
     In an exemplary embodiment of the EWOD device, at least one edge the substrate assembly incorporating the thin film electronics is co-incident with the active area. 
     In an exemplary embodiment of the EWOD device, one of the first substrate assembly or the second substrate assembly includes thin film electronics for applying an electrowetting voltage into the channel, the thin film electronics defining an active area within the channel; and a portion of the active area extends beyond the substrate assembly including the thin film electronics. 
     In an exemplary embodiment of the EWOD device, the second substrate assembly is a lower substrate assembly and includes the thin film electronics. 
     Another aspect of the invention is a related method of inputting fluid into the EWOD device. In exemplary embodiments, the method of inputting fluid comprising the steps of: inputting a nonpolar fluid into the channel via one of the fluid input ports; inputting a polar fluid into one of the fluid input ports defined by the spacer, and the configuration of the spacer at the one of the fluid input ports directs the polar fluid to the channel; and applying an electrowetting voltage to move the polar fluid into the channel. The method of input fluid may include one or more of the following features, either individually or in combination. 
     In an exemplary embodiment of the method of inputting fluid, the spacer contacts only a portion of the first and second substrate assemblies to form a spacerless region within the EWOD device, the method further including the steps of: incompletely filling the channel with the nonpolar fluid to form an air bubble within the spacerless region; and inputting a polar fluid into one of the fluid input ports defined by the spacer that is at a boundary of where the air bubble and the nonpolar fluid meet. 
     In an exemplary embodiment of the method of inputting fluid, the spacer contacts only a portion of the first and second substrate assemblies to form a spacerless region within the EWOD device, the method further including the steps of: incompletely filling the channel with the nonpolar fluid to form a main body of nonpolar fluid and an air bubble within the spacerless region; forming an input droplet comprising a droplet of the polar fluid encased within a shell of the nonpolar fluid; inputting the input droplet into one of the fluid input ports defined by the spacer that is spaced apart from the main body of the nonpolar fluid; and applying an electrowetting voltage to move the input droplet to come in contact with the main body of the nonpolar fluid. 
     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 enhanced 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 —array element electrodes 
           12 A—individual array element electrode 
           12 B—individual array element electrode 
           14 —liquid droplet 
           16 —top substrate 
           18 —spacer 
           20 —nonpolar surround fluid 
           22 —insulator layer 
           24 —first hydrophobic coating 
           26 —contact angle 
           28 —second hydrophobic coating 
           30 —reference electrode 
           36 —AM-EWOD device 
           44 —lower substrate 
           46 —thin film electronics 
           48 —array element electrodes 
           50 —electrode or element array 
           52 —liquid droplet 
           54 —upper substrate 
           56 —spacer 
           62 —upper substrate 
           64 —lower substrate 
           66 —spacer 
           67 —active area of the device 
           68 —oil 
           70 —air bubble 
           72 —pipette 
           74 —polar fluid 
           80 / 80   a - f —EWOD device 
           82 —upper substrate assembly 
           84 —lower substrate assembly 
           86 —spacer 
           88 —EWOD channel 
           90 —regions of spacer 
           92 —spacerless region 
           93 —active area 
           94 —oil 
           96 —spacer teeth 
           97 —spacer base region 
           98 —fluid input ports 
           100 —air bubble 
           102 —pipetted polar fluid 
           104 —thin film drive electronics 
           106 —active area 
           108 —inactive border region 
           114 —ridge 
           118 —EWOD electrode portion 
           120 —droplet 
           122 —exit port 
           125 —EWOD device 
           130 —input droplet 
           132 —polar fluid droplet 
           134 —oil shell