Abstract:
A method of manipulating droplet in a programmable EWOD microelectrode array comprising multiple microelectrodes, comprising: constructing a bottom plate with multiple microelectrodes on a top surface of a substrate covered by a dielectric layer; the microelectrode coupled to at least one grounding elements of a grounding mechanism, a hydrophobic layer on the top of the dielectric layer and the grounding elements; manipulating the multiple microelectrodes to configure a group of configured-electrodes to generate microfluidic components and layouts with selected shapes and sizes, comprising: a first configured-electrode with multiple microelectrodes arranged in array, and at least one second adjacent configured-electrode adjacent to the first configured-electrode, the droplet disposed on the top of the first configured-electrode and overlapped with a portion of the second adjacent-configured-electrode; and manipulating one or more droplets among multiple configured-electrodes by sequentially activating and de-activating one or more selected configured-electrodes to actuate droplets to move along selected route.

Description:
CROSS REFERENCE TO RELATED APPLICATIONS 
       [0001]    The present application claims benefit of priority under 35 U.S.C. 119(e) to: U.S. Patent Application 61/312,240, entitled “Field-Programmable Lab-on-a-Chip and Droplet Manipulations Based on EWOD Micro-Electrode Array Architecture” and filed Mar. 9, 2010; U.S. Patent Application 61/312,242, entitled “Droplet Manipulations on EWOD-Based Microelectrode Array Architecture” and filed Mar. 9, 2010; U.S. Patent Application 61/312,244, entitled “Micro-Electrode Array Architecture” and filed Mar. 10, 2010. The foregoing applications are hereby incorporated by reference into the present application in their entireties. 
         [0002]    The present application also incorporates by reference in its entirety co-pending U.S. Patent Application ______, entitled “Field Programmable Lab-on-a-Chip Based on Microelectrode Array Architecture”, and filed on the same date as the present application, namely, Feb. 17, 2011; co-pending U.S. Patent Application ______, entitled “Microelectrode Array Architecture”, and filed on the same date as the present application, namely, Feb. 17, 2011. 
     
    
     FIELD OF THE INVENTION 
       [0003]    The present invention relates to EWOD-based microfluidic systems and methods. More specifically, the present invention relates to the methods and system for droplet manipulation employing EWOD microelectrode array architecture technique. 
       BACKGROUND OF THE INVENTION 
       [0004]    Microfluidics technology has grown explosively over the last decade for the potential to carry out certain chemical, physical or biotechnological processing techniques. Microfluidics refers to the manipulation of minute quantities of fluid, typically in the micro- to nano-liter range. The use of planar fluidic devices for performing small-volume chemistry was first proposed by analytical chemists, who used the term “miniaturized total chemical analysis systems” (μTAS) for this concept. An increasing number of researchers from many disciplines other than analytical chemistry have embraced the fundamental fluidic principle of μTAS as a way of developing new research tools for chemical and biological applications. To reflect this expanded scope, the broader terms “microfluidics” and “Lab-on-a-chip (LOC)” are now often used in addition to μTAS. 
         [0005]    The first generation microfluidic technologies are based on the manipulation of continuous liquid flow through microfabricated channels. Actuation of liquid flow is implemented either by external pressure sources, integrated mechanical micropumps, or by electrokinetic mechanisms. Continuous-flow systems are adequate for many well-defined and simple biochemical applications, but they are unsuitable for more complex tasks requiring a high degree of flexibility or complicated fluid manipulations. Droplet-based microfluidics is an alternative to the continuous-flow systems, where the liquid is divided into discrete independently controllable droplets, and these droplets can be manipulated to move in channels or on a substrate. By using discrete unit-volume droplets, a microfluidic function can be reduced to a set of repeated basic operations, i.e., moving one unit of fluid over one unit of instance. A number of methods for manipulating microfluidic droplets have been proposed in the literature. These techniques can be classified as chemical, thermal, acoustical, and electrical methods. Among all methods, electrical methods to actuate droplets have received considerable attention in recent years. 
         [0006]    Electrowetting-on-dielectric (EWOD) is one of the most common electrical methods. Digital microfluidics such as the Lab-on-a-chip (LOC) generally means the manipulation of droplets using EWOD technique. The conventional EWOD-based LOC device generally includes two parallel glass plates. The bottom plate contains a patterned array of individually controllable electrodes, and the top plate is coated with a continuous ground electrode. Electrodes are preferably formed by a material like indium tin oxide (ITO) that have the combined features of electrical conductivity and optical transparency in thin layer. A dielectric insulator coated with a hydrophobic film is added to the plates to decrease the wettability of the surface and to add capacitance between the droplet and the control electrode. The droplet containing biochemical samples and the filler medium are sandwiched between the plates while the droplets travel inside the filler medium. In order to move a droplet, a control voltage is applied to an electrode adjacent to the droplet and at the same time the electrode just under the droplet is deactivated. 
         [0007]    Unfortunately, the conventional LOC systems employing EWOD technique built to date are still highly specialized to particular applications. The current LOC systems rely heavily on the manual manipulation and optimization of the bioassays. Moreover, current applications and functions in the EWOD-LOC system are time-consuming and require costly hardware design, testing and maintenance procedures. The most disadvantages about the conventional EWOD-LOC systems are the design of “hardwired” electrodes. “Hardwired” means the shapes, the sizes, locations, and the electrical wiring traces to the controller of the electrodes are physically confined to permanently etched structures. Regardless of their functions, once the electrodes are fabricated, their shapes, sizes, locations and traces can&#39;t be changed. So therefore it may result in high non-recurring engineering costs, as well as the limited ability to update the functionality after shipping or partially re-configuring the LOC design. 
         [0008]    There is a need in the art for a system and method for reducing the labor and cost associated with generating the microfluidic systems with the droplet manipulation. EWOD microelectrode array architecture technique can provide the field-programmability that the electrodes and the overall layout of the LOC can be software programmable. A microfluidic device or embedded system is said to be field-programmable or on-site programmable if its firmware (stored in non-volatile memory, such as ROM) can be modified “in the field,” without disassembling the device or returning it to its manufacturer. This is often an extremely desirable feature, as it can reduce the cost and turnaround time for replacement of buggy or obsolete firmware. The ability to update the functionality after shipping, partial re-configuration of the portion of the design and the low non-recurring engineering costs relative to LOC design can offer advantages for many other applications. 
         [0009]    The art raises the LOC designs to the application level to relieve LOC designers from the burden of manual optimization of bioassays, time-consuming hardware design, costly testing and maintenance procedures. 
         [0010]    Also, based on the novel EWOD Microelectrode Array Architecture, the art to manipulate droplets in LOC systems can be dramatically improved. There are various embodiments of present invention in the advanced manipulations of droplets in creating, transportation, mixing and cutting based on the EWOD Microelectrode Array Architecture. 
       SUMMARY 
       [0011]    Disclosed herein is a method of manipulating droplet in a programmable EWOD microelectrode array comprising multiple microelectrodes. In one embodiment, the method includes: (a) constructing a bottom plate comprising an array of multiple microelectrodes disposed on a top surface of a substrate covered by a dielectric layer; wherein each of the microelectrode is coupled to at least one grounding elements of a grounding mechanism, wherein a hydrophobic layer is disposed on the top of the dielectric layer and the grounding elements to make hydrophobic surfaces with the droplets; (b) manipulating the multiple microelectrodes to configure a group of configured-electrodes to generate microfluidic components and layouts with selected shapes and sizes, wherein the configured-electrodes including: a first configured-electrode comprising multiple microelectrodes arranged in array, and at least one second adjacent configured-electrode adjacent to the first configured-electrode, the droplet being disposed on the top of the first configured-electrode and overlapped with a portion of the second adjacent-configured-electrode; and, (c) manipulating one or more droplets among the multiple configured-electrodes by sequentially applying driving voltages activating and de-activating one or more selected configured-electrodes to sequentially activate/deactivate the selected configured-electrodes to actuate droplets to move along selected route. 
         [0012]    Still In another embodiment, a method of manipulating droplet in a programmable EWOD microelectrode array comprising multiple microelectrodes, the method including: (a) constructing a bottom plate comprising an array of multiple microelectrodes disposed on a top surface of a substrate covered by a dielectric layer; wherein each of the microelectrode is coupled to at least one grounding elements of a grounding mechanism, wherein a hydrophobic layer is disposed on the top of the dielectric layer and the grounding elements to make hydrophobic surfaces with the droplets; (b) manipulating the multiple microelectrodes to configure a group of configured-electrodes to generate microfluidic components and layouts with selected shapes and sizes, wherein the configured-electrodes including: a first configured-electrode comprising multiple microelectrodes arranged in array, and at least one second adjacent configured-electrode adjacent to the first configured-electrode, the droplet being disposed on the top of the first configured-electrode and overlapped with a portion of the second adjacent-configured-electrode; (c) deactivating the first configured-electrode and activating the second adjacent configured-electrode to pull the droplet from the first configured-electrode onto the second configured-electrode, and; (d) manipulating one or more droplets among the multiple configured-electrodes by sequentially applying driving voltages activating and de-activating one or more selected configured-electrodes to sequentially activate/deactivate the selected configured-electrodes to actuate droplets to move along selected route. 
         [0013]    In another embodiment, a method of manipulating droplet in a programmable EWOD microelectrode array comprising multiple microelectrodes, the method including: (a) constructing a bottom plate comprising an array of multiple microelectrodes disposed on a top surface of a substrate covered by a dielectric layer; wherein each of the microelectrode is coupled to at least one grounding elements of a grounding mechanism, wherein a hydrophobic layer is disposed on the top of the dielectric layer and the grounding elements to make hydrophobic surfaces with the droplets; (b) manipulating the multiple microelectrodes to configure a group of configured-electrodes to generate microfluidic components and layouts with selected shapes and sizes, wherein the configured-electrodes including: a first configured-electrode comprising multiple microelectrodes arranged in array, and at least one second adjacent configured-electrode adjacent to the first configured-electrode, the droplet being disposed on the top of the first configured-electrode and overlapped with a portion of the second adjacent-configured-electrode; (c) configuring a third neighboring configured-electrode not overlapped with the droplet on the first configured-electrode, and, (d) manipulating one or more droplets among the multiple configured-electrodes by sequentially applying driving voltages activating and de-activating one or more selected configured-electrodes to sequentially activate/deactivate the selected configured-electrodes to actuate droplets to move along selected route. 
         [0014]    Still in another embodiment, The EWOD Microelectrode Array Architecture of the present invention employs the “dot matrix printer” concept that a plurality of microelectrodes (e.g., “dots”) are grouped and are simultaneously activated/deactivated to form varied shapes and sizes of electrodes to meet the requirements of fluidic operational functions in field applications. 
         [0015]    In another embodiment, all EWOD microfluidic components can be generated by grouping the multiple microelectrodes, including, but not limit to, reservoirs, electrodes, mixing chambers, droplet pathways and others. Also physical layouts of the LOC for the locations of I/O ports, reservoirs, electrodes, pathways and electrode networks all can be done by configurations of microelectrodes. The grouped microelectrodes after the configuration are configured electrodes to distinguish it from the conventional electrodes. 
         [0016]    In another embodiment, the varied shapes of sizes of configured-electrodes such as reservoirs, electrodes, mixing chambers, droplet pathways and physical layouts of the LOC for the locations of I/O ports, reservoirs, electrodes, pathways and electrode networks of the microfluidic system are able to be software programmed, re-configured and field-programmed to meet the requirements of operational functions in field applications. 
         [0017]    In other embodiments, the bi-planar structure can be employed in the design of EWOD Microelectrode Array Architecture in the manipulation of droplets in which the upper top plate is implemented in the system. 
         [0018]    Still in another embodiment, the design of the EWOD Microelectrode Array Architecture in the manipulation of droplets can be based on a coplanar structure in which the EWOD actuations can occur in the single plate configuration without the top plate. 
         [0019]    In another embodiment, the method of creating a LOC structure to accommodate the widest range of droplet sizes and volumes by a coplanar structure with a removable, adjustable and transparent top plate to accommodate the widest range of droplet sizes and volumes under the EWOD Microelectrode Array Architecture. 
         [0020]    In yet other embodiments, all typical EWOD microfluidic operations can be performed by configuring and controlling of the “configured-electrodes” under the EWOD Microelectrode Array Architecture. “Microfluidic operations” means any manipulation of a droplet on a droplet microactuator. A microfluidic operation may, for example, include: loading a droplet into the droplet microactuator; dispensing one or more droplets from a source droplet; splitting, separating or dividing a droplet into two or more droplets; transporting a droplet from one location to another in any direction; merging or combining two or more droplets into a single droplet; diluting a droplet; mixing a droplet; agitating a droplet; deforming a droplet; retaining a droplet in position; incubating a droplet; disposing of a droplet; transporting a droplet out of a droplet microactuator; and/or any combination of the foregoing. 
         [0021]    In yet another embodiment, besides the conventional control of the configured electrodes to perform typical microfluidic operations, special control sequences of the microelectrodes can offer advanced microfluidic operations in manipulations of droplets. Advanced microfluidic operations based on the EWOD Microelectrode Array Architecture may include: transporting droplets diagonally or in any directions; transporting droplets through the physical gaps by Interim bridging” technique; transporting droplets by Electrode Column Actuation; Washing out dead volumes; transporting droplets in lower driving voltage situation; transporting droplets in controlled low speed; performing precise cutting; performing diagonal cutting; performing coplanar cutting; merging droplets diagonally; deforming droplets to speed mixing; improving mixing speed by uneven back-and-forth mixer; improving mixing speed by circular mixer; improving mixing speed by multilaminates mixer; and/or any combination of the foregoing. 
         [0022]    While multiple embodiments are disclosed, still other embodiments of the present invention will become apparent to those skilled in the art from the following detailed description, which shows and describes illustrative embodiments of the invention. As will be realized, the invention is capable of modifications in various aspects, all without departing from the spirit and scope of the present invention. Accordingly, the drawings and detailed description are to be regarded as illustrative in nature and not restrictive. 
     
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         [0023]      FIG. 1A  is a cross-section view generally illustrating the conventional sandwiched EWOD system. 
           [0024]      FIG. 1B  is a top view generally illustrating the conventional EWOD two-dimensional electrode array. 
           [0025]      FIG. 2  is a diagram generally illustrating the microelectrode array that can be configured into various shape and size of configured-electrodes. 
           [0026]      FIG. 3A  is the diagram of different shapes of configured-electrodes and the LOC layout using the microelectrode array architecture. 
           [0027]      FIG. 3B  is a conventional physically etched structure. 
           [0028]      FIG. 3C  is the diagram of configured-electrodes for the enlarged section of the reservoir and configured-electrodes. 
           [0029]      FIG. 4  is a hybrid structure with a removable, adjustable and transparent top plate to accommodate the widest range of droplet sizes and volumes. 
           [0030]      FIGS. 5A ,  5 B and  5 C are diagrams of the “ground girds” coplanar structure. 
           [0031]      FIGS. 6A and 6B  are diagrams of “ground pads” coplanar structure. 
           [0032]      FIGS. 7A ,  7 B and  7 C are diagrams of “programmed ground pads” coplanar structure. 
           [0033]      FIG. 8  is a diagram showing the hybrid plate. 
           [0034]      FIGS. 9A ,  9 B and  9 C show the loading of the samples. 
           [0035]      FIGS. 9D and 9E  show the self-positioning of loaded samples onto the reservoir. 
           [0036]      FIG. 10  is a diagram showing the creation of droplet under EWOD Microelectrode Array Architecture. 
           [0037]      FIG. 11A  is a diagram showing the droplet creating using droplet aliquots technique. 
           [0038]      FIG. 11B  is a diagram showing the sample preparation by droplet aliquots technique. 
           [0039]      FIG. 12  is a diagram showing the transportation of droplet based on the EWOD Microelectrode Array Architecture and the ability being actuated in all directions. 
           [0040]      FIGS. 13A ,  13 B and  13 C are diagrams showing the transportation of a droplet using interim bridging technique based on the EWOD Microelectrode Array Architecture. 
           [0041]      FIGS. 14A and 14B  are diagrams showing the electrode column actuation under the EWOD Microelectrode Array Architecture. 
           [0042]      FIGS. 15A ,  15 B and  15 C are diagrams showing the cutting of a droplet based on the EWOD Microelectrode Array Architecture. 
           [0043]      FIGS. 16A ,  16 B and  16 C are diagrams showing the precise cutting of a droplet based on the EWOD Microelectrode Array Architecture. 
           [0044]      FIGS. 17A ,  17 B,  17 C and  17 D are diagrams showing the diagonal cutting of a droplet based on the EWOD Microelectrode Array Architecture. 
           [0045]      FIGS. 18A ,  18 B and  18 C are diagrams showing the coplanar cutting of a droplet based on the EWOD Microelectrode Array Architecture. 
           [0046]      FIGS. 19A and 19B  are diagrams showing the merge/mixing of two droplets based on the EWOD Microelectrode Array Architecture. 
           [0047]      FIGS. 20A ,  20 B, and  20 C are diagrams showing the quick mixing of droplets by uneven-geometry movements based on the EWOD Microelectrode Array Architecture. 
           [0048]      FIGS. 21A and 21B  are diagrams showing the uneven back-and-forth mixer based on the EWOD Microelectrode Array Architecture. 
           [0049]      FIG. 22  is a diagram showing the circular mixer based on the EWOD Microelectrode Array Architecture. 
           [0050]      FIGS. 23A ,  23 B,  23 C,  23 D,  23 E, and  23 F are diagrams showing the multilaminates mixer based on the EWOD Microelectrode Array Architecture. 
           [0051]      FIGS. 24A ,  24 B and  24 C are illustrations of the creation of liquids by continuous-flow actuations. 
           [0052]      FIGS. 24D and 24E  are illustrations of the cutting of liquid by continuous-flow actuations. 
           [0053]      FIGS. 25A ,  25 B and  25 C are illustrations of the merge/mixing of liquids by continuous-flow actuations. 
       
    
    
     DETAILED DESCRIPTION 
       [0054]    Referring to  FIG. 1A , a conventional electrowetting microactuator mechanism (in small scale for illustration purposes only) is illustrated in  FIG. 1A . EWOD-based digital microfluidic device  100  consists of two parallel glass plates  120  and  121 , respectively. The bottom plate  121  contains a patterned array  130  of individually controllable electrodes, and the top plate  120  is coated with a continuous ground electrode  140 . Electrodes are preferably formed by a material, such as indium tin oxide (ITO) that has the combined features of electrical conductivity and optical transparency in thin layer. A dielectric insulator  170 , e.g., parylene C, coated with a hydrophobic film  160  such as Teflon AF, is added to the plates to decrease the wettability of the surface and to add capacitance between the droplet and the control electrode. The droplet  150  containing biochemical samples and the filler medium, such as the silicone oil or air, are sandwiched between the plates to facilitate the transportation of the droplet  150  inside the filler medium. In order to move a droplet  150 , a control voltage is applied to an electrode  180  adjacent to the droplet and at the same time the electrode just under the droplet  150  is deactivated. 
         [0055]      FIG. 1B  is a top view generally illustrating the conventional EWOD on a two dimensional electrode array  190 . A droplet  150  is moving from electrode  130  into an activated electrode  180 . The black color of electrode  180  indicates when a control voltage is applied. The EWOD effect causes an accumulation of charge in the droplet/insulator interface, resulting in an interfacial tension gradient across the gap  135  between the adjacent electrodes  130  and  180 , which consequently causes the transportation of the droplet  150 . By varying the electrical potential along a linear array of electrodes, electrowetting can be used to move nanoliter-volume liquid droplets along this line of electrodes. The velocity of the droplet can be controlled by adjusting the control voltage in a range from 0-90 V, and droplets can be moved at speeds of up to 20 cm/s. Droplets  151  and  152  can also be transported, in user-defined patterns and under clocked-voltage control, over a 2-D array of electrodes without the need for micropumps and microvalves. 
         [0056]    EWOD based microfluidic devices use the interfacial tension gradient across the gap between the adjacent electrodes to actuate the droplets. The designs of electrodes include the desired shapes, sizes of each of the electrode and the gaps between each of the two electrodes. In the EWOD based design, the droplet pathways generally are composed of a plurality of electrodes that connect different areas in the design. These electrodes can be used either for transporting procedure or for other more complex operations such as mixing and cutting procedures in the droplet manipulation. 
         [0057]    The present invention employs the “dot matrix printer” concept that each microelectrode in the EWOD Microelectrode Array Architecture is a “dot” which can be used to form all EWOD microfluidic components. In other words, each of the microelectrodes in the microelectrode array can be configured to form various microfluidic components in different shapes and sizes. According to customer&#39;s demand, multiple microelectrodes can be deemed as “dots” that are grouped and can be activated simultaneously to form different configured-electrodes and perform microfluidic operations. Activate means to apply necessary electrical voltages to the electrodes that the EWOD effect causes an accumulation of charge in the droplet/insulator interface, resulting in an interfacial tension gradient across the gap between the adjacent electrodes, which consequently causes the transportation of the droplet. Deactivate means to remove the applied electrical voltages from the electrodes. 
         [0058]      FIG. 2  illustrates one embodiment of the EWOD microelectrode array architecture technique of the present invention. In this embodiment, the microelectrode array  200  is composed of a plurality (30×23) of identical microelectrodes  210 . This microelectrode array  200  is fabricated based on the standard microelectrode specification (shown here as microelectrode  210 ) and fabrication technologies that are independent from the ultimate LOC applications and the detail microfluidic operation specifications. In another word, this microelectrode array  200  is a “blank” or “pre-configuration” LOC. Based on the application needs, then this microelectrode array can be configured or software programmed into the desired LOC. As shown in  FIG. 2 , each of the configured-electrode  220  is composed of 100 microelectrodes  210  (i.e., 10×10 microelectrodes). “Configured-electrode” means the 10×10 microelectrodes  210  are grouped together to perform as an integrated electrode  220  and will be activated or deactivated together at the same time. Normally, the configuration data is stored in non-volatile memory (such as ROM) and can be modified “in the field,” or “on-site” in any designated location without disassembling the device or returning it to its manufacturer.  FIG. 2  shows a droplet  250  sits on the center configured-electrode  220 . 
         [0059]    As shown in  FIG. 2 , the sizes and shapes of the configured-electrodes of the present invention can be designed based on application needs. Examples of the control of the sizes of the configured-electrodes are configured-electrodes  220  and  240 . Configured-electrode  220  has the size of 10×10 microelectrodes and configured-electrode  240  has the size of 4×4 microelectrodes. Besides the configuration of the sizes of the configured-electrodes, different shapes of the configured-electrodes also can be configured by using the microelectrode array. While configured-electrode  220  is square, configured-electrode  230  is composed of 2×4 microelectrodes in rectangular shape. Configured-electrode  260  is left-side-toothed-square, and configured-electrode  270  is round shape. 
         [0060]    Also, as shown in  FIG. 2 , the volume of the droplet  250  is proportional to the size of the configured-electrode  220 . In other words, by controlling the size of the configured-electrode  220 , the volume of the droplet  250  is also limited to fit into the designed size of the configured-electrode  220 ; therefore the field-programmability of the shape and size of the “configured-electrodes” means the control of droplet volumes. Different LOC applications and microfluidic operations will require different droplet volumes, and a dynamic programmable control of the droplet volumes is a highly desirable function for LOC designers. 
         [0061]    As shown in  FIG. 3A , the shapes of the configured-electrodes of the present invention can be designed based on application&#39;s needs. The shapes of the configured-electrodes are made of a plurality of microelectrodes. Depending on the design needs, the group of microelectrodes are configured and activated as a group to form the desired shape of the configured-electrode. In the present invention, the shapes of the configured-electrodes can be square, square with tooth edges, hexagonal, or any other shapes. Referring to  FIG. 3A , the shapes of configured-electrodes of the transportation path  340 , detection window  350  and the mixing chamber  360  are square. The reservoir  330  is special-shaped large sized configured-electrode. The waste reservoir  320  is tetragon shaped. 
         [0062]      FIGS. 3B and 3C  show the enlarged version of the reservoir  330  from  FIG. 3A .  FIG. 3B  is illustrated as a physically etched reservoir structure  331  manufactured by conventional EWOD-LOC systems. The components show permanently etched reservoir  331  and the four permanently etched electrodes  371 . In comparison of  FIG. 3B  (conventional design),  FIG. 3C  is a field-programmed LOC structure with similar sized configured reservoir  332  grouped electrodes  372 . The configured reservoir  332  can be made by grouping multiple microelectrodes  311  into desired size and shape to make such reservoir component. The grouped electrodes  371  contain 4×4 microelectrodes  311 . 
         [0063]    After defining the shapes and sizes of the necessary microfluidic components, it&#39;s also important to define the locations of the microfluidic components and how these microfluidic components connected together as a circuitry or network.  FIG. 3A  shows where the physical locations of these microfluidic components are positioned and how these microfluidic components are connected together to perform as a functional LOC. These microfluidic components are: configured-electrodes  370 , reservoirs  330 , waste reservoir  320 , mixing chamber  360 , detection window  350  and transportation paths  340  that connect different areas of the LOC. If it&#39;s a field-programmable LOC then after the layout design, there are some unused microelectrodes  310 . Designers can go for a hardwired version to save cost after the FPLOC is fully verified then unused microelectrodes  310  can be removed. 
         [0064]    The conventional EWOD-based LOC design is based on a bi-planar structure that has a bottom plate containing a patterned array of electrodes, and a top plate coated with a continuous ground electrode. In one embodiment of the present invention, the LOC device employing EWOD microelectrode array architecture technique is based on a coplanar structure in which the actuations can occur in a single plate configuration without the top plate. The coplanar design can accommodate a wider range of different volume sizes of droplets without the constrained of the top plate. The bi-planar structure has a fixed gap between the top plates and has the limitation to accommodate wide range of the volume size of droplets. Still in another embodiment, the LOC devices employing EWOD microelectrode array architecture technique based on the coplanar structure still can add a passive top plate to seal the test surface for the protection of the fluidic operations or for the purpose of protecting the test medium for a longer shelf storage life. 
         [0065]    In another embodiment, a removable, adjustable and transparent top plate is employed in the coplanar structure for the EWOD microelectrode array architecture technique to optimize the gap distance between the top plate  410  and the electrode plate  420  as shown in  FIG. 4 . The electrode plate  420  is implemented by the EWOD microelectrode array architecture technique that the side view of the configured-electrode for droplet  430  includes three microelectrodes (shown in black). The configured-electrode for droplet  440  includes six microelectrodes and the configured-electrode for droplet  450  includes eleven microelectrodes. This embodiment is especially useful in the application such as field-programmable LOC. While EWOD microelectrode array architecture provides the field-programmability in configuring the shapes and the sizes of the configured-electrode, a system structure that can accommodate the widest ranges of sizes and volumes of the droplets is highly desirable. Because the wider the droplet sizes and volumes a field-programmable LOC can accommodate, the more applications can be implemented. The optimized gap distance can be adjusted to fit the desired sizes of the droplets. In the present invention, the optimized gaps can be implemented in three approaches: First, all the droplets can be manipulated without touching the top plate  410 . This approach is generally applied to the coplanar structure. In a second approach, all droplets can be manipulated by touching the top plate  410  that droplets are sandwiched between the top plate  410  and the electrode plate  420 . The second approach is generally applied to bi-planar structure. The third approach or a hybrid approach incorporates the functions of coplanar structure and an adjustable gap between the top cover  410  and the coplanar electrode plate  420 . This hybrid approach can be used to provide the droplets with the widest range. As shown in  FIG. 4 , the droplet  430  and droplet  440  sit within the gap are manipulated without touching the top plate  410 . The droplet  450  is manipulated to be sandwiched between the top plate  410  and the electrode plate  420 . This invention is not limited to the EWOD microelectrode array architecture technique. It can also be applied to other conventional electrode plates while the applicable ranges of the droplet sizes may be limited. 
         [0066]    The plate structure of the microelectrode of Microelectrode Array Architecture can be designed by using scaled-down bi-planar structure based on the popular configuration of EWOD chip today. A bi-planar EWOD based microelectrode structure (in small scale for illustration purposes only) is illustrated in  FIG. 1A . Three microelectrodes  130  and two parallel plates  120  and  121  are shown in the figure. The bottom plate  121  contains a patterned array of individually controllable electrodes  130 , and the top plate  120  is coated with a continuous ground electrode  140 . A dielectric insulator  170  coated with a hydrophobic film  160  is added to the plates to decrease the wettability of the surface and to add capacitance between the droplet and the control electrode. The droplet  150  containing biochemical samples and the filler medium, such as the silicone oil or air, are sandwiched between the plates to facilitate the transportation of the droplet  150  inside the filler medium. 
         [0067]    In one embodiment of the present invention, the LOC device employing EWOD microelectrode array architecture technique is based on a coplanar structure in which the actuations can occur in a single plate configuration without the top plate. The coplanar design can accommodate a wider range of different volume sizes of droplets without the constrained of the top plate. The bi-planar structure has a fixed gap between the top plates and has the limitation to accommodate wide range of the volume size of droplets. Still in another embodiment, the LOC devices employing EWOD microelectrode array architecture technique based on the coplanar structure still can add a passive top plate to seal the test surface for the protection of the fluidic operations or for the purpose of protecting the test medium for a longer shelf storage life. 
         [0068]    In the present invention, the microelectrode plate structure can be physically implemented in many ways especially in the coplanar structure.  FIG. 5A  shows the “ground grids” coplanar microelectrode structure comprises one driving-microelectrode  510 , ground lines  511 , and gaps  515  between the driving-microelectrode  510  and the ground lines  511 . When the electrode is activated, the driving-microelectrode  510  is charged by a DC or square-wave driving voltage. The ground lines  511  are on the same plate with the driving-microelectrode  510  to achieve the coplanar structure. The gap  515  is to ensure no vertical overlapping between  510  and  511 . 
         [0069]      FIG. 5B  is the conventional droplet operation unit includes permanently etched electrodes  520 ,  521 , ground lines  531 , (in vertical and in horizontal directions). These two etched electrodes  520 ,  521  are each separated by the ground lines  531  in horizontal and vertical directions. The droplet  540  sits in the electrode  520 . As shown in  FIG. 5B , the droplet  540  is too small to touch the surrounded ground lines  531  and the actuation of the droplet  540  can&#39;t be performed. This could be potential problems in droplet manipulation often observed in conventional droplet system. The general remedy is to load a larger size droplet  550  but it is often difficult to control the desired droplet size manually. Also, limited by the ground lines  531  in the conventional system, electrodes  520  and  521  can&#39;t have the interdigitated perimeters to improve droplet manipulations. 
         [0070]      FIG. 5C  shows the improved droplet operation unit of the current invention in a coplanar structure. The configured electrode  520 ′ comprises a plurality of field-programmable microelectrodes  510 . The configured electrode can be software programmed according to the size of the droplet. In this example, the configured electrode  520 ′ includes 9 (3×3) microelectrodes  510 . In  FIG. 5C , the droplet  541  sits on the configured electrode  520 ′. The droplet  541  is similar to the size of droplet  540  ( FIG. 5B ) for comparison purposes. In  FIG. 5C , the configured electrode  520 ′ comprises a plural numbers of cross-sectioned ground lines  511 . In the present invention, the effective droplet manipulations can be achieved since the droplet  541  physically overlaps with the configured electrode  520 ′ and the plural ground lines  511 . 
         [0071]      FIG. 6A  illustrates another implementation of the “ground pads” coplanar microelectrode. The driving-microelectrode  610  is in the middle with the ground pads  611  at the four corners and the gap  615  between  610  and  611 . Instead of the ground lines in the embodiment shown in  FIG. 5A , this embodiment uses ground pads to achieve the coplanar structure. In comparison to the conventional implementation, fundamentally our invention provides a group grounding (there are 21 ground pads  611  overlap with droplet  651  in  FIG. 6B ) that is more reliable than the basic one-to-one relationship of conventional implementation. If one droplet depends only on one ground pad then the size of the droplet would be critical to make sure a reliable droplet manipulation because the overlap between the droplet and the ground pad is a must. A sea of ground pads don&#39;t have this constrain; regardless the size of the droplet, many ground pads would be overlapped with the droplet as shown in  FIG. 6B . The driving force for the droplet is basically proportional to the charge accumulated across the biased activating electrode and the ground pad. And typically the charge accumulation is also proportional to the surface area of the electrode and ground pad. A small size ground pad will have significant degrading on the driving force unless a special treatment of the ground pad is applied to improve other physical parameters and it will complicated the fabrication processes. In our invention the group of ground pad can be easily adjusted to optimize the total surface area of the ground pads. In addition, the diving force of the droplet for a coplanar structure eventually will be balanced at around the middle point of the ground pad and the driving electrode. So there is a chance that the droplet never can reach the second ground pad and that cause an unreliable droplet operation. This is especially true for a smaller droplet. Our invention using group grounding so consistent overlaps of ground pads, microelectrodes, and droplets guarantee the reliable droplet operations. Also, in our invention the miniature microelectrode (typically is less than 100×100 μm 2 ) is beyond the feasibility of PCB technology and required microfabrication techniques derived from semiconductor integrated circuit manufacturing. 
         [0072]      FIG. 7A  illustrates another embodiment of the “programmed ground pads” coplanar microelectrode structure. There are no ground lines or ground pads on the same plate with microelectrodes. Instead, some microelectrodes are used as the ground pads to achieve a coplanar electrode structure.  FIG. 7A  shows 4×4 identical square microelectrodes  710  with gap  715  in between. In this embodiment, any one of the microelectrodes  710  can be configured to act as the ground electrode by physically connected to the electrical ground. In this embodiment, the microelectrodes  710  at the four corners are configured as ground electrodes  711 . This invention has the advantage of group grounding vs. a one-to-one electrode and grounding structure in the conventional implementation. Also, the field-programmability and the miniature microelectrodes provide more flexibility and more granularities in the dynamic configuration of the “configured-electrodes” and the “configured-ground pads”. As indicated in  FIG. 7B , because of the one-to-one electrode and grounding structure in the prior art, the droplet  750  can only move on the x-axis direction and droplet  751  can only move on the y-axis direction. In this conventional coplanar structure configuration, the droplet  750  would be centered between the activated electrode  720  and the ground electrode which is marked as black because of the distribution of accumulated charges between the electrode  720  and the ground pads. The only way to move the droplet  750  is to deactivate electrode  720  and to activate the adjacent electrode  730 ; in this way, the droplet  750  will be pulled into the direction along the line as indicated by the arrow  740 . In comparison, droplet  752  sits on a coplanar surface employing the EWOD microelectrode array architecture can move in any directions as indicated in  FIG. 7C . When “configured-electrode”  760  is activated droplet  752  moves upward. The same thing happens, when “configured-electrode”  761  is activated droplet  752  moves leftward. And when interim “configured-electrode”  762  is activated droplet  752  moves diagonally and the activation of “configured-electrode”  763  (with the deactivation of “configured-electrode”  762 ) pulls droplet  752  diagonally onto “configured-electrode”  763 . For the illustrating purpose, each “configured-electrode”  790  has the ground microelectrodes on the four corners but this is not a fixed layout. Interim steps including changes on the ground electrodes or the activating electrodes can be implemented for the best results of the manipulations of the droplet. 
         [0073]    In another embodiment of the present invention, the LOC device employing EWOD microelectrode array architecture technique is based on a hybrid plate structure in which the actuations can occur either in a coplanar configuration or in a bi-planar configuration.  FIG. 8  illustrates a switch  810  that can be controlled to switch the EWOD microelectrode structure between the coplanar mode and the bi-planar mode. In a coplanar mode the continuous ground electrode  840  on the cover plate  820  is connected to the ground and the ground grids  880  on the electrode plate  821  is disconnected from the ground. On the other hand, in a bi-planar mode the ground grids  880  on the electrode plate  821  is connected to the ground and the ground electrode  840  on the cover plate  820  is disconnected from the ground. In another embodiment, the “ground grids” can be replaced by the “ground pads” or the “programmed ground pads” of the as described in previous sections. Also, in one embodiment, the coplanar ground schemes might not be disconnected as long as the extra grounding doesn&#39;t cause any issues in bi-planar structure operations. 
         [0074]    Disclosed herein is the droplet creating procedure in the droplet manipulation. The samples and reagents are loaded from the input ports to the reservoirs and then the liquid droplets are extruded from the reservoirs. The reservoirs can be created in the form of large electrode areas that enable the liquid droplet to access to and egress. In the EWOD-based microfluidic system, the creating procedure of the droplet is the most critical component. The system may improve the design of droplet creation procedure since the implementation of the fluidic input port is challenging due to the huge discrepancy between the scales of mini-liters sample amount and micro-liters or even nano-liters sample amount. Loading samples and reagents onto the chip requires an interface between the microfluidic device and the outside large scaled devices. As indicated in  FIG. 9A , conventionally this interface is composed of an input port  910  which mounted on a through hole of the top plate  915  and a reservoir  920 . Samples or reagents  930  are loaded from the input ports into reservoirs and then droplets  940  of samples or reagents are created from the reservoirs. In  FIG. 9A , the sample input port  910  must appropriately match with the location of the reservoir  920  to position the sample  930  correctly. This traditional approach can lead to incorrect or messy sample loading by human error. 
         [0075]    One embodiment is based on the coplanar structure that the cover can be added after the samples or reagents are loaded onto the LOC so there is no need for fixed input ports. This is especially important for the EWOD microelectrode array architecture because the field-programmability of the architecture can configure shapes, sizes and locations of the reservoirs and the fixed input ports.  FIG. 9B  shows the loading of the sample  950  by a needle  960  directly onto the coplanar electrode plate  970 . The loading of the sample don&#39;t have to be very precise because if necessary the locations of the reservoirs can be adjusted by software programming to compensate the physical loading deviation.  FIG. 9C  indicates a passive cover  980  can be added into after the sample  950  is loaded into the electrodes  970 . 
         [0076]    In another embodiment, the flexibility of the EWOD Microelectrode Array Architecture makes it possible to self-adjust the position of the loaded samples or reagents to the reservoirs. This means the need of a precisely positioned input port and the difficulties to handle the samples and reagents through the input port to the reservoir can be avoided.  FIG. 9D  shows the loaded samples are broken into droplet  951  and droplet  952  and both are not precisely positioned on the top of the reservoir  941 . In one embodiment, droplet  952  is not necessary to be able to overlap with the reservoir  941 . For a conventional LOC, it&#39;s difficult to re-position the droplet  952  into the reservoir  941 . 
         [0077]    IN one embodiment, the self-positioning can be done even if the sample droplet  952  is loaded away from the reservoir  941 . This can be achieved by activating an interim configured-electrode  961  to pull the droplet  952  to overlap with the reservoir  941 . Next, deactivate the interim configured electrode  961  and activate the reservoir  941 . In  FIG. 9E , the sample droplet  953  can be correctly positioned inside the reservoir  941 .  9 E FIG. 10  represents the droplet creation procedure under the EWOD Microelectrode Array Architecture. In the conventional procedure, special shaped reservoir  1030  and an overlapped electrode  1035  must present in order to create droplets. In the current invention, the overlapped electrode  1035  does not necessarily be present. The shape of the reservoir  1030  can be a square-shaped reservoir  1015  and don&#39;t need an overlapped electrode  1035 . In another embodiment, the shape of the reservoir  1015  can be any other shape depending on the design needs by designing the array of the microelectrodes. As shown in  FIG. 10 , the creation of the droplet refers to the process of extruding the droplet  1050  out from the square-shaped reservoir  1015 . To start the droplet creation procedure, interim electrode  1030  is activated first as the pull-back electrode and then another interim electrode  1035  is activated to extrude the liquid. Subsequently, through the activation of adjacent serial configured-electrodes  1040  by extruding a liquid finger from the reservoir  1015  and eventually creating droplet  1050 . Each of the configured-electrodes  1040  is composed of a configured 4×4 microelectrode square. In one embodiment, the dimensions of the configured-electrodes  1040  can be in a range from tens of micro-meters to several mini-meters but not limited to this range. The shape of the configured-electrodes can be square or other shapes. In one embodiment, the reservoirs can be square, round or special-shaped. 
         [0078]      FIG. 11A  illustrates the embodiment of the droplet aliquots creation procedure. By manipulating the microelectrodes, the configured electrodes  1120  can be activated. Each small droplet  1115  is about the size of the configured electrode that can be extracted from the reservoir  1110 . The configured electrodes  1120  comprising a group of microelectrodes are therefore activated to collect the desired amount of droplets as shown in  FIG. 11A . Conventionally, droplet sizes are approximated to the sizes of the electrodes and a more precise way to control the volumes of the droplets doesn&#39;t exist. In the current droplet aliquots creation system it can be used to do more precise control of the volumes of the droplets. Also, in another embodiment, the volume of the bigger droplet  1130  can be measured to count the number of smaller droplets  1115  created from droplet  1130 . 
         [0079]      FIG. 11B  illustrates another embodiment of the sample preparation using droplet aliquots technique. One of the common sample preparation steps is the removing of blood cells from the full blood to get plasma for the immunoassay. As shown in  FIG. 11B , using the droplet aliquots technique through microelectrodes  1140  to create smaller droplet which is too small to carry some or any of the blood cells  1180  then move the small droplets  1145  through the small-scaled vertical gap  1170  to form a desire droplet  1150 . The combination of the droplet aliquots technique and the small gap  1170  can efficiently move the small droplets  1145  from the reservoir/droplet  1160  through the channel  1170  to form a bigger droplet  1150  while blood cells  1180  are blocked. The physical obstacle here is used to help droplet aliquots technique and it could be different shapes than square to create smaller droplet with microelectrode. It is not used as the main cause of the removal of the blood cells. By using droplet aliquots technique, this sample preparation invention not only can remove the particles from the droplet but also can prepare the right-sized droplets for diagnostic test. 
         [0080]      FIG. 12  shows the droplet transportation using the EWOD microelectrode array architecture. In one embodiment, there are 9 adjacent configured electrodes  1231 .  1232 , to  1239 . Each of the configured electrodes is composed of a configured 10×10 microelectrode squares. The droplet  1250  lies on top of the center configured-electrode  1235 . In one embodiment of the current system, the droplet can be transported either in the north-south or east-west directions by the manipulations of the configured electrodes. For example, by activating configured electrode  1234  and deactivating configured electrode  1235 , droplet  1250  can travel from configured electrode  1235  onto configured-electrode  1234 . In another embodiment, the droplet  1250  can be transported in a diagonal direction according to users&#39; needs. For example, droplet  1250  can be transported diagonally from configured-electrode  1235  onto anyone of configured-electrodes  1231 ,  1233 ,  1237 , or  1239 , even though these four configured-electrodes  1231 ,  1233 ,  1237 , and  1239  have no physical overlapping with droplet  1250 . In order to move the droplet  1250  diagonally, one embodiment is to activate configured electrode  1260  as the interim step, and then subsequently activate the desired configured electrode  1233  and then deactivate the interim configured electrode  1260  so therefore so to move the droplet  1250  diagonally into the desired configured electrode  1233 . As shown in  FIG. 12 , the droplet  1250  can be moved in all eight directions in a square-electrode setting, including north-south, east-west, north-west, north-east, south-east or south-west directions. In one embodiment, the sizes of the interim configured electrode  1260  can include multiple microelectrodes according to users&#39; needs. In another embodiment, the shapes of the interim configured electrode  1260  can be varied to facilitate the droplet transportation. Still in another embodiment, the transportation direction of droplet  1250  is not limited to the eight directions. Even the adjacent configured electrode falls outside of the eight directions an interim configured electrode can be created and activated to transport the droplet into desired location. 
         [0081]    Another embodiment in the droplet transportation and movement of the droplet with the EWOD Microelectrode Array Architecture including the Interim bridging technique is illustrated in  FIGS. 13A-13C . Through droplet cutting and evaporation, the droplet can be too small to be actuated reliably by the electrodes.  FIG. 13A  shows two configured-electrodes  1330 ,  1340 , respectively, which are separated by a gap  1360 . The droplet  1350  sits on the left-side configured-electrode  1330 . The gap  1360  between the two configured-electrodes  1330  and  1340  is wide enough to segregate the two configured-electrodes  1330 ,  1340 . The droplet  1350  sits on the left-side configured-electrode  1330  would not touch the next adjacent configured-electrode  1340 . 
         [0082]    In  FIG. 13A , the droplet  1350  cannot be moved directly from electrode  1330  to next adjacent electrode  1340 , since there is no physical overlapping between droplet  1350  and electrode  1340  to change the surface tension. This problem is often seen in conventional EWOD transportation.  FIG. 13B  illustrates one embodiment of the transportation of the droplet  1350  from  FIG. 13A  into the desired configured-electrode  1340 . In this procedure, the microelectrodes covered by the “toothed” area  1370  are activated. The toothed configured electrode  1370  covers partially the left-side configured electrode  1330 , gap  1360 , and the entire adjacent configured-electrode  1340 . As shown in  FIG. 13B , the “toothed” configured-electrode  1370  has a physical overlap with droplet  1350 . So trigger the activation of configured-electrode  1370  will move the droplet  1350  onto the top of configured electrode  1370 .  FIG. 13C  illustrates the completion of the droplet transportation to the desired configured electrode  1340 . After the droplet  1350  is transported to the desired configured-electrode  1370 , the “toothed” configured-electrode  1370  is de-activated. The configured-electrode  1340  is then activated to position and locate the droplet  1350  into the desired square-shaped electrode  1340 . 
         [0083]    Another embodiment in droplet transportation and movement with the EWOD Microelectrode Array Architecture includes the electrode column actuation manipulation. Through droplet cutting and evaporation, the droplet can be too small to be actuated reliably by the electrodes. As illustrated in  FIG. 14A , the droplet  1450  is much smaller than the electrode  1410  and no physical overlapping between the droplet  1450  and the adjacent electrode  1411 . In this situation even if electrode  1411  is activated the droplet  1450  still cannot be moved into electrode  1411  so the droplet can be easily stuck inside the system. One embodiment to effectively flush out the stuck droplets is to use the electrode column actuation. In  FIG. 14B , the actuating electrodes are arranged into columns to perform the electrode column actuation. In one embodiment, the ach configured electrode column  1420  is composed of 1×10 microelectrodes. Three configured electrode columns are grouped together to perform the electrode column actuation as marked black in  FIG. 14B . The default column width is one microelectrode but can be other numbers depending on the applications. In another embodiment, the most effective electrode column actuation with a group of columns has the width no less than the radius of the droplet  1450 . Still in another embodiment, the length of the column depends on the application and normally the longer the better. 
         [0084]      FIG. 14B  shows how the three-configured-electrode column can be manipulated to facilitate the droplet transportation. The configured electrode column  1421  before the leading configured-electrode column  1420  can be activated, and the tailing configured-electrode column  1422  is deactivated. In this embodiment, regardless the sizes of the droplets, the three configured electrode column provides a maximum effective length of the contact line. As a result, the droplet  1450  can be moved efficiently and smoothly because the capillary force on the droplet  1450  is consistent and maximized. The droplet  1450  can be moved in a much lower driving voltage than the conventional EWOD droplet operations. This electrode column actuation technique can be used to transport droplets with smooth movement in much lower driving voltage. Also, because the consistent capillary force of this technique, it can be used to do the control of the droplet speed especially in low speed situations by advancing the configured-electrode column in low speed. In another embodiment, under the marginal driving voltages, the electrode column actuation can be applied to actuate the droplets. Still in another embodiment, slowly but steadily moving DI water droplet (1.1 mm diameter) in 10 cSt silicon oil has been observed below 8 Vp-p 1 k Hz square driving voltage with 80 μm gap. Still in another embodiment, the length of the configured electrode column can be configured to be the full length of the LOC. A single sweep of the electrode column actuation can wash out all dead droplets in the LOC.  FIG. 14C  shows that while the activated configured electrode columns (marked as black) keep moving to the right and eventually move out of the configured-electrode  1410 , the small droplet  1450  is also carried out of configured-electrode  1410 . 
         [0085]      FIGS. 15A-15C  show one embodiment for performing a typical three-electrode cutting of a droplet under the EWOD microelectrode array architecture.  FIG. 15A  shows three configured electrodes  1510 ,  1511 ,  1512  line together horizontally. The droplet  1550  ready to be cut sits on the center configured electrode  1511 . In  FIG. 15A , the configured electrode  1511  is activated to hold the droplet  1550 . The droplet  1550  overlaps with the portions of adjacent configured electrodes  1510  and  1512 .  FIG. 15B  shows the droplet cutting stage by activating the configured electrodes  1510 ,  1512  at the same time and deactivate the configured electrode  1511 . By the electrode manipulation the droplet  1550  is been pulled toward left-right directions to electrodes  1510  and  1512 . In one embodiment, the hydrophilic forces induced by the two outer configured-electrodes  1510  and  1512  stretch the droplet while the hydrophobic forces in the center pinch off the liquid into two daughter droplets  1551 ′ and  1552 ′ as shown in  FIG. 15C . 
         [0086]    One embodiment of the droplet cutting is illustrated in  FIGS. 16A-16C .  FIGS. 16A-16C  shows three configured electrodes  1610 ,  1611 ,  1612  line together horizontally. The droplet  1650  ready to be cut sits on the center configured electrode  1611 . Instead of using outer two configured-electrodes  1610  and  1612  to cut the droplet  1650 , the electrode column actuation technique is used to slowly but firmly pull the droplet  1650  toward configured-electrodes  1610  and  1612  as shown in  FIG. 16A . In this embodiment, two configured electrode columns  1615  and  1616  (marked as black in  FIG. 16A ) are used and activated to pull the droplet apart. Each of the two configured electrode columns includes five columns of electrodes.  FIG. 16B  illustrates the two electrode columns keep moving apart by advancing one microelectrode column each over a time, so to slowly pull and move the droplet  1650  toward opposite direction. The hydrophilic forces induced by the two electrode columns  1615  and  1616  are applied to stretch the droplet  1650 . When electrode columns  1615  and  1616  reach the outer edges of the configured-electrodes  1610  and  1612 , deactivate these configured-electrode columns  1615  and  1616 . The configured-droplets  1610  and  1612  are activated to pinch off the liquid into two sub-droplets  1651  and  1652  as shown in  FIG. 16C . 
         [0087]      FIGS. 17A-17C  illustrates the embodiment of performing a diagonal cutting performed with the EWOD microelectrode array architecture. The diagonal cutting starts with moving the droplet to be cut onto a interim configured-electrode  1712  which is centered at the joint corner of the four configured-electrodes  1710 ,  1711 ,  1713  and  1714  in  FIG. 17A . The moving of the droplet  1750  can be achieved by activating the electrodes. After the droplet completely centered at the joint corner of the four configured-electrodes, the interim configured-electrode  1712  is deactivated and configured electrodes  1710 ,  1711  are activated so to stretch the droplet  1750  into a liquid column as indicated in  FIG. 17B . To pinch off the liquid into two daughter droplets, the inner corners of configured electrodes  1710  and  1711  are deactivated to produce the necessary hydrophobic forces in the middle of droplet  1750 .  FIG. 17C  shows the L-shaped interim configured electrodes  1715  and  1716  are activated to further stretches the droplet  1750  with only a thin neck in-between. The hydrophobic forces in the middle subsequently help to pinch off droplet  1750  into two sub-droplets  1751  and  1752 . Finally, configured-electrodes  1710  and  1711  are activated again to center-position the sub-droplets  1751  and  1752  as illustrated in  FIG. 17D . 
         [0088]    The diagonal cutting of the droplet cutting is efficient and effective because the two pulling electrodes possess longer length of the electrode contact. The pulling capillary forces on the droplet are greater than the conventional cutting. As a result, the cutting voltage can be reduced and more uniform droplet cutting can be achieved. For a conventional cutting, it may require voltages that exceed the saturation voltage (i.e., voltage corresponds to contact angle saturation). To obtain more reliable EWOD droplet operations, extra care must be used in setting the conditions for uniform splitting so not to exceed the saturation voltage. Thus, the diagonal cutting is a good candidate for droplet cutting to keep the cutting voltages below the saturation voltage. Also, the diagonal cutting is less constrained by the droplet size. A conventional cutting requires a bigger droplet that can be physically overlapped with the outer two electrodes. The diagonal cutting can virtually cut any size of droplet. 
         [0089]    In one embodiment, the droplet cutting procedure can be applied to the coplanar structure when the droplet cutting is performed on the open surface under the EWOD Microelectrode Array Architecture.  FIGS. 18A-18C  illustrate the droplet cutting procedure on an open surface under the EWOD Microelectrode Array Architecture.  FIG. 18A  illustrates a droplet  1850  sits on the left-side configured electrode  1840 . The droplet  1850  will be cut into two sub-droplets  1870  as shown on  FIG. 18C . The droplet cutting procedure generally involves the next two procedures. First, stretch the droplet-to-be-cut  1850  into a thin liquid column  1860  by activating the configured-electrode  1830  under appropriate voltages. This can be seen in  FIG. 18B . Such “thin” liquid column generally refers to the liquid column with smaller width than the starting droplet diameter. Next, activate the two preselected configured-electrodes  1840  and  1820  to cut and to center-position droplets  1870  into these two configured-electrodes  1840  and  1820  as shown in  FIG. 18C . The key for the coplanar cutting is to have enough overlaps between the droplet and the outer two configured-electrodes to have enough capillary force to overcome the curvature of the droplet to perform the cutting. In one embodiment, a passive cutting is presented when the liquid column  1860  is cut into multiple droplets by hydrodynamic instability. In another embodiment, both the passive and the active cutting are employed. While the droplet is stretched into a thin liquid column, either the passive force or active force can be employed to break the starting droplet into two smaller droplets. When use the passive force, the calculation of the length of liquid column is important. When use active force, the optimized length is not important. Either passive cutting or active cutting, at the final step of the cutting procedure, configured electrodes  1840  and  1820  are normally activated in order to position the droplets into the desired configured-electrodes. In another embodiment, either an active or a passive cutting procedure is performed under the open surface structure by using EWOD Microelectrode Array Architecture.  FIG. 18C  illustrates the completion of cutting when the droplet  1850  is cut into two sub-droplets  1870 . 
         [0090]    One embodiment of performing a basic merge or mixing operation under the EWOD microelectrode array architecture as shown in  FIGS. 19A-19B . In the present discussion, the terms merge and mixing have been used interchangeably to denote the combination of two or more droplets. This is because the merging of two droplets does not in all cases directly or immediately result in the complete mixing of the components of the initially separate droplets. In  FIG. 19A , two droplets  1950  and  1951  are initially positioned at each of the corresponding configured electrodes  1910  and  1912 , respectively, and separated by at least one intervening configured electrode  1911 . Both droplets  1950  and  1951  have partial overlaps with the central configured electrode  1911 . As shown in  FIG. 19B , by deactivating the two configured electrodes  1910  and  1912  and activating the central configured electrode, the droplets  1950  and  1951  can be moved toward each other across the central configured-electrode  1911  and then merged into a bigger droplet  1953 . 
         [0091]    In EWOD Microelectrode Array Architecture, mixing of analytes and reagents is a critical step. The droplets act as virtual mixing chambers, and mixing occurs by transporting two droplets into the same electrode. The ability to mix liquids rapidly while utilizing minimum area greatly improves the throughput. Conventionally, an effective mixing of droplets might need eight (2×4) electrodes to move the mixed droplet in certain way among these eight electrodes to speed up the mixing. A way to mix the droplets efficiently without the requirements of using big real estate for the mixing operation is highly desirable. However, as microfluidic devices are approaching the sub nano-liter regime, reduced volume flow rates and very low Reynolds numbers can make mixing liquids difficult to achieve under reasonable time scales. Improved mixing relies on two principles: the ability to create turbulent flow at such small scales, or alternatively, the ability to create multilaminates to achieve fast mixing. The EWOD Microelectrode Array Architecture can provide active droplet-based mixing at least an order of magnitude faster than passive mixing by diffusion. 
         [0092]      FIGS. 20A-20C  illustrate the active mixing procedure of the droplet manipulation by uneven-geometry movement to create turbulent flow based on the EWOD Microelectrode Array Architecture. As shown in  FIG. 20A , the droplets  2050 ,  2070  can be deformed to desired shapes by the manipulation of the configured electrodes. By activating the configured electrodes  2051  and  2071  as shown in  FIG. 20B , the droplets are shown as droplet  2050 ′ and droplet  2070 ′. The center configured electrode  2060  then is activated in order to pull the droplets  2050 ′,  2070 ′ into the mixing configured electrode  2060  (marked in black) as shown in  FIG. 20C . In  FIG. 20B , the black areas indicate two activated configured electrodes  2051  and  2071 . These activated electrodes can apply to deform and pull these two droplets  2050 ′ and  2070 ′ into the center configured electrode  2060 . This interim activating step shown in  FIG. 20B  also helps a smooth mixing movement of the two droplets. The shapes of the black area and the deformed droplets in  FIGS. 20B-20C  are for illustration purposes only. In another embodiment, the shapes can be any types based on the needs. 
         [0093]      FIGS. 21A and 21B  illustrate the microelectrode array mixer for improving the mixing speed. In one embodiment, an uneven back-and-forth mixer can be used to speed up the droplet mixing. This can be done by activating a group of microelectrodes to create an irreversible pattern that breaks the symmetry of the two circulations to improve the speed of mixing. The initial state is illustrated as in  FIG. 21A  that a droplet  2150  contains both sample and reagent sitting on top of configured electrode  2140 . The first step for the uneven back-and-forth mixing is to activate configured electrode  2160  to deform the droplet  2150  to the direction of the arrows as shown in  FIG. 21B . Then configured electrode  2160  is de-activated and configured electrode  2140  is activated to pull the droplet back to the original position as indicated in  FIG. 21A . The back-and-forth mixing can be done multiple times to achieve the optimized mixing results. Also, the shapes of the configured-electrode  2140  and the deformed droplets in  FIGS. 21A and 21B  are for illustration purposes only. In one embodiment, the shapes can be any types of designs as long as they have the ability to create turbulent flows, or alternatively, the ability to create multilaminates. 
         [0094]    Still in another embodiment of EWOD droplet based mixing procedure,  FIG. 22  illustrates a circular mixer for improving the mixing speed. This can be done by activating a sequence of the smaller groups of microelectrodes to create an irreversible horizontal circulation that breaks the symmetry of the vertical laminar circulation to speed up the mixing. One embodiment, as shown in  FIG. 22 , is to form eight configured-electrodes ( 2210 ,  2220 ,  2230 ,  2240 ,  2250 ,  2260 ,  2270  and  2280 ) that enclose the droplet  2290  and then activate these configured electrodes one-by-one in sequence and in a circular manner. For example, in the first step, the configured electrode  2210  is activated for a short period of time to cause surface tension change and to create circulation inside the droplet  2290  on the configured electrode  2210 . Next, the configured-electrode  2210  is deactivated followed by activating the next adjacent configured-electrode  2220 . The circular activating procedure is repeated through entire eight configured electrodes ( 2210  to  2280 ) to create the horizontal circulation inside the droplet  2290 . This circulation flow activation can be done multiple times based on the needs. In another embodiment, the circulation flow can be done clockwise, counter-clockwise or an alternative mix of the two to achieve the best mixing results. Still in another embodiment, the shapes of the configured-electrodes  2210  to  2280  can be other types and the circulation are for illustration purposes only. Still in another embodiment, such circulation mixing can be any types of designs as long as they have the ability to create turbulent flow, or alternatively, the ability to create multilaminates. 
         [0095]    In one embodiment, a small footprint (2×2 configured-electrodes) mixer to create multilaminates to speed up the mixing procedure in EWOD microelectrode array architecture is achieved. This multilaminates mixer is especially useful for low aspect ratio (&lt;1) situation. The aspect ratio is the ratio of the gap between electrode plate and the ground plate and the dimension of the electrode. Low aspect ratio means more difficult to create turbulent flow inside the droplet and the ability to create multilaminates becomes more important. One embodiment is illustrated in  FIGS. 23A-23E . Diagonal mixing and diagonal cutting are used in this special mixer. The diagonal movement of the droplets can be done by the EWOD microelectrode array architecture. In  FIG. 23A , the black droplet  2351  positioned at configured electrode  2314  will be mixed with the white droplet  2350  positioned at configured-electrode  2311 . An interim configured electrode  2310  will be the mix chamber and will be activated to pull in both droplets  2351  and  2350 . To start the multilaminates mixing, step one is to merge the two droplets diagonally. The diagonal direction of the droplet merge can be either 45 degree or 135 degree. The subsequent step of diagonal cutting will be perpendicular to this merge operation.  FIG. 23B  indicates the first merge of droplet  2351  and droplet  2350  into a black-and-white droplet  2352 . Because of the low Reynolds number and the low aspect ratio, droplet  2352  has purely diffusion-based static mixing which results in a long mixing time, so the mixed droplet is shown as half white and half black. The second step is to do the diagonal cutting. Ninety degree from the starting diagonal mixing of droplet  2352  is illustrated in  FIG. 23C . While the interim configured-electrode  2310  is deactivated, configured electrodes  2312  and  2313  and other interim configured-electrodes are activated to diagonally cut droplet  2352  into two daughter droplets  2353  and  2354  as shown in  FIG. 23C . The details of the diagonal cutting are discussed in the above-described Diagonal Cutting procedure. Because of the slow mixing rate, so the two daughter droplets  2353  and  2354  keep the black/white laminates with the same orientation after the diagonal cutting. Then, the third step of the multilaminates mixing is to move the two droplets back onto the starting configured-electrodes to repeat the diagonal mixing and cutting. In  FIG. 23D , droplets  2354  is moved from configured electrode  2312  onto next adjacent configured electrode  2311 . Droplets  2353  is moved from configured electrode  2313  onto next adjacent configured-electrode  2314 . This can be done by activating the electrodes  2311 ,  2314  and deactivate electrodes  2312  and  2313 . Cares are needed to avoid the merge of droplets  2353  and  2354  during movement. For example, the deactivations of electrodes and activations of electrodes might cause a physical contact of the two droplets  2353 ,  2354  while they are moving and so the two droplets would merge together. In one embodiment, interim configured-electrodes  2315  and  2316  are activated first to create the safeguard zone between the two droplets to prevent accidental merge during their movement toward the desired electrodes. After droplets  2353  and  2354  are moved into configured-electrodes  2316  and  2315 , move the two droplets into configured-electrodes  2311  and  2314 . The procedure can be repeated to create the necessary numbers of multilaminates to speed up the mixing.  FIG. 23E  shows four-laminated droplet  2355  as the result of repeated steps to diagonally merge droplets  2353  and  2354  (from  FIG. 23D ) into droplet  2355 .  FIG. 23F  illustrates eight-laminated droplet  2356  after repeated cycles of the multilaminates mixing. 
         [0096]    In various embodiments, EWOD Microelectrode Array Architecture can perform continuous-flow microfluidic operations instead of droplet-based microfluidic operations. Continuous microfluidic operations provide very simple in control but very effective way of doing microfluidic operations.  FIGS. 24A-C  illustrate the creation of a certain volume of liquid  2430  from the reservoir  2410 . As shown in  FIG. 24A , a small line of microelectrodes formed a bridge  2415  between the targeted configured-electrode  2460  and the reservoir  2410 . When the bridge  2415  and the targeted configured-electrode  2460  are activated that causes a liquid flow from the reservoir into the targeted configured-electrode  2460 .  2430  indicates the liquid flows from the bridge into the configured-electrode  2460 . The bridge here is a single line of microelectrodes. This bridge configuration has the characteristics of both continuous-flow and droplet-based systems. It has all the benefits of a channel that once the bridge configured-electrode is activated the liquid will flow through it without extra controls and concerns on the activating timing and speeds. But it also has all the advantages of droplet-based system that once the bridge  2415  is deactivated all liquid will be pulled back to either the reservoir or the targeted configured-electrode  2460  and it has no dead-volume in the channel. Once the targeted configured-electrode  2460  is filled up then deactivated the bridge  2415  to cut the liquid  2430  from the reservoir  2410  as shown in  FIG. 24B . The liquid fill-up of the configured-electrode  2460  is automatic that once all microelectrodes of the bridge and the configured-electrode electrode are filled up with liquid then the liquid flow from the reservoir  2410  will stop, so the timing control of the procedure is not critical. The creation of liquid  2430  can be precisely controlled by activating the appropriate microelectrodes  2460  and the breaking point of the bridge. As shown in  FIG. 24B , liquid  2430  is breaking out from the reservoir  2410  by deactivating microelectrode  2416  first then the bridge is deactivated. This procedure will make sure most of the liquid formed the bridge will be pull back to the reservoir  2410  and the liquid  2430  will be precisely controlled by the number of microelectrodes of the configured-electrode  2460 . In  FIG. 24B , the configured-electrode  2460  is composed of 10×10 microelectrodes. Other sizes and shapes of the configured-electrodes can be defined to create different liquid sizes and shapes.  FIG. 24C  shows the disappearing of the liquid bridge and the liquid  2430  is created by activating reservoir  2410  and the configured-electrode  2460 . 
         [0097]    In one embodiment, the same creating procedure of liquid can be used to perform the cutting of the liquid into two sub-liquids as illustrated in  FIG. 24D . After deactivating configured-electrode  2460 , configured-bridge-electrode  2417  and targeted configured-electrode  2471  are activated and liquid flows from the bridge into the area of  2470 . Deactivating the configured-bridge-electrode  2417 , then activating configured-electrodes  2461  and  2471  breaks up and forms the two sub-liquids  2470  and  2430  as illustrated in  FIG. 24E . This cutting process can generate the two sub-liquids in different sizes as long as the size of the configured-electrodes  2461  and  2471  are pre-calculated to the desired sizes. 
         [0098]    In another embodiment,  FIGS. 25A-C  illustrate the mixing procedure by the continuous-flow microfluidic operations.  FIG. 25A  shows the activating of bridges  2515  and  2525  and the activating of configured-electrodes  2516  and  2526 , liquids are flowing from reservoirs  2510  and  2520  through the bridges into the mixing chamber  2530 . Here liquids associate with configured-electrodes  2516  and  2526  are in de-formed shapes for better mixing and also liquids also are in different size for a ratio mixing. Gap is between configured-electrodes  2516  and  2526  to prevent the premature mixing. Once the liquid fill up both configured-electrodes  2516  and  2526 , then configured-electrode  2530  (10×10-microelectrodes) is activated and the two liquid will be mixed as indicated in  FIG. 25B . Then two bridge-electrodes are deactivated as illustrated in  FIG. 25C . 
         [0099]    In this simple mixing microfluidic operations, actually all fundamental microfluidic operations are demonstrated: (1) Creating: liquids  2516  and  2526  are created from reservoirs  2510  and  2520  in a precise way, (2) Cutting: liquid  2516  is cut off from liquid  2510  and liquid  2526  is cut from liquid  2520 , (3) Transporting: Bridges  2515  and  2525  transport liquids to the mixing chamber, and (4) Mixing: liquid  2516  and  2526  are mixed at  2530 . It&#39;s very obvious that this continuous-flow technique not only can be used to perform all microfluidic operations but also in a more precise way because the resolution of the precision is depend on the small microelectrode. 
         [0100]    The shape of the microelectrode in FPLOC can be physically implemented in different ways. In one embodiment of the invention,  FIG. 26A  illustrates an array of square microelectrodes and one of them is highlighted as  2601 . And 6×6 microelectrodes form the configured-electrode  2602 .  FIG. 26A  totally have a 3×2 configured-electrodes. In another embodiment,  FIG. 26B  shows an array of hexagon microelectrodes and one of them is highlighted as  2603 . And 6×6 microelectrodes form the configured-electrode  2604  and there are 3×2 configured-electrodes in  FIG. 26B . The interdigital edge of the hexagon microelectrode has the advantage in moving the droplet across the gap between the configured-electrodes. Yet in another embodiment,  FIG. 26C  shows an array of square microelectrodes that are arranged in a wall-brick layout and one of them is highlighted as  2605 . And 6×6 microelectrodes form the configured-electrode  2606  and there are 3×2 configured-electrodes in  FIG. 26C . The interdigital edge of the hexagon microelectrode has the advantage in moving the droplet across the gap between the configured-electrodes, but this only happens on the x-axis. There are many other shapes of the microelectrodes can be implemented and not only limited to the three shapes discussed here. 
         [0101]    Although the present invention has been described with reference to preferred embodiments, persons skilled in the art will recognize that changes may be made in form and detail without departing from the spirit and scope of the invention.