Patent Publication Number: US-2021178397-A1

Title: Apparatus and method for forming droplets having predetermined volume by electrowetting

Description:
FIELD OF THE INVENTION 
     Embodiments of the present invention relate generally to electrowetting-on-dielectric (EWOD) technology, and more particularly relate to apparatuses and methods for forming microdroplets having a predetermined volume from one or more bulk droplets by electrowetting techniques. 
     BACKGROUND OF THE INVENTION 
     Electrowetting-on-dielectric (EWOD) is a liquid driving mechanism to change a contact angle of an aqueous droplet between two electrodes on a hydrophobic surface. A bulk liquid droplet as large as several millimeters (i.e., several microliters in volume) can be moved by an array of electrodes disposed on a substrate, such as an inorganic substrate (e.g., silicon/glass substrate) or organic substrate (e.g., a cyclic olefin polymer/polycarbonate substrate). 
       FIG. 1A  is a perspective view of a schematic diagram illustrating an EWOD device  10  that may be used to explain embodiments of the present disclosure. The EWOD device includes a substrate structure  11  having a substrate  12 , an insulating layer  13  on the substrate, and an array of electrodes  14  within or under the insulating layer. The array of electrodes  14  includes a first set of electrodes  14   a  arranged in parallel to each other and spaced apart from each other in a first direction, and a second set of electrodes arranged in parallel to each other and spaced apart from each in a second direction substantially perpendicular to the first direction. The first and second set of electrodes are spaced apart from each other within the insulating layer  13 , which may include a plurality of dielectric layers of the same material or different materials. The EWOD device also includes an input-output circuit  15  in the substrate and operative to interface with an external control circuit to provide control voltages having time-varying voltage waveforms to the array of electrodes  14 . 
     Referring to  FIG. 1A , a liquid droplet  16  disposed on the surface of the insulating layer  13  may be moved along a certain direction by turning off/off control voltages at electrodes below the droplet and at adjacent electrodes. 
       FIG. 1B  is a cross-sectional view of the EWOD device shown in  FIG. 1A  taken along the line B-B′. The cross sectional view of the second set of electrodes  14   b  is shown. The first set of electrodes  14   a  (not shown) may be disposed above or below the second set of electrodes  14   b  and spaced apart from the second set of electrodes by one or more dielectric layers. 
     BRIEF SUMMARY OF THE INVENTION 
     Embodiments of the present disclosure provides an apparatus, system, and method for mixing a droplet with reagents, forming extremely small drops (particles, microdroplets, or samples) from the mixed droplet, reading the DNA concentration of the samples by optical detection or measuring the pH value of each sample through integrated ion-sensitive field-effect transistor (ISFET) sensors, thereby calculating the DNA concentration of the droplet. It should be noted that although the embodiments describe apparatuses and processes of measuring a pH value of a droplet, the present disclosure is not limited thereto. Those of skill in the art will appreciate that the apparatuses and processes described herein may be applied to pH measurements of any aqueous and non-aqueous liquids based on pH changes within the extremely small drops (microdroplets. 
     In one aspect embodiment of the present disclosure, an apparatus for forming a plurality of microdroplets from a droplet includes a substrate, a dielectric layer on the substrate and having a plurality of hydrophilic surface regions spaced apart from each other by a hydrophobic surface, and a plurality of electrodes in the dielectric layer. The electrodes are configured to form an electric field across the droplet in response to voltages provided by a control circuit to move the droplet across the dielectric layer in a lateral direction while leaving portions of the droplet on the hydrophilic surface regions to form the plurality of microdroplets on the hydrophilic surface regions. In some embodiments, the apparatus may further include one or more sensors associated with one of the hydrophilic surface regions. A sensor includes an ion sensitive field effect transistor containing an ion sensing film configured to be exposed to a solution containing in a microdroplet and provide a signal associated with a concentration level of the solution of the microdroplet, and a reference electrode configured to apply a reference voltage to the solution. 
     In another aspect, a substrate structure includes a first substrate, a dielectric layer having a hydrophobic surface on the first substrate, a control circuit, a plurality of conductive wirings, and a plurality of electrodes in communication with the control circuit through the conductive wirings and configured to form electric fields in response to voltages provided by the control circuit. The dielectric layer includes a first region for receiving a droplet, a second region for receiving one or more reagents, the second region being in communication with the first region and configured to mix the droplet with the one or more reagents to obtain a mixed droplet, and a third region in communication with the second region and comprising a plurality of hydrophilic surface regions spaced apart from each other by the hydrophobic surface. A portion of the droplet forms a plurality of microdroplets on the hydrophilic surface regions when the droplet moves over the hydrophilic surface regions. 
     In some embodiments, the apparatus also includes a fourth region in communication with the third region and configured to collect a remaining portion of the droplet after the remaining portion of the droplet has moved across the third region. 
     In some embodiments, the substrate structure further includes a second substrate and an electrically conductive layer on the second substrate facing the hydrophobic surface of the dielectric layer, and a space between the electrically conductive layer and the dielectric layer forming a channel for the droplet. 
     In some embodiments, the electrically conductive layer is operative as a common electrode, and the plurality of electrodes comprise a first array of parallel stripes along a first direction from the first region to the third region, and a second array of parallel stripes in the second region along a second direction perpendicular to the first direction. 
     In some embodiments, the third region further includes one or more sensors associated with one of the plurality of hydrophilic surface regions. A sensor includes an ion sensitive field effect transistor including an ion sensing film configured to be exposed to a solution containing in a microdroplet and provide a signal associated with a concentration level of the solution of the microdroplet, and a reference voltage electrode configured to apply a reference voltage to the solution. 
     In yet another aspect, a method for forming a plurality of samples having a substantially uniform size from a droplet may include providing a substrate structure having a substrate, a dielectric layer on the substrate, a plurality of electrodes in the dielectric layer, wherein the dielectric layer has a hydrophobic surface and a plurality of hydrophilic surface regions surrounded by the hydrophobic surface, discharging the droplet on a surface of the dielectric layer, and applying time-varying voltage waveforms to the electrodes to move the droplet across the hydrophilic surface regions of the dielectric layer to form the plurality of samples on the hydrophilic surface regions. 
     This summary is provided to introduce the different embodiments of the present disclosure in a simplified form that are further described in detail below. This summary is not intended to be used to limit the scope of the claimed subject matter. Other features, details, utilities, and advantages of the claimed subject matter will be apparent from the following detailed description. 
     Definitions 
     The terms “wafer” and “substrate” are to be understood as including silicon-on-insulator (SOI) or silicon-on-sapphire (SOS) technology, doped and undoped semiconductors, epitaxial layers of silicon supported by a base semiconductor foundation, and other semiconductor structures. Furthermore, when reference is made to a “wafer” or “substrate” in the following description, previous process may have been utilized to form regions or junctions in the base semiconductor structure or foundation. In addition, the semiconductor need not be silicon-based, but could be based on silicon-germanium, germanium, or gallium arsenide. 
     The term “computer readable medium” as used herein refers to any medium that participates in providing instructions to the processor of the controller for execution. A computer readable medium may take many forms, including but not limited to, nonvolatile media, volatile media, and transmission media. Nonvolatile media includes, for example, optical disks, magnetic disks, and magneto-optical disks, such as the hard disk or the removable media drive. Volatile media includes dynamic memory, such as the main memory. Moreover, various forms of computer readable may be involved in carrying out one or more sequences of one or more instructions to the processor of the controller for execution. For example, the instructions may initially be carried on a magnetic disk of a remote computer. The remote computer can load the instructions for implementing all or a portion of the present invention remotely into a dynamic memory and send the instructions over a network to the controller. 
     The term “hydrophobic” refers to a material having a contact angle of water in air of greater than or equal to 90 degrees. In certain embodiments, hydrophobic surfaces may feature contact angle greater than 90 degrees, such 120 degrees, 150 degrees, etc. In contrast, the term “hydrophilic” refers to a material having a contact angle of water in air or immiscible liquid such as oil of less than 90 degrees. 
     The term “droplet” has its normal meaning in the art and refers to a liquid with boundaries formed at least in part by surface tension having a certain volume, e.g., between about several milliliters (10 −3 ) to about several microliters (10 −6 ). A droplet may be a water-based (aqueous) droplet including any organic or inorganic species such as, biological molecules, proteins, living or dead organisms, reagents, and any combination thereof. A droplet may be a non-aqueous liquid. A droplet may be spherical or non-spherical and have a size ranging from 1 micron to of about several millimeters. A droplet may be partitioned into multiple very small portions (small droplets) that are spaced apart from each other and having a substantially uniform size. The very small portions may have a volume of between 1 microliter (10 −6  L or mL) and 100 nanoliter (10 −9  liter or nL), between 100 nL and 10 10 nL, between 10 nL and 100 pL (10 −12  liter), 100 pL and 10 pL. In certain embodiments, the very small portions may have a volume of a few picoliters. The very small portions are alternatively referred to as microdoplets in the present disclosure. 
     The term “reagent” refers to a molecule or a compound of different molecules being able to induce a specific reaction with a species present in a droplet. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         FIG. 1A  is a simplified perspective view of a schematic diagram illustrating an EWOD device that may be used to explain embodiments of the present disclosure. 
         FIG. 1B  is a simplified cross-sectional view of the EWOD device shown in  FIG. 1A  taken along the line B-B′. 
         FIG. 2A  is a simplified cross-sectional view of a portion of an EWOD device according to an embodiment of the present disclosure. 
         FIG. 2B  is a simplified cross-sectional view of a portion of an EWOD device according to another embodiment of the present disclosure. 
         FIG. 2C  is a simplified cross-sectional view of a portion of an EWOD device according to yet another embodiment of the present disclosure. 
         FIG. 2D  is a simplified plan view of an EWOD device according to an exemplary embodiment of the present disclosure. 
         FIG. 2E  is a simplified cross-sectional view illustrating electric fields generated by electrodes according to an embodiment of the present disclosure. 
         FIG. 2F  is a simplified cross-sectional view illustrating electric fields generated by electrodes according to another embodiment of the present disclosure. 
         FIG. 2G  is a simplified cross-sectional view illustrating electric fields generated by electrodes according to yet another embodiment of the present disclosure. 
         FIGS. 3A to 3C  are simplified top views of a droplet moving across a surface of a dielectric layer according to embodiments of the present disclosure.  FIG. 3A  is a simplified top view illustrating that a droplet is discharged on a first electrode of an array of electrodes according to an embodiment of the present disclosure.  FIG. 3B  is a simplified top view illustrating that the droplet is moved to a second (adjacent) electrode under the effect of electric fields by the EWOD device according to an embodiment of the present disclosure. 
         FIG. 3C  is a simplified top view illustrating that the droplet is moved out of the array of electrodes while leaving a residue on the second electrode according to an embodiment of the present disclosure. 
         FIG. 4A  is a cross-sectional view of a portion of an EWOD device according to an embodiment of the present disclosure. 
         FIG. 4B  is a cross-sectional view of a portion of an EWOD device according to another embodiment of the present disclosure. 
         FIG. 4C  is a perspective view of a portion of an EWOD device shown in  FIG. 4A . 
         FIG. 4D  is a perspective view of a portion of an EWOD device shown in  FIG. 4B . 
         FIG. 4E  is a perspective view of a portion of an EWOD device according to an embodiment of the present disclosure. 
         FIG. 5  is a cross-sectional view of an ISFET device according to an embodiment of the present disclosure. 
         FIG. 6A  is a simplified top view of an integrated lab-on-a-chip device according to an embodiment of the present disclosure. 
         FIG. 6B  is a simplified top view of an example arrangement of electrodes of the integrated lab-on-a-chip device in  FIG. 6A  according to an embodiment of the present disclosure 
         FIG. 7  is a simplified flowchart illustrating a method for forming a plurality of samples from a droplet according to an embodiment of the present disclosure. 
         FIG. 8  is a simplified flowchart illustrating a method for operating an integrated lab-on-a-chip apparatus according to an embodiment of the present disclosure. 
         FIG. 9  is a simplified schematic diagram of a computer system that can be used to control the EWOD device and the lab-on-a-chip device according to an embodiment of the present disclosure. 
         FIG. 10  is a cross sectional view of device structure having differential surface regions according to some embodiments of the present disclosure. 
         FIGS. 11A-11F  are cross-sectional views illustrating a method for forming a device structure of  FIG. 10  having differential surface regions according to some embodiments of the present disclosure. 
         FIGS. 12A-12C  are cross-sectional views illustrating a method for forming a device structure of  FIG. 10  having differential surface regions according to alternative embodiments of the present disclosure. 
     
    
    
     In accordance with common practice, the described features and elements are not drawn to scale but are drawn to emphasize features and elements relevant to the present disclosure. 
     DETAILED DESCRIPTION 
     It will be appreciated that for simplicity and clarity of illustration, elements shown in the Figures have not necessarily been drawn to scale. For example, the dimensions of some of the elements are exaggerated relative to each other for clarity. Further, where considered appropriate, reference numerals have been repeated among the Figures to indicate corresponding elements. 
     In the following detailed description, reference is made to the accompanying drawings which form a part hereof, and in which is shown by way of illustration specific embodiments in which the invention may be implemented. The term “upper”, “lower”, “vertical”, “horizontal”, “depth”, “height”, “width”, “top”, “bottom”, etc., is used with reference to the orientation of the Figures being described. Because components of embodiments of the present invention can be positioned in a number of different orientations, the term is used for purposes of illustration and is not limiting. 
     The use of the terms first, second, etc. do not denote any order, but rather the terms first, second, etc. are used to distinguish one element from another. Furthermore, the use of the terms a, an, etc. does not denote a limitation of quantity, but rather denote the presence of at least one of the referenced items. 
     As used herein, turning off an electrode refers to lowering the voltage of that electrode to a level below a common voltage, such as connecting an electrode to a ground potential. Alternatively, turning off an electrode may also refer to setting that electrode in a floating state. Conversely, turning on an actuation electrode refers to increasing the voltage of that actuation electrode to a level above the common voltage. The common voltage may be any voltage that is shared by a number of circuit elements of the EWOD device, e.g., the ground potential. 
     As used herein, a droplet is an encapsulated liquid. A droplet may be spherical or non-spherical. A droplet may be partitioned into multiple very small portions (microdroplets) that are spaced apart from each other and having a substantially uniform size. The very small portions of the droplet are alternatively referred to as microdroplets in the present disclosure. 
       FIG. 2A  is a simplified cross-sectional view of a portion of an EWOD device  20 A according to an embodiment of the present disclosure. Referring to  FIG. 2A , the EWOD device  20 A includes a first substrate  22 , a dielectric layer  23  on the substrate  21 , a set of actuation electrodes  24  (e.g.,  24   a ,  24   b ,  24   c ) within the dielectric layer  23 , a common electrode  27  attached to a second substrate  28  and facing toward the actuation electrodes  24 . 
     The common electrode  27  may be grounded or have other common voltage. The dielectric layer  23  and the common electrode  27  are spaced apart from each other by a spacer  29 . Referring to  FIG. 2A , a liquid droplet  26  is disposed between the actuation electrodes  24  and the common electrode  27  and is moving along a lateral direction across the surface of the dielectric layer  23  by means of changing or varying the voltage levels applied to the actuator electrodes in relation to the common electrode. In an embodiment, the EWOD device  20 A may further include a control circuit (not shown) configured to provide control voltages to the common electrode and the actuation electrodes. By turning on and off voltages applied to the actuation electrodes, the control circuit can move the liquid droplet  26  in a lateral direction across the surface of the dielectric layer  23 . For example, an electric field is generated by applying a first voltage to the actuation electrode  24   a  below the droplet  26  and a second voltage to the adjacent actuation electrode  24   b , the generated electric field causes the droplet  26  to move toward the actuation electrode  24   b . The moving speed of the droplet  26  can be controlled by the magnitude of a voltage difference between the adjacent actuation electrodes. In one embodiment, the form of the liquid droplet  26  can be changed by varying the voltage difference between the actuation electrodes  24  and the common electrode  27  where the droplet  26  is disposed therebetween. It is understood that the number of actuation electrodes in the set of actuation electrodes can be any integer number. In the example shown in  FIG. 2A , three actuation electrodes are used in the set of actuation electrodes. But it is understood that the number is arbitrary chosen for describing the example embodiment and should not be limiting. 
     Referring to  FIG. 2A , two substrate structures may be separately formed. For example, a first substrate structure may be formed including the substrate  22 , the dielectric layer  23 , and the actuation electrodes  24  within the dielectric layer  23 . The substrate  22  may be a thin-film transistor (TFT) array substrate formed by conventional thin-film transistor manufacturing processes. A second substrate structure may include a substrate  28  and a common electrode layer  27  on the substrate  28 . A spacer  29  may be formed either on the first substrate structure or the second substrate structure. In certain embodiments, the spacer  29  has a height in the range between several micrometers to several millimeters. In general, the height of the spacer  29  is less than the diameter of the droplet such that the droplet disposed on the dielectric layer  23  has physical contact with the second substrate structure. The first and second substrate structures are then bonded together to form the EWOD device  20 A. In other words, the space or air gap between the first substrate structure and the second substrate structure is determined by the height or thickness of the spacer  29 . The space or air gap forms a channel for the droplet. 
     In the embodiment shown in  FIG. 2A , the common electrode  27  and the set of actuation electrodes  24  (e.g.,  24   a ,  24   b ,  24   c ) are connected to voltages provided by a control circuit (not shown) through the input-output circuit  15  shown in  FIG. 1A . In some embodiments, the common electrode may be connected to a ground potential or a stable DC voltage. The control circuit applies time varying voltages through the input-output circuit to the set of actuation electrodes through respective electronic switches (that can be, e.g., thin film transistors or MOS circuitry in the substrate or off-chip) to generate an electric field across the droplet to move the droplet along a path. In some embodiments, the surface of the common electrode  27  is covered by an insulating layer made from a hydrophobic material. In other embodiments, the surface of the dielectric layer  23  is coated with a thin hydrophobic film having a submicron thickness. 
       FIG. 2B  is a simplified cross-sectional view of a portion of an EWOD device  20 B according to another embodiment of the present disclosure. Referring to  FIG. 2B , the EWOD device  20 B includes a substrate  22   b , a dielectric layer  23   b  on the substrate  21   b , a set of actuation electrodes  24  ( 24   a ,  24   b ,  24   c ) within the dielectric layer  23   b , and a set of common electrodes  27  (only one electrode  27   b  is shown) overlying the dielectric layer  23   b . The common electrode  27   b  and the actuation electrodes are spaced apart from each other by a portion of the dielectric layer. Similar to  FIG. 2A , the droplet  26  can be moved along a path in the lateral direction across the surface of the dielectric layer  23   b  by applying a first voltage at the actuation electrode (e.g.,  24   a ) below the droplet  26  and a second voltage at the adjacent actuation electrode (e.g.,  24   b ). The movement and direction of the droplet  26  is thus controlled by the control circuit (not shown) which applies voltages to certain actuation electrodes through a set of electronic switches (MOS circuitry in the substrate  22   b , not shown). Different to the EWOD  20 A shown in  FIG. 20A , the EWOD device  20 B has the common electrode  27   b  close to the actuation electrodes  24 , and the droplet  26  is not sandwiched between the common electrode  27  and the actuation electrodes  24 . The EWOD device  20 B also differs from the EWOD  20 A by not having the spacer  29 . 
     Referring to  FIG. 2B , the set of actuation electrodes  24  and the set of common electrodes  27  may be two layers of strip electrodes intersected with each other on different planes on the substrate. The actuator electrodes  24  and the common electrodes  27  are operative to move the droplet  26  across the surface of the dielectric layer  23   b . In some embodiments, the common electrode  27   b  has a surface that is covered by an insulating layer made from a hydrophobic material. In other embodiments, the surface of the dielectric layer  23  is coated with a thin hydrophobic film having a submicron thickness. 
       FIG. 2C  is a cross-sectional view of a portion of an EWOD device  20 C according to yet another embodiment of the present disclosure. Referring to  FIG. 2C , the EWOD device  20 C includes a substrate  22   c , a dielectric layer  23   c  on the substrate  22   c , a set of actuation electrodes  24  (e.g.,  24   a ,  24   b ,  24   c ) within the dielectric layer  23   c , a set of common electrode (e.g., one common electrode  27   c  is shown) overlying the dielectric layer  23   c . The common electrode  27   c  and the actuation electrodes are spaced apart from each other by a portion of the dielectric layer. In some embodiments, the common electrode  27   c  has a surface that is covered by an insulating layer made from a hydrophobic material or a thin film of submicron hydrophobic coating on the surface of dielectric layer  23 . The EWOD device  20 C may further include a second substrate  28   c  spaced apart from the substrate  21   c  through a spacer  29   c . Similar to  FIG. 2A , the droplet  26  can be moved along a path within the channel formed by a space or air gap between the surface of the dielectric layer and the second substrate  28   c . The movement of the droplet is controlled by voltages applied to the electrodes through electronic switches by a control circuit (not shown). 
       FIG. 2D  is a simplified plan view of an EWOD  20 D according to an exemplary embodiment of the present disclosure. Referring to  FIG. 2D , the actuation electrodes are arranged in an array having routing channels for routing electrical signals from a control circuit  28  to the actuation electrodes  24  and to the common electrodes  27   c . The spacer  29   c  is shown to have a circular cross-section, however, the circular cross-sectional shape is not limiting and any other cross-sectional shapes are equally suitable such as square, rectangular, oval, elliptic and other shapes. Similarly, the actuation electrodes are shown to have a square shape, but the square shape is not limiting and other shapes are equally suitable such as rectangular, circular, oval, elliptic and other shapes. In one embodiment, the spacer  29   c  is spaced at a distance to leave sufficient space to allow free movement of the droplet. In order words, the spacer  29   c  is dimensioned and spaced such a way that it does not hinder movements of the droplet across the surface of the dielectric layer. It will be understood that although the routing channels are shown as coplanar with the array of electrodes, one of skill in the art will appreciate that the routing channels and the control circuit can be disposed in the substrate and in different layers of the dielectric layer. It will also be understood that the actuation electrodes  24  and the common electrodes  27   c  can have their relative positions transposed, i.e., the common electrodes may be disposed below the actuation electrodes. 
     In another embodiment, the EWOD device may have a single array of electrodes. 
     In other words, the common electrodes and the actuation electrodes are coplanar, i.e., the common electrodes and the actuation electrodes are arranged in a same plane within the dielectric layer. For example, a plurality of actuation electrodes and a plurality of common electrodes are arranged alternatively adjacent to each other, the control circuit may apply DC or AC voltages and ground potential sequentially to the actuation electrodes and common electrodes to control the movement of the droplet. In yet another embodiment, each electrode in the array of electrodes is individually controlled by a control circuit through a set of electronic switches such that each electrode can be an actuator electrode at a first time period and a common electrode at a second time period. 
       FIG. 2E  is a simplified cross-sectional view illustrating exemplary electric fields  20 E generated by electrodes according to an embodiment of the present disclosure. Referring to  FIG. 2E , a common electrode  27  may be driven by a ground potential (gnd), and actuation electrodes  24   a ,  24   b ,  24   c  may be driven by a DC or AC voltage (Ve) in a time-sequential manner. For example, an DC voltage is first applied to the electrode  24   a , the voltage difference between the actuation electrode  24   a  and the common electrode  27  generates an electric field E that moves the droplet  26  along the microchannel defined by the electrode  27  and the dielectric layer  23 . By setting a voltage at an electrode adjacent to the droplet  26 , the droplet  26  can be moved to that electrode along the lateral direction between the dielectric layer  23  and the common electrode  27 . This structure is similar or the same as the EWOD device shown in  FIG. 2A . In one embodiment, the DC voltages may be provided by a control circuit through the input-output circuit  15  shown in  FIG. 1A ). The main electric field is perpendicular to the surface of the dielectric layer  23 . 
       FIG. 2F  is a simplified cross-sectional view illustrating exemplary electric fields  20 F generated by electrodes according to another embodiment of the present disclosure. Referring to  FIG. 2F , a control circuit (not shown) may apply DC or AC voltages to the electrodes  24   a ,  24   b ,  24   c , and  27   b  in a time-sequential manner to generate electric field patterns that control the movement of the droplet  26 . For example, a voltage set at an electrode adjacent to the droplet will move the droplet onto that electrode. This structure is the same or similar to the EWOD device shown in  FIG. 2B or 2C . 
       FIG. 2G  is a simplified cross-sectional view illustrating electric fields  20 G generated by electrodes according to another embodiment of the present disclosure. Referring to  FIG. 2F , a control circuit (not shown) may apply DC or AC voltages to the electrodes  24   a ,  24   b , and  24   c  in a time-sequential manner to generate electric field patterns that control the movement of the droplet  26 . In this embodiment, the electrodes  24   a ,  24   b , and  24   c  may be operative alternatively as actuator electrodes and common electrodes. The half-cylindrical field is formed between the electrodes  24   a  and  24 B. Referring to  FIGS. 2E, 2F , and  2 G, the electric filed E and the resulting electric force F are a function of the voltage difference between the electrodes and the size of the electrodes. By varying the voltage difference between adjacent electrodes in a time-sequential manner, an electric field and the resulting force are generated causing the transport of the droplet  26  along the direction of the electric force. In some embodiments, the not active electrodes (e.g., electrode  24   c ), which do not contribute to the movement (or transport) of the droplet, can be left floating, i.e., not connected. In the example shown in  FIG. 2G , the droplet  26  will stay between the electrodes  24   a  and  24   b , i.e., between the voltage Ve and ground. 
       FIGS. 3A to 3C  are top sequential views of a droplet moving across a surface of a dielectric layer according to an embodiment of the present disclosure. Referring to  FIG. 3A , a droplet  26  is disposed on an EWOD device as described above in any of the EWOD devices  20 A,  20 B, and  20 C. The EWOD device includes a substrate having an array of thin-film transistors or MOS circuitry, a dielectric layer on the substrate, and an array of actuation electrodes (and/or common electrodes) within the dielectric layer, the actuations electrodes and the common electrodes are connected to a control circuit through conductive wirings in the routing channels and receive control signals from the control circuit via the thin-film transistors. The droplet  26  is disposed on a surface of the dielectric layer above a first actuator electrode  34   a . By turning off (or floating) the first actuation electrode below the droplet and turning on the actuation electrode next to it, the droplet can be moved toward the next electrode. In one embodiment, the surface portion of the dielectric layer above the array of the actuation electrodes can be modified with a predefined feature which has more attraction to a liquid (e.g., the liquid droplet) than a hydrophobic surface. The feature may have a dimension ranging from micrometer to nanometer corresponding to microliter and nanoliter in volume, respectively. The feature  35  may be manufactured on the dielectric layer accurately thousands or million times on the dielectric layer using currently available submicron semiconductor manufacturing processes. 
     As used herein, turning off an actuation electrode refers to lowering the voltage of that actuation electrode to a level same as a common voltage which is applied to the common electrode. Conversely, turning on an actuation electrode refers to increasing the voltage of that actuation electrode to a level above the common voltage. The EWOD devices can operate with DC (DC electrowetting) or AC (AC electrowetting) voltages as long as a potential between the electrodes is at a DC voltage level to form an electric field for moving the droplet. In certain embodiments, when an adjacent electrode is completely or partially turned on, the droplet disposed adjacent to it will be moved onto that turned-on electrode and wets the features disposed on the turned-on electrode. As used herein, the term “feature” refers to a region or a structure in or on which a liquid material (e.g., a drop) is deposited or formed. By moving the droplet to a next turned-on electrode using a time-varying voltage waveform provided by a control circuit, the droplet will move from electrode to electrode, thereby leaving residual tiny drops (very small or tiny drops or microdroplets)  26   a  in or on the features. The volume of the residual tiny drops is completely determined by the feature dimension (size) as well as the contact angle of the liquid droplet on the surface in the environment (e.g., air or oil).  FIG. 3B  is a top view illustrating that the droplet  26  is moving from the first electrode  34   a  to the second electrode  34   b  having nine features  35  according to an embodiment of the present disclosure.  FIG. 3C  is a top view illustrating that the remaining droplet  26   b  is moving from the second electrode to the third electrode  34   c , thereby leaving residual tiny drops (microdroplets)  26   a  in or on the features, in accordance with an embodiment of the present disclosure. To prevent evaporation of microdroplets in the air, the droplet can be surrounded by other immiscible liquid like silicone oil. It is understood that the number of features on the electrode can be any integer number. In the example shown in  FIGS. 3A to 3C , nine features are used in the second electrode. But it is understood that the number is arbitrarily chosen for describing the example embodiment and should not be limiting. It is also understood that each electrode (e.g., first, second, third electrodes) may have the same number of features, or they may have different number of features. Referring to  FIGS. 3A to 3C , the features are shown to have a square shape, however, it in understood the shown shape is not limiting and any other shapes are equally suitable such as circular, rectangular, oval, elliptic, polygonal, and other shape. 
     It is noted that the electrodes according to embodiments of the present disclosure can be arranged in various configurations and the electrodes can have many shapes. For example, the electrodes can have a polygonal shape (e.g., square, rectangular, triangular, and the like), a circular shape, an oval shape, etc. The configuration can be a checker-board configuration, or other geometric configurations. 
     The patterned features can be implemented in different ways. In one embodiment, the surface of the dielectric layer is selectively partitioned into hydrophobic regions and hydrophilic regions. Within a certain range of a surface area ratio between the hydrophobic regions and hydrophilic regions, and as well as the interfacial tension between a droplet and the environment (oil or air), the droplet can be moved away from the hydrophobic regions while leaving residual small drops (microdroplets) in the hydrophilic regions. The volume of a residual small drop (microdroplet) is defined by the dimension of a hydrophilic feature as well as the contact angle of the droplet on the surface in the environment (air or oil).  FIG. 4A  is a cross-sectional view of a portion of an EWOD device  40 A according to an embodiment of the present disclosure. Referring to  FIG. 4A , the EWOD device  40 A includes a substrate structure having a first substrate (not shown) with a first dielectric layer  43   a  disposed thereon and a second substrate (not shown) with a second dielectric layer  47   a  disposed thereon. A droplet  26  is disposed between the first dielectric layer  43   a  having a first surface  44  and the second dielectric layer  47   a  having a second surface facing the first surface. In one embodiment, the first surface of the first dielectric layer includes a plurality of hydrophilic regions  48   a  that protrude above the surface of the first dielectric layer  43   a . The surface of the first dielectric layer  43   a  is coated with a hydrophobic film, i.e., the protruding hydrophilic regions  48   a  are surrounded by an interstitial hydrophobic surface region. The protruding hydrophilic regions  48   a  may be arranged in a pattern such that a residual small droplet (microdroplet) of the droplet disposed on each of the hydrophilic regions has a desired volume. The protruding hydrophilic regions each may have a polygonal shape (e.g., square, rectangular), oval, circular, elliptic, and other shapes. The term “protruding hydrophilic region” may also be referred to as a “spot” or “island” in the present disclosure. In some embodiments, the second dielectric layer  47   a  is made of glass that is coated with a hydrophobic film. In some embodiments, the first dielectric layer and the second dielectric layer are formed separately, and a spacer such as the one shown and described in  FIGS. 2A, 2C, and 2E  is formed either on the first dielectric layer or on the second dielectric layer. The first and second dielectric layers are then bonded together to form the structure shown in  FIG. 4A  having a space therebetween operative as a channel for the droplet  26 . In some embodiments, the second dielectric layer  47   a  is coated with an electrical conducting layer of a metal material that serves as a common electrode (e.g., ground electrode) and a hydrophobic film on the conducting layer. The protruding hydrophilic regions may be formed on the surface of the first dielectric layer  43   a  using conventional semiconductor manufacturing techniques. Some of these semiconductor manufacturing techniques will be described in detail in the example section further below. The EWOD  40 A also includes an array of electrodes (not shown) embedded within the first dielectric layer, the second dielectric layer, or in both first and second dielectric layers that generate a moving electric field in response to time-sequentially varying voltages provided by a control circuit. The droplet  26  is moved by the moving electric field across the surface of the first dielectric layer while leaving residual small portions (microdroplets)  26 ′ of the droplet  26  on the protruding hydrophilic regions  48   a.    
       FIG. 4B  is a cross-sectional view of a portion of an EWOD device  40 B according to another embodiment of the present disclosure. Referring to  FIG. 4B , the EWOD device  40 B includes a substrate structure that is similar to the substrate structure shown in  FIG. 4A  with the difference that the first dielectric layer  43   b  includes a plurality of grooves (microwells)  49  having a certain depth instead of the protruding regions. In one embodiment, the second surface of the second dielectric layer is hydrophobic, and the first dielectric layer includes a plurality of grooves (microwells)  49  having a certain depth. The terms “grooves,” “recesses,” and “microwells” are used interchangeably herein. Each of the grooves also have an opening determined by a length and a width. In some embodiments, the length and the width are less than 1 micron. By carefully choosing the shape of microwells or/and alter the inside surface (sidewalls and/or bottom) of the microwells from hydrophobic to hydrophilic chemically (i.e., by surface treatment) or electrically (i.e., by electrowetting), a portion of the aqueous droplet would spontaneously wet into a microwell and has tendency to stay inside the microwell even after the electrical field has been removed. Within a certain range of the area ratio between the flat hydrophobic surface of the first dielectric layer and the microwells as well as the interfacial tension between the droplet and environment (oil or air), the droplet can be moved away from the microwells while leaving (depositing) residual small droplets (microdroplets) inside the microwells. The volume of residual small droplets (microdroplets) is determined by the predetermined microwell dimension and contact angle of the droplet on the opening of the microwell. Similar to the structure described above in connection with  FIG. 4A , the second dielectric layer  47   b  may be coated with an electrical conducting layer of a metal material that serves as a common electrode (e.g., ground electrode) and a hydrophobic film on the conducting layer. The recessed hydrophilic regions may be formed in the first dielectric layer  43   b  using conventional semiconductor manufacturing techniques. The EWOD  40 B also includes an array of electrodes (not shown) embedded within the first dielectric layer, the second dielectric layer, or in both first and second dielectric layers that generate a moving electric field in response to time-sequentially varying voltages provided by a control circuit (not shown). The droplet  26  is moved by the moving electric field across the surface of the first dielectric layer while leaving residual small portions (microdroplets)  26 ′ of the droplet  26  on the surface of the microwells. In some embodiments, an array of microwells can be obtained using lithographically patterning and etching processes. Some of these semiconductor manufacturing techniques will be described in detail in the example section further below. 
       FIG. 4C  is a perspective view of a portion of an EWOD device  40 A shown in  FIG. 4A . Referring to  FIG. 4C , a set of round cylindrical spots (protruding hydrophilic regions)  48   a  are arranged in an array. As used herein, the terms “spots,” “islands,” “protruding regions” are interchangeably used. The spots  48   a  are spaced apart and isolated from each other by an interstitial hydrophobic surface  44 . It is noted that, although the spots  48   a  are shown having round cylindrical shape in the example shown, those of skill in the art will appreciate that the spots  48   a  may also have other shapes, such as rectangular, square, or oval cylindrical shape. 
       FIG. 4D  is a perspective view of a portion of an EWOD device  40 B shown in  FIG. 4B . Referring to  FIG. 4D , a set of round cylindrical grooves (microwells)  49  are arranged in an array. The microwells  49  are filled with a hydrophobic material and spaced apart from each other by an interstitial hydrophobic surface  46 . In some embodiments, the hydrophobic material filling the microwells  49  has an upper surface flush with an upper surface of the interstitial hydrophobic surface  46 . It is noted that, although the microwells  49  are shown as round cylindrical wells, the microwells  49  may also be rectangular, square, or oval cylindrical wells depending upon applications. 
       FIG. 4E  is a perspective view of a portion of an EWOD device according to an embodiment of the present disclosure. Referring to  FIG. 4E  a set of hydrophilic surfaces  491  is surrounded (flanked) by hydrophobic interstitial regions  461  on the surface of the dielectric layer  43 , i.e., the hydrophilic surfaces  491  are flush with the surface of the hydrophobic interstitial regions  461  on the surface of the dielectric layer  43 . 
     In accordance with the present disclosure, the large number of microdroplets having a uniform size can be used to perform droplet digital PCR on a microfluidic chip. With a small volume of each sample and below certain DNA concentration meeting the Poisson distribution requirement, each sample of the droplet (microdroplet) would have either one DNA molecule or no DNA molecule. By thermo-cycling the samples (microdroplets) with a conventional PCR or incubating them under a certain temperature with an isothermal PCR, a single DNA molecule within a target region can be amplified on each sample within the environment (e.g., oil). After reading the final droplet&#39;s DNA concentration by optical detection or pH measurement through integrated on-chip ion-sensitive field-effect transistor (ISFET) sensors, we can quantify the absolute numbers of a targeted DNA in the array of samples (microdroplets) and then use the absolute DNA quantification to calculate the DNA concentration in the bulk droplet. The terms “sample,” “residual small droplet,” “small portions of the droplet,” and “microdroplet” are used interchangeably herein and refer to a small droplet formed from a bulk droplet according to embodiments of the present disclosure. 
     In accordance with the present disclosure, a droplet containing multiple different DNA targets can be dispensed on a region of a single microfluidic chip, the droplet is then moved by electrowetting to a next region which produces a multitude of samples (copies of the DNA targets) from the droplet for detection or measurement of the samples. In some embodiments, the next region where the droplet is moved to may include a plurality of hydrophilic regions spaced apart by an interstitial hydrophobic surface. The plurality of hydrophilic regions may be the one shown and described in connection with  FIGS. 4A and 4B . Each of the hydrophilic regions may include an ion-sensitive field-effect transistor (ISFET) sensor configured to measure a pH value of the samples disposed thereon. That is, the single microfluidic chip may include an array of ISFET sensors each being associated with one of the samples of the droplet. This arrangement of the single microfluidic chip enables the formation of a plurality of samples (microdroplets) from a droplet by electrowetting and the measurement of the samples by an array of ISFET devices that are integrated on-chip. In accordance with the present disclosure, the array of ISFET sensors embedded in the hydrophilic regions facilities a concurrent measurement of different targets from a droplet with high sensitivity and accuracy. 
       FIG. 5  is a cross-sectional view of an ISFET device  50  according to an embodiment of the present disclosure. Referring to  FIG. 5 , the ISFET device  50  is a metal oxide semiconductor (MOS) transistor, which can be a p-channel MOS field effect transistor (MOSFET) or an n-channel MOSFET transistor that is manufactured using standard CMOS manufacturing processes. In the following description, an n-channel MOS transistor (NMOS) transistor is used according to an exemplary embodiment of the present disclosure. However, it should be noted that the selection of NMOS or PMOS is merely a choice depending upon a chosen process or substrate, and is not limiting. Referring to  FIG. 5 , the ISFET device  50  has a substrate  52 , a source region S and a drain region D formed in the substrate, a dielectric layer  53  on the substrate, and a floating gate G formed within or on the dielectric layer  53 . The ISFET device further includes a sensing membrane  55  on the floating gate G and below a microdroplet  56 , and a reference electrode  57  entirely or partially immersed in the microdroplet  56  and spaced apart from the sensing membrane  55 . The sensing membrane may include any material that provides sensitivity to hydrogen ion concentration (pH), such as silicon nitride, silicon oxynitride, and the like. As known to those of skill in the art, other sensing membranes may be used that are sensitive to other ions. 
     Still referring to  FIG. 5 , the ISFET device  50  also includes a voltage source Vgs configured to provide a voltage Vgs between the sample and the source region S, a voltage source Vds configured to provide a voltage Vds between the source region and the drain region. When the voltage Vgs is greater than the threshold voltage Vth of the ISFET device, the channel between the source and drain regions will conduct current. The amount of current Ids flowing between the source region and the drain region represents a concentration or a pH value of the sample  56 . In one embodiment, the source region and the substrate have the same potential, e.g., ground potential. 
     In the example shown in  FIG. 5 , one ISFET device is used for measuring ion concentration of the microdroplet  56 . But it is understood that the number is only chosen for describing the example embodiment and should not be limiting. In some embodiments, more than one ISFET device may be used for measuring ion concentration of the microdroplet  56 . In other words, each spot or microwell can have a number of ISFET devices. 
     In some embodiments, each spot of the array of spots or each microwell of the array of microwells shown in respective  FIGS. 4C and 4D  may have more than one ISFET device to increase the measurement sensitivity and accuracy. The number of ISFET devices available for each spot or microwell depends from conventional CMOS manufacturing processes and application requirements. Power supply voltages supplied to the ISFET devices through electrical connections may be implemented using conventional CMOS manufacturing processes and will not be described herein for the sake of brevity. 
       FIG. 6A  is a simplified top view of an integrated lab-on-a-chip device  60 A according to an embodiment of the present disclosure. Referring to  FIG. 6A , the integrated lab-on-a-chip device  60 A includes a substrate structure having a droplet receiving region  61  configured to receive one or more droplets  26 , a reagent receiving region  62  configured to receive one or more reagents  63 , a mixing region  64  configured to mix the droplet  26  with the one or more reagents  63  to obtain a mixed droplet  263 , and an array of EWOD devices configured to partition a droplet (mixed or not mixed) into a plurality of microdroplets and amplify the microdroplets. In one embodiment, the array of EWOD devices may feature first heating elements configured to heat the microdroplets to a first temperature for amplification of the microdroplets and second heating elements for annealing the amplified microdroplets. In one embodiment, the lab-on-a-chip device  60  may further include an array of sensors each being associated with a sample and configured to measure a concentration or a pH value of the microdroplets. In one embodiment, the droplet receiving region  61  may have the device structure shown in  FIGS. 1A and 1B . In one embodiment, the reagent receiving region  62  may have the device structure shown in  FIGS. 1A and 1B . In other words, the integrated lab-on-a-chip device  60  may be operable to move the one or more droplets and the one or more reagents toward the mixing region  64  and control the mixing of the droplets with the reagents according to a user provided software program. In one embodiment, the array of EWOD devices may include a plurality of EWOD devices arranged in a regular pattern, each of the EWOD devices may have the structure similar or the same as the device structure shown in  FIGS. 2A to 2C . In some embodiments, each of the EWOD devices may include a plurality of ISFETs. An example of an ISFET device is shown in  FIG. 5 . The integrated lab-on-a-chip device  60 A may further include a waste (collection) region  66  for collecting the residual portion of the droplet after the microdroplets have been formed in the array of EWOD devices and/or the microdroplets after they have been processed and measured. In the example shown in  FIG. 6A , two electrodes are used in the droplet receiving regions  61 , eight electrodes are used in the upper portion of the reagent receiving region  62 , eight electrodes are used in the lower portion of the reagent receiving region  62 , eight electrodes are used in the mixing region  64 , and an array of four EWOD devices are used. But it is understood that these number are arbitrary chosen for describing the example embodiment and should not be limiting. 
     In some embodiments, the integrated lab-on-a-chip device  60 A may also include a control circuit  67  configured to provide control signals to the droplet receiving region  61 , the reagent receiving region  62 , the mixing region  64 , the array of EWOD devices, and the waste region  66  for moving the droplet  61 , the reagents  63 , the mixed droplet  263 , the partitioned droplet (i.e., microdroplets) and the residual portion of droplet after passing through the array of EWOD devices. In one embodiment, the integrated lab-on-a-chip device  60 A may include an input/output (IO) port  68  configured to interface with a host. In one embodiment, the host may be a separate or external processor configured to provide control signals to the integrated lab-on-a-chip device  60 . In another embodiment, the host may be integrated with the integrated lab-on-a-chip device  60  in a same package. One of ordinary skill in the art would recognize many variations, modifications, and alternatives. Referring still to  FIG. 6A , the control circuit  67  may be disposed remotely from the integrated lab-on-a-chip device  60 A and communicates with the integrated lab-on-a-chip device  60 A via an input-output port or a serial interface port. In one embodiment, the integrated lab-on-a-chip device  60 A may also include a first heating block “heater  1 ” formed within the substrate structure below the surface of the mixing regions  64  for maintaining and/or varying an incubation temperature for the mixed droplet  263 . In one embodiment, the integrated lab-on-a-chip device  60 A may further include a second heating block “heater  1 ” formed within the substrate structure below the surface of the array of EWOD devices for maintaining and/or varying an incubation temperature for the microdroplets. The first and second heating blocks are formed of metal or polysilicon wires, metal or polysilicon layer(s), polysilicon layer(s) that can convert an electric energy of signals received from the control circuit  67  into a thermal energy. 
       FIG. 6B  is a simplified top view of an example arrangement of electrodes  60 B of the integrated lab-on-a-chip device  60 A according to an embodiment of the present disclosure. Referring to  FIG. 6B , the electrodes  60 B of the integrated lab-on-a-chip device of  FIG. 6A  includes a first array of electrically conductive stripes  611  having, e.g., stripes  611   a ,  611   b ,  611   c ,  611   d ,  611   e ,  611   f , and  611   g  arranged in parallel to each other along a first direction from the first region (the droplet receiving region) toward the third region (the array of EWOD devices). The first array of stripes  611  is controlled by a control circuit  67  and configured to generate moving electric fields to transport the droplet along the first direction. The electrodes  60 B also includes a second array of parallel stripes  612  having, e.g., stripes  612   a ,  612   b ,  612   c ,  612   d , arranged in parallel to each other along a second direction perpendicular to the first direction. The second array of stripes  612  is configured to generate moving electric fields to transport the droplet along the second direction toward the second region (the mixing region). The first array of stripes intersect the second array of stripes and are spaced apart from the second array of stripes by an insulating layer, i.e., the first and second arrays of stipes are arranged in different layers separated by at least one insulating layer. In the example shown in  FIG. 6B , the second array of stripes  612  are arranged in the reagent receiving region for transporting the reagents toward the mixing region. But it is understood that the second array of stripes  612  can also be arranged in the droplet receiving region, in the mixing regions, and in the array of EWOD devices to generate moving electric fields and electric forces to move the droplet, the mixed droplet, and the microdroplets along the second direction. In some other embodiments, the electrodes  60 B may include an array of electrodes similar to the one shown and described in connection with  FIGS. 1A and 1B . One of ordinary skill in the art would recognize many variations, modifications, and alternatives. 
       FIG. 7  is a simplified flowchart illustrating a method  70  for forming a plurality of microdroplets from a droplet according to an embodiment of the present disclosure. 
     Referring to  FIG. 7 , the method  70  may include, at  701 , providing a substrate structure having a lower substrate, a dielectric layer on the lower substrate, a plurality of electrodes in the dielectric layer. In one embodiment, the dielectric layer has a hydrophobic surface and a plurality of protruding hydrophilic surface regions (spots) having substantially the same size spaced apart by the hydrophobic surface. In another embodiment, the dielectric layer has a hydrophobic surface and a plurality of microwells (small grooves) having substantially the same size in the dielectric layer. Each of the microwells has a hydrophilic bottom and hydrophilic sidewalls (collectively referred to as a hydrophilic surface region). At  703 , the method may include dispensing a droplet on a surface region of the dielectric layer. At  705 , the method may include applying time-varying control voltage signals by a control circuit (e.g., host) to the electrodes to move the droplet across the hydrophobic surface of the dielectric layer while leaving small residual portions on the hydrophilic surface regions to form a plurality of microdroplets thereon, the microdroplets have substantially the same size. 
       FIG. 8  is a simplified flowchart illustrating a method  80  for operating an integrated lab-on-a-chip apparatus according to an embodiment of the present disclosure. In some embodiments, the integrated lab-on-a-chip apparatus may be the same or similar to the integrated lab-on-a-chip device  60  shown in  FIG. 6 . That is, the integrated lab-on-a-chip apparatus includes a droplet receiving region configured to receive a droplet, a reagent receiving region configured to receive one or more reagents, a mixing region configured to mix a droplet with one or more reagents, and an array of EWOD devices configured to partition a received droplet (a droplet of the droplet receiving region that may be mixed with one or more reagents or a droplet that is not mixed with any reagent) into a plurality of microdroplets, the mixed droplet incubates with a constant or/and variable temperature controlled by heaters underneath the surface of the mixing region  64 , and the microdroplets further incubate at a constant or/and variable incubation temperature by heaters underneath the surface of the EWOD devices. In one embodiment, the lab-on-a-chip apparatus may further include an array of sensors associated with the microdroplets and configured to measure an ion concentration or a pH value of the microdroplets. Referring to  FIG. 8 , the method  80  may include, at  801 , dispensing a droplet on a surface of the droplet receiving region. At  803 , the method may include moving the droplet toward an EWOD device of the array of EWOD devices across the mixing region, where the droplet is partitioned into a plurality of microdroplets whose pH value before or/and after incubation may be determined by an ISFET sensor formed on the EWOD device. At  807 , the method may collect and discard any unused (residual) portion of the droplet and the microdroplets in a waste region of the integrated lab-on-a-chip apparatus. In one embodiment, the method may also include, at  802 , dispensing one or more reagent droplets onto a surface of the reagent receiving region. At  802 , the method may further include moving the one or more reagent droplets toward the mixing region for mixing the reagent droplets with the droplet under a user-defined software program prior to moving the droplet to the EWOD devices for partitioning the droplet into multiple microdroplets. In one embodiment, the method may further include, at  802 ′, incubating the mixed droplets for reactions at a constant or variable temperature. In one embodiment, the method may also include, at  804 , incubating the microdroplets in the EWOD array at an incubation temperature. In one embodiment, the incubation temperature in the EWOD array region is maintained at a constant incubation temperature. In one embodiment, the incubation temperature in the EWOD array region may be variable. In some embodiments, after discarding the residual portion of the droplet and the microdroplets that have been measured, the method may repeat at step  801 . 
       FIG. 9  is a simplified schematic diagram of a mobile computing device  90  that can be used to control the EWOD device and the lab-on-a-chip device according to an embodiment of the present disclosure. Referring to  FIG. 9 , the mobile computing device  90  may include a monitor  910 , computing electronics  920 , user output devices  930 , user input devices  940 , a communications interface  950 , and the like. 
     The computing electronics  920  may include one or more processors  960  that communicates with a number of peripheral devices via a bus subsystem  990 . These peripheral devices may include user output devices  930 , user input devices  940 , a communications interface  950 , and a storage subsystem, such as random access memory (RAM)  970  and a disk drive  980 . 
     The user input devices  930  may include all any types of devices and interfaces for inputting information to the computer device  920 , e.g., a keyboard, a keypad, a touch screen, a mouse, a trackball, a track pad, a joystick and other types of input devices. 
     The user output devices  940  may include any types of devices for outputting information from the computing electronics  920 , e.g., a display (e.g., monitor  910 ). 
     The communications interface  950  provides an interface to other communication networks and devices. The communications interface  950  may serve as an interface for receiving data from and transmitting data to other systems. For example, the communications interface  950  may include a USB interface for communicating with an EWOD device or a lab-on-a chip device. 
     The RAM  970  and the disk drive  980  are examples of tangible media configured to store data such as embodiments of the present disclosure, including executable computer code, human readable code, or the like. Other types of tangible media include floppy disks, removable hard disks, optical storage media such as CD-ROMS, DVDs and bar codes, semiconductor memories such as flash memories, non-transitory read-only-memories (ROMS), battery-backed volatile memories, networked storage devices, and the like. The RAM  970  and the disk drive  980  may be configured to store the basic programming and data constructs that provide the functionality of the present invention. 
     Software code modules and instructions that provide the functionality of the present disclosure may be stored in the RAM  970  and the disk drive  980 . These software modules may be executed by the processors  960 . 
     Referring still to  FIG. 9 , an EWOD device  91  and a lab-on-a-chip device  92  each may include an interface port  94  configured to provide communications with the mobile computing device  90 . In some embodiments, the mobile computing device  90  may provide instructions and control signals via the interface port  94  to control the signal levels of the electrodes in the EWOD device  91  and the lab-on-a-chip device  92 . In some embodiments, the EWOD device  91  may include a substrate structure as described in one of the  FIGS. 2A-2C, 3A-3C, 4A-4E , and one or more of ISFET devices of  FIG. 5 . The EWOD device  91  is designed to accept a droplet and provide a pH value associated with the droplet. In some embodiments, the EWOD device  91  may be part of the lab-on-a chip device  92 . 
     EXAMPLES 
     The following examples are offered to illustrate, but not to limit the claimed invention. 
     Example 1 
     This example discloses the preparation of the patterned features on a surface of a dielectric layer.  FIG. 4A  shows a cross-sectional view of a dielectric layer  43   a . The dielectric layer  43   a  may be deposited on a substrate (e.g., substrate  22  shown in  FIG. 2A ) by a conventional deposition (e.g., chemical vapor deposition) process. The dielectric layer may include silicon oxide, silicon nitride, fluorinated silicate glass (FSG), or organo-silicate glass (OSG). In one embodiment, the dielectric layer is coated with a hydrophobic material. The hydrophobic material may be coated using a spin-spray process where the hydrophobic material is sprayed onto the surface of the dielectric layer. In another embodiment, the surface of the dielectric layer is exposed to a hydrophobic solution at a certain temperature and for a certain time duration. In yet another embodiment, the hydrophobic material may be formed on the dielectric layer by a deposition (e.g., chemical vapor deposition) process. The hydrophobic material may include organic or inorganic materials such as octadecyltrichlorosilane, perfluorodecyltrichlorosilane, other fluorinated layers such as tetrafluoroethylene, and the like. Thereafter, a patterned photoresist layer is formed on the dielectric layer (which is coated with a hydrophobic material) to exposed a surface portion of the dielectric layer. The dielectric layer is then subjected to a wet hydrophilicity increasing surface treatment such as a spin-spray process where a hydrophilicity increasing solution is sprayed onto the surface of the dielectric layer using the patterned photoresist layer as a mask. In another embodiment, the exposed surface of the dielectric layer is in contact with a hydrophilicity increasing solution at a certain temperature and for a certain time duration. The hydrophilicity increasing solution includes a surfactant or wetting agent including one of cationic surfactants, anionic surfactants, and nonionic surfactants. Thereafter, the patterned photoresist layer is removed to obtain the hydrophilic regions  48   a  on the surface of the dielectric layer, as shown in  FIG. 4A  or  FIG. 4C . 
     In another embodiment, after forming a patterned photoresist layer on the dielectric layer, an etch process (wet etching, dry etching, or a combination of wet and dry etching) is performed on the dielectric layer using the patterned photoresist as a mask to form a plurality of recessed grooves in the dielectric layer. Thereafter, a hydrophilicity increasing solution is then deposited filling the grooves. The patterned photoresist layer is then removed by a chemical mechanical polishing (CMP) process to obtain the structure as shown in  FIG. 4B  or  FIG. 4D . 
     Example 2 
     This example discloses the formation of the electrodes within the dielectric layer. In one embodiment, a first dielectric layer is formed on a substrate. A patterned photoresist layer is then formed on the dielectric layer. A metal layer or a doped polysilicon layer is then deposited on the first dielectric layer using the patterned photoresist layer as a mask. The patterned photoresist layer is then removed. Thereafter, a second dielectric layer is formed on the metal layer to form the structure as shown in  FIG. 2A  to  FIG. 2C . 
     In another embodiment, a first dielectric layer is formed on a substrate. A metal layer is then deposited on the first dielectric layer. Thereafter, a patterned photoresist layer is formed on the metal layer. An etch process is then performed on the metal layer using the patterned photoresist layer as a mask. The patterned photoresist layer is then removed. Thereafter, a second dielectric layer is formed on the metal layer to form the structure as shown in  FIGS. 2A to 2C . 
     Example 3 
     This example discloses a novel apparatus and method of generating a large array of extremely small drops (microdroplets) having a predetermined volume in the range between nanoliter (10 −9  liter or nL) and picoliter (10 −12  liter or pL) from a droplet by electrowetting. The novel apparatus may be the one similar or the same as the EWOD device shown in  FIG. 4A  or  FIG. 4B . The transport mechanism of a dispensed droplet is shown with reference to  FIGS. 3A through 3C . The droplet is moved over a surface of a dielectric layer having an array of raised hydrophilic regions (spots) or over a surface of a dielectric layer having an array of microwells each having a hydrophilic bottom and sidewalls. The raised (protruded) hydrophilic regions (spots) and/or the microwells are spaced apart from each other by an interstitial hydrophobic surface. In one embodiment, the raised hydrophilic regions and/or the microwells have a circular or oval shape. In another embodiment, the raised hydrophilic regions and/or the microwells have a polygonal (rectangular, square, hexagonal) shape. In one embodiment, the raised hydrophilic regions (spots) and/or the microwells have a height in the range between 1 nanometer and 100 microns, preferably between 1 micron and 10 microns, and more preferably below 10 nanometers. In one embodiment, the raised hydrophilic regions and/or the microwells have a square shape with a width or length in the range between 1 nanometer and 100 microns, preferably between 1 micron and 10 microns, and more preferably below 10 nanometers. In one embodiment, the raised hydrophilic regions and/or the microwells have a circular shape with a diameter in the range between 1 nanometer and 100 microns, preferably between 1 micron and 10 microns, and more preferably below 10 nanometers. 
     Example 4 
     This example discloses the formation of a device structure having hydrophobic/hydrophilic interstitial surfaces with dimensions (sizes) from micrometers to nanometer. Example embodiments are shown in  FIGS. 4A and 4C .  FIG. 10  is a cross sectional view of device structure  1000  having differential surface regions according to some embodiments of the present disclosure. The device structure  1000  includes a substrate  1001 . A surface layer  1002  including a plurality of first thin film regions  1011  and a plurality of second thin film regions  1021  are disposed on the substrate  1001 . The substrate  1001  includes MOS circuitry (e.g., control circuit  15 ) and electrodes (e.g., electrodes  14 ) that are actuated or controlled by the control circuitry  15  as shown in  FIG. 1A . 
     A first covering layer  1012  is formed on the top surfaces of the first thin film regions  1011 , and a second covering layer  1022  is formed on the top surfaces of the second thin film regions  1021 . In some embodiments, a differential surface layer is formed by alternating regions of first covering layer  1012  and regions of second covering layer  1022 . 
     According to some embodiments, methods are provided for selecting the first material and the second material to adjust the hydrophobicity of the first covering layer and the second covering layer to form a differential hydrophobic/hydrophilic surface. 
     In some embodiments, the differential hydrophobic/hydrophilic surface can have alternating nonpolar molecular regions that repels water and polar molecular regions which can form ionic or hydrogen bond with water molecular. In some embodiments, a method includes first forming alternating regions of inorganic SiO 2  and metal oxide material including one or more of various metal oxides, such anodized aluminum (Al 2 O 3 ), tantalum oxide (Ta 2 O 5 ), niobium oxide (Nb 2 O 5 ), zirconium oxide (ZrO 2 ), and titanium oxide (TiO 2 ), etc. These alternating regions can be formed using standard semiconductor thin film deposition and photolithography process on silicon (Si) or glass substrate, as described further below. 
     Next, the metal oxide surface can be treated to modify the surface property. One method is to selectively coat the metal oxide surface using polyvinylphosphonic acid (PVPA), which is a type of hydrophilic polymer inside the native pH of the phosphonic acid (pH=2), at a temperature in the temperature range of 80° C. to 100° C. In a specific embodiment, the treatment can be carried at, for example, 90° C. In some cases, the treatment can be relatively fast, for example, in less than 2 minutes (min). This step can be followed by a dry annealing step intended to support formation of covalent bonds. A dry anneal process can be carried at a suitable temperature, e.g., at 80° C. for about 10 min. This reaction can be selective, i.e., no reaction will take place on the SiO 2  surface. 
     Another method is to selectively coat the metal oxide regions by self-assembled monolayers (SAMs) based on the adsorption of the alkyl phosphate ammonium salts from aqueous solution. SAM formation does not occur on SiO 2  surfaces under the same conditions. The coated surface hydrophobicity can be adjusted by the formulation in the aqueous SAM forming solution, and the contact angle of water can range from 50 to 110 degrees. In some cases, the contact angle of water can range from 20 to 130 degrees. Covalent bonds can be formed during the annealing, and unreacted materials rinsed by DI water. 
     After the PVPA or phosphate treatment, the substrate can be dried and treated with a hydrophobic silane such as Fluorinated Alkyl-Silanes, dialkyl-Silanes etc. Alternatively, the substrate can be dried and treated with a hydrophilic silane such as Hydroxyakyl-terminated silanes in solution or chemical vapor deposition. These treatments can form stable covalent bonds with the SiO 2  surface and change the surface to be hydrophobic or hydrophilic without affecting metal oxide surface&#39;s hydrophobicity. 
     With the highly selective surface treatment of inorganic SiO 2  and metal oxide surfaces with different organic chemicals of different hydrophobicity, the differential hydrophobic/hydrophilic surface can be made with well-defined pattern by the semiconductor processes. 
       FIGS. 11A-11F  are cross-sectional views illustrating a method for forming a device structure of  FIG. 10  having differential surface regions according to some embodiments of the present disclosure. The method for forming a device structure having differential surfaces includes providing a substrate, and forming a surface layer having alternating first thin film regions and second thin film regions on the substrate. The surface layer is exposed to a first material to form a first covering layer on the first thin film regions, but not on the second thin film regions. The surface layer is then exposed to a second material to form a second covering layer on the second thin film regions, but not on the first thin film regions which are now covered by the first covering layer. The method includes selecting the first material and the second material to adjust the hydrophobicity of the first covering layer and the second covering layer. 
       FIG. 11A  shows a thin film layer formed on a substrate. Referring to  FIG. 11A , a thin film layer  1120  is formed on a substrate  1101 . Substrate  1101  may be made of any suitable material, for example, a glass or a semiconductor. A semiconductor substrate can include various semiconductor materials, such as silicon, III-V group on silicon, graphene-on-silicon, silicon-on-insulator, combinations thereof, and the like. The substrate  1101  can be a bare wafer. The substrate  1101  can also include various devices and circuit structures. For example, the substrate  1101  can include a CMOS circuitry layer (substrate  12  including, e.g., the control circuit and other electronic switches), and a dielectric layer (e.g., dielectric layer  13  including the electrode layer  14 ). 
     Referring to  FIG. 11A , the thin film layer  1120  is formed on the substrate  1101 . In an embodiment, the thin film layer  1120  contains inorganic silicon oxide, e.g., SiO 2 . In some embodiments, the thin film layer  1120  can include silicon, silicon nitride, metal oxides, etc., or combinations thereof. The thin film layer  1120  can also include other materials that can be silanized. The thin film layer  1120  can be formed on substrate  1101  using any conventional semiconductor thin film deposition techniques. 
       FIG. 11B  shows a second thin film layer  1110  formed on a first thin film layer  1120 . In some embodiments, the second thin film layer  1110  can include metal oxides or metals. Suitable metal oxides can include, for example, anodized aluminum (Al 2 O 3 ), tantalum oxide (Ta 2 O 5 ), niobium oxide (Nb 2 O 5 ), zirconium oxide (ZrO 2 ), and titanium oxide (TIO 2 ), etc. The thin film layer  1110  can also include metal materials, such as tungsten, titanium, titanium nitride, silver, tantalum, tantalum oxide, hafnium, chromium, platinum, tungsten, aluminum, gold, copper, combinations or alloys thereof, and the like. The thin film layer  1110  can also be formed using conventional semiconductor thin film deposition techniques. 
       FIG. 11C  shows a patterned mask layer  1130  formed on the thin film layer  1110 . The mask layer  1130  includes openings exposing regions of the thin film layer  1110 . The mask layer  1130  may be applied according to any suitable method, such as spin coating, dipping, and/or the like. The mask layer  1130  may also be of any suitable material, such as a photoresist. As shown in  FIG. 11C , the mask layer  1130  is patterned with the openings according to conventional semiconductor lithography techniques. In some embodiments, the mask layer  1130  can be a hard mask, which is a patterned layer of suitable thin film material having suitable etch selectivity to serve as an etching mask. 
     After the patterned mask layer  1130  is formed, an etch process can be carried out to remove the exposed portions  1117  in the thin film layer  1110  to obtain thin film regions  1111 . The etch process can be performed according to conventional semiconductor process techniques. Subsequently, the patterned mask layer  1130  can be removed by conventional semiconductor process techniques. The resulting device structure is illustrated in  FIG. 11D . 
       FIG. 11E  shows a covering layer  1112  selectively formed on the thin film regions  1111 . The selective covering layer formation is carried out by exposing the device structure to a suitable first material, such that covering layer  1112  is formed on the top surfaces of the thin film regions  1111 , but not on the top surfaces of the thin film regions  1121 . Depending on the materials for thin film regions  1111  and  1121 , various materials can be used, as described in detail below. After the treatment of the material, an annealing process can be performed to selectively form a covering layer  1112  on the thin film regions  1111 . The annealing process can be carried out at a temperature in a range of 70°−90° C. for 5-15 minutes. As an example, a dry anneal process can be carried out at a temperature of 80° C. for 10 minutes. The unreacted material can be removed by a rinse process in DI (deionized) water. 
       FIG. 11F  shows a second covering layer  1122  selectively formed on the top surfaces of the second thin film regions  1121 . The selective covering layer is formed by exposing the device structure to a suitable second material, such that covering layer  1122  is formed on the top surfaces of the thin film regions  1121 , but not on the top surfaces of thin film regions  1111  with the covering layer  1112 . Depending on the materials for thin film regions  1111  and  1121 , various materials can be used, as described in detail below. After the process with the second material,  FIG. 11F  shows a cross-sectional view of a device structure  1100 , similar to the device structure  1000  of  FIG. 10 , having a surface layer  1102  with differential surface regions  1112  and  1122  according to some embodiments of the invention. As shown, the device structure  1100  includes a substrate  1101 . The surface layer  1102  including a plurality of the first thin film regions  1111  and a plurality of second thin film regions  1121  are disposed on substrate  1101 . 
     A first covering layer  1112  is formed on the top surfaces of the first thin film regions  1111 , and second covering layer  1122  is formed on the top surfaces of the second thin film regions  1121 . In this embodiment, a differential surface layer is formed by alternating regions of the first covering layer  1112  and regions of second covering layer  1122 . 
     In some embodiments, the differential surface regions can include alternating hydrophilic surfaces and hydrophobic surfaces. In some embodiments, the differential surface regions can include alternating surfaces of positive charges and surfaces of negative charges. In the description below, thin film regions  1111  are referred to as the first thin film regions, and thin film regions  1121  are referred to as the second thin film regions. The covering layers  1112  are referred to as the first covering layers, which are formed by reaction between the first thin film regions with a first material. The covering layers  1122  are referred to as the second covering layers, which are formed by reaction between the second thin film regions with a second material. 
     In some embodiments, the first thin film regions can include thin films of metal oxides or metals, as described above. Then the metal oxides or metals can receive a treatment and be exposed to phosphonic acids, such as PVPA (Polyvinylphosphonic acid). In some embodiments, the treatment can be carried out at a temperature ranging from 80° C. to 100° C. for 1-3 minutes. For example, the treatment can be carried out at 90° C. for 2 minutes. This treatment can form a hydrophilic covering layer. 
     In some embodiments, the metal oxides or metals can be exposed to phosphates in a SAM (self-assembled monolayer) process. For example, a SAM process using ammonium salt of hydroxy dodecyl phosphate, OH-DDPO 4 (NH4) 2 , can form a hydrophobic covering layer with a contact angle of about 110 degrees. In another example, a SAM process using 12-Hydroxy dodecyl phosphate, (OH-DDPO 4 ), can form a hydrophiliic covering layer with a contact angle of about 50 degrees. In still other examples, a SAM process using a mixture of the different phosphate compounds can form a covering layer with different hydrophobicity, with contact angles ranging from 50 to 110 degrees. Further, with suitable combination of different phosphates, a covering layer with different hydrophobicity can be formed, with contact angles that can ranges from, for example, 20 to 130 degrees. 
     After the first covering layers formed on the metal oxides or metals thin films, a second covering layer can be formed selectively on the regions of the second thin film regions of, e.g., inorganic silicon oxide. For example, a hydrophobic covering layer can be formed by treating the device in a hydrophobic silane, such as fluorinated Alkyl-Silanes, dialkyl-Silanes, etc. Alternatively, a hydrophilic covering layer can be formed by treating the device in a hydrophilic silane, such as such as Hydroxyakyl terminated silanes, etc. With appropriate selection of the silane compounds, the second covering layer can be formed only on the second thin film regions of, e.g., inorganic silicon oxide, and not on the first covering layer already formed on the first thin film materials. Besides inorganic silicon oxide, the second thin film regions can also include materials such as silicon, silicon nitride, metals oxides, or combinations thereof. 
     In the process described above, the alternating first thin film regions  1110  and second thin film layer  1120  are formed in a sequence such that the first thin film regions  1110  are formed on the second thin film regions  1120 . In some other embodiments, the second thin film regions  1120  can be formed on the first thin film regions  1110 , as illustrated below with reference to  FIGS. 12A-12C . 
       FIGS. 12A-12C  are cross-sectional views illustrating a method for forming a device structure of  FIG. 10  having differential surface regions according to alternative embodiments of the present disclosure. 
       FIG. 12A  shows a cross-sectional view of a device structure having alternating surface regions of thin film layer  1210  and thin film layer  1220 . Referring to  FIG. 12A , a surface layer including a plurality of first thin film regions  1211  and a plurality of second thin film regions  1221  are formed on substrate  1201 . 
     The device structure in  FIG. 12A  is similar to that in  FIG. 11D , with the first thin film layer  1210  corresponding to the first thin film layer  1110  in  FIG. 11D , and the second thin film layer  1220  corresponding to the second thin film layer  1120  in  FIG. 11D . One difference between the structures in  FIGS. 11D and 12A  is that the first thin film layer  1210  is below the second thin film layer  1220 . The device structure in  FIG. 12A  can be formed using a similar process as described in  FIGS. 11A-11C , with the sequence of thin film layer formation reverse. 
       FIG. 12B  shows a covering layer  1212  selectively formed on the thin film regions  1211 . The selective covering layer formation is by exposing the device structure to a suitable first material, such that the covering layer  1212  is formed on the top surfaces of the thin film regions  1210 , but not on the top surfaces of the thin film regions  1220 . The treatment process and annealing process are similar to those described in connection with  FIG. 11E , with the covering layer  1212  corresponding to the covering layer  1112  in  FIG. 11E . 
       FIG. 12C  shows a second covering layer  1222  selectively formed on the top surfaces of the second thin film regions  1221 . The selective covering layer formation is by exposing the device structure to a suitable second material, such that covering layer  1222  is formed on the top surfaces of the thin film regions  1221 , but not on the top surfaces of thin film region  1211  with the covering layer  1212 . The treatment process is similar to that described in connection with  FIG. 11F . After the process with the second material,  FIG. 28C  shows a cross-sectional view of a device structure  2800 , having a surface layer  2802  with differential surface regions  2812  and  2822 . As shown in  FIG. 12C , the device structure  1200  includes a substrate  1201 . The surface layer  1202  including a plurality of first thin film regions  1211  and a plurality of second thin film regions  1221  are disposed on substrate  1201 . 
     A first covering layer  1212  is formed on the top surfaces of the first thin film regions  1211 , and a second covering layer  1222  is formed on the top surfaces of the second thin film regions  1221 . In this embodiment, a differential surface layer is formed by alternating regions of first covering layer  1212  and regions of second covering layer  1222 . 
     In alternative embodiments, the processes illustrated in  FIGS. 11A-11F  and  FIGS. 12A-12C  can also be modified. For example, the sequence of surface layer formation can be reversed with proper selection of thin film materials and the compounds for surface treatment. In some embodiments, in  FIGS. 11D-11F , the covering layer  1122  can be formed on the thin film regions  1121  in the structure of  FIG. 11D  first, and then the covering layer  1112  can be formed on the thin film regions  1111 . Similarly, in  FIGS. 12A-12C , the covering layer  1222  can be formed on the thin film regions  1221  in the structure of  FIG. 12A  first, and then the covering layer  1212  can be formed on the thin film regions  1211 . 
     Although the processes described herein are described with respect to a certain number of steps being performed in a certain order, it is contemplated that additional steps may be included that are not explicitly shown and/or described. Further, it is contemplated that fewer steps than those shown and described may be included without departing from the scope of the described embodiments (i.e., one or some of the described steps may be optional). In addition, it is contemplated that the steps described herein may be performed in a different order than that described. 
     For example, the first and second thin film regions can be either metal or metal oxide, or silicon oxide. Even though in the above example, a covering layer formed by phosphonic acid or phosphate on metal oxides is formed first, followed by a covering layer formed by silane on silicon oxide. In some embodiments, a first covering layer can be formed first by silane on silicon oxide, and then a second covering layer can be formed by phosphonic acid or phosphate on metal oxides. In some embodiments, the treatment by phosphonic acid or phosphate on metal oxides is followed by an annealing process as described above. 
     Example 5 
     This example discloses a novel apparatus and method of mixing a droplet with one or more reagents and transporting the mixed droplet to an array of EWOD to obtain a plurality of extremely small drops (microdroplets). In one embodiment, the extremely small drops (microdroplets) generated by the novel apparatus and method have a uniform size. The novel apparatus may be an integrated lab-on-a-chip device similar or the same as shown in  FIG. 6 . In some embodiments, the novel apparatus may include different regions such as a droplet discharge region for receiving a droplet, a reagent discharge region for receiving a reagent, a mixing region for mixing the droplet with the reagent, and an array of IWOD devices for generating a plurality of samples having a uniform size. In one embodiment, the apparatus may also include temperature-regulating elements for performing a conventional PCR or incubate the microdroplets under a predetermined temperature (isothermal PCR). In one embodiment, the IWOD regions each may feature an array of raised (protruding) hydrophilic surface regions or spots (as shown in  FIGS. 4A and 4C ) or an array of recessed hydrophilic surface regions or microwells (as shown in  FIGS. 4B and 4D ). In some embodiments, each of the spots or microwells include one or more ISFET for measuring an ion concentration or a pH value of an associated microdroplet. In one embodiment, the apparatus may include an interface port configured to communicate with an external host that collects the measured pH values of the samples and calculate a DNA concentration of the droplet. 
     It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes. 
     It is to be understood that the above description is intended to be illustrative and not restrictive. Many embodiments will be apparent to those of skill in the art upon reviewing the above description. The scope of the invention should, therefore, be determined not with reference to the above description, but instead should be determined with reference to the appended claims along with their full scope of equivalents. 
     While the foregoing disclosure shows illustrative aspects of the disclosure, it should be noted that various changes and modifications could be made herein without departing from the scope of the disclosure as defined by the appended claims. The functions, steps and/or actions of the method claims in accordance with the aspects of the disclosure described herein need not be performed in any particular order. Furthermore, although elements of the disclosure may be described or claimed in the singular, the plural is contemplated unless limitation to the singular is explicitly stated. 
     For all flowcharts herein, it will be understood that many of the steps can be combined, performed in parallel or performed in a different sequence without affecting the functions achieved.