Device and method for forming droplets with a predetermined volume by electrowetting

Abstract

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 (E) across the droplet in response to a voltage provided by a control circuit to move the droplet across the dielectric layer in a lateral direction (F) while leaving portions of the droplet on the hydrophilic surface regions to form a plurality of microdroplets on the hydrophilic surface regions.

Description

[0001] This application is a divisional application of patent application No. 201980051553.0, filed on August 1, 2019, by Shenzhen BGI Genomics Co., Ltd., entitled "Apparatus and Method for Forming Droplets with a Predetermined Volume by Electrowetting". Technical Field

[0002] Embodiments of the present invention generally relate to electrowetting over a dielectric (EWOD) technology, and more specifically to apparatus and methods for forming microdroplets of a predetermined volume from one or more bulk droplets by electrowetting technology. Background Technology

[0003] Electrowetting on a dielectric (EWOD) is a liquid-driven mechanism used to change the contact angle of a water droplet between two electrodes on a hydrophobic surface. Bulk droplets as large as a few millimeters (i.e., a few microliters in volume) can be moved by an array of electrodes disposed on a substrate (e.g., an inorganic substrate, such as a silicon / glass substrate) or an organic substrate (e.g., a cyclic olefin polymer / polycarbonate substrate).

[0004] Figure 1A This is a perspective view illustrating an EWOD device 10 that can 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 electrode array 14 within or beneath the insulating layer. The electrode array 14 includes a first set of electrodes 14a arranged parallel to each other and spaced apart from each other in a first direction, and a second set of electrodes arranged parallel to each other and spaced apart from each other in a second direction substantially perpendicular to the first direction. The first and second sets of electrodes are spaced apart from each other within the insulating layer 13, which may include multiple dielectric layers having the same or different materials. The EWOD device also includes an input-output circuit 15 in the substrate, which interfaces with external control circuitry to provide a control voltage having a time-varying voltage waveform to the electrode array 14.

[0005] Reference Figure 1A By turning off / off the control voltage at the electrode below the droplet and the adjacent electrode, the droplet 16 arranged on the surface of the insulating layer 13 can be moved in a specific direction.

[0006] Figure 1B yes Figure 1A The EWOD device shown is a cross-sectional view taken along line B-B'. A cross-sectional view of the second set of electrodes 14b is shown. The first set of electrodes 14a (not shown) may be disposed above or below the second set of electrodes 14b and spaced apart from the second set of electrodes by one or more dielectric layers. Summary of the Invention

[0007] Embodiments of this disclosure provide apparatus, systems, and methods for: mixing droplets with reagents to form microdroplets (particles, microdroplets, or samples) from the mixed droplets; reading the DNA concentration of the sample by optical detection or measuring the pH of each sample by an integrated ion-sensitive field-effect transistor (ISFET) sensor, thereby calculating the DNA concentration of the droplets. It should be noted that although the embodiments describe apparatus and processes for measuring the pH of droplets, this disclosure is not limited thereto. Those skilled in the art will understand that, based on pH changes within the microdroplets, the apparatus and methods described herein can be applied to pH measurements of any aqueous and non-aqueous liquids.

[0008] In one embodiment of this disclosure, an apparatus for forming a plurality of microdroplets from a droplet includes: a substrate; a dielectric layer disposed on the substrate and having a plurality of hydrophilic surface regions spaced apart from each other by hydrophobic surfaces; and a plurality of electrodes disposed in the dielectric layer. The electrodes are configured to form an electric field across the droplet in response to a voltage provided by a control circuit, to laterally move the droplet across the dielectric layer 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. The sensors include: an ion-sensitive field-effect transistor including an ion-sensing membrane configured to be exposed to a solution contained in the microdroplets and to provide a signal associated with a concentration level of the solution of the microdroplets; and a reference electrode configured to provide a reference voltage to the solution.

[0009] In another aspect, a substrate structure includes: a first substrate; a dielectric layer having a hydrophobic surface on the first substrate; control circuitry; a plurality of conductive wirings; and a plurality of electrodes communicating with the control circuitry via the conductive wirings and configured to form an electric field in response to a voltage provided by the control circuitry. The dielectric layer includes: a first region for receiving droplets; a second region for receiving one or more reagents, the second region being in communication with the first region and configured to mix the droplets with the one or more reagents to obtain mixed droplets; and a third region in communication with the second region and including a plurality of hydrophilic surface regions spaced apart from each other by the hydrophobic surface. As the droplets move over the hydrophilic surface regions, a portion of the droplets forms a plurality of microdroplets on the hydrophilic surface regions.

[0010] In some embodiments, the device further includes a fourth region connected to the third region and configured to collect the remaining portion of the droplet after the remaining portion of the droplet has moved across the third region.

[0011] In some embodiments, the substrate structure further includes a second substrate and a conductive layer located on the second substrate and facing the hydrophobic surface of the dielectric layer, and the space between the conductive layer and the dielectric layer forms a channel for the droplet.

[0012] In some embodiments, the conductive layer operates as a common electrode, and the plurality of electrodes include a first parallel strip array along a first direction from the first region to the third region and a second parallel strip array located in the second region and along a second direction perpendicular to the first direction.

[0013] In some embodiments, the third region further includes one or more sensors associated with one of the plurality of hydrophilic surface regions. The sensors include: an ion-sensitive field-effect transistor comprising an ion-sensing membrane configured to be exposed to a solution contained in the microdroplet and to provide a signal associated with a concentration level of the solution in the microdroplet; and a reference voltage electrode configured to apply a reference voltage to the solution.

[0014] In another aspect, a method for forming a plurality of samples having substantially uniform size from a droplet may include: providing a substrate structure having: a substrate; a dielectric layer located on the substrate; a plurality of electrodes located 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 onto the surface of the dielectric layer; and applying a time-varying voltage waveform 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.

[0015] This summary is provided to illustrate different embodiments of the present disclosure in a simplified form, which will be described in detail below. This summary is not intended to limit the scope of the claimed subject matter. Other features, details, utility, and advantages of the claimed subject matter will become apparent from the following detailed description.

[0016] definition

[0017] The terms "wafer" and "substrate" should be understood to include silicon-on-insulator (SOI) or silicon-on-sapphire (SOS) technologies, doped and undoped semiconductors, epitaxial layers of silicon supported by a substrate semiconductor base, and other semiconductor structures. Furthermore, when "wafer" or "substrate" is mentioned in the following description, prior processes may have been used to form regions or junctions within the substrate semiconductor structure or base. Additionally, the semiconductor need not be silicon-based, but can be based on silicon-germanium, germanium, or gallium arsenide.

[0018] As used herein, the term "computer-readable medium" refers to any medium that participates in providing instructions to the processor of a controller for execution. Computer-readable media can take many forms, including but not limited to non-volatile media, volatile media, and transmission media. Non-volatile media include, for example, optical discs, magnetic disks, and magneto-optical disks, such as hard disks or removable media drives. Volatile media include dynamic memory, such as main memory. Furthermore, various forms of computer-readable media may be involved when the processor of the controller executes one or more sequences of one or more instructions. For example, instructions may initially be carried on a disk of a remote computer. The remote computer may remotely load all or part of the instructions used to implement the invention into dynamic memory and send the instructions to the controller via a network.

[0019] The term "hydrophobic" refers to a material that has a contact angle with water in air greater than or equal to 90 degrees. In some embodiments, a hydrophobic surface may have a contact angle greater than 90 degrees, such as 120 degrees, 150 degrees, etc. Conversely, the term "hydrophilic" refers to a material that has a contact angle with water in air or an immiscible liquid such as oil less than 90 degrees.

[0020] The term "droplet" has its normal meaning in this technical field and refers to a droplet with a certain volume (e.g., about a few milliliters) having a boundary at least partially formed by surface tension. -3 ) to about a few microliters (10 -6 A droplet is a liquid between 1 microliter and 100 microliters. Droplets can be water-based (aqueous) droplets, including any organic or inorganic substance such as biomolecules, proteins, living or dead organisms, reagents, and any combination thereof. Droplets can also be non-aqueous liquids. Droplets can be spherical or non-spherical, and their size ranges from 1 micrometer to approximately several millimeters. Droplets can be divided into multiple very small portions (small droplets) spaced apart from each other and having a substantially uniform size. The volume of a very small portion can be as small as 1 microliter (10⁻¹⁰). -6 L or mL) and 100 nanoliters (10 -9 Between 100 nL and 10 nL, and between 10 nL and 100 pL (10 nL or nL) -12 The volume ranges from 100 pL to between 10 pL. In some embodiments, very small fractions can have a volume of several picoliters. In this disclosure, very small fractions are alternatively referred to as microdroplets.

[0021] The term "reagent" refers to a molecule or compound of different molecules that can induce a specific reaction with substances present in a droplet. Attached Figure Description

[0022] Figure 1A This is a simplified perspective view illustrating an EWOD device that can be used to explain embodiments of this disclosure.

[0023] Figure 1B yes Figure 1A The simplified cross-sectional view of the EWOD device shown is taken along line B-B'.

[0024] Figure 2A This is a simplified cross-sectional view of a portion of an EWOD device according to one embodiment of the present disclosure.

[0025] Figure 2B This is a simplified cross-sectional view of a part of an EWOD device according to another embodiment of this disclosure.

[0026] Figure 2C This is a simplified cross-sectional view of a part of an EWOD device according to yet another embodiment of this disclosure.

[0027] Figure 2D This is a simplified plan view of an EWOD device according to an exemplary embodiment of the present disclosure.

[0028] Figure 2E This is a simplified cross-sectional view showing the electric field generated by the electrodes according to one embodiment of the present disclosure.

[0029] Figure 2F This is a simplified cross-sectional view showing the electric field generated by the electrodes according to another embodiment of the present disclosure.

[0030] Figure 2G This is a simplified cross-sectional view showing the electric field generated by the electrodes according to yet another embodiment of the present disclosure.

[0031] Figures 3A to 3C This is a simplified top view of a droplet moving on the surface of a dielectric layer according to an embodiment of the present disclosure. Figure 3A This is a simplified top view showing the discharge of droplets on the first electrode of the electrode array according to one embodiment of the present disclosure. Figure 3B This is a simplified top view illustrating, according to one embodiment of the present disclosure, the movement of a droplet to a second (adjacent) electrode by an EWOD device under the influence of an electric field. Figure 3C This is a simplified top view illustrating, according to one embodiment of the present disclosure, the removal of a droplet from an electrode array while leaving a residue on a second electrode.

[0032] Figure 4A This is a cross-sectional view of a portion of an EWOD device according to one embodiment of the present disclosure.

[0033] Figure 4B This is a cross-sectional view of a portion of an EWOD device according to another embodiment of this disclosure.

[0034] Figure 4C yes Figure 4AA perspective view of a portion of the EWOD device shown.

[0035] Figure 4D yes Figure 4B A perspective view of a portion of the EWOD device shown.

[0036] Figure 4E This is a perspective view of a portion of an EWOD device according to one embodiment of the present disclosure.

[0037] Figure 5 This is a cross-sectional view of an ISFET device according to one embodiment of the present disclosure.

[0038] Figure 6A This is a simplified top view of an integrated lab-on-a-chip device according to one embodiment of the present disclosure.

[0039] Figure 6B It is based on one implementation scheme of this disclosure. Figure 6A A simplified top view of an exemplary arrangement of electrodes in an integrated on-chip laboratory device.

[0040] Figure 7 This is a simplified flowchart illustrating a method for forming multiple samples from droplets according to one embodiment of the present disclosure.

[0041] Figure 8 This is a simplified flowchart illustrating a method for operating an integrated on-chip laboratory device according to one embodiment of the present disclosure.

[0042] Figure 9 This is a simplified schematic diagram of a computer system that can be used to control EWOD devices and on-chip laboratory equipment according to one embodiment of this disclosure.

[0043] Figure 10 This is a cross-sectional view of a device structure with different surface regions according to some embodiments of this disclosure.

[0044] Figures 11A to 11F This illustrates some embodiments of the present disclosure for forming surface regions with differences. Figure 10 A cross-sectional view of the method for constructing the device structure.

[0045] Figures 12A to 12C This illustrates an alternative embodiment of the present disclosure for forming surface regions with differences. Figure 10 A cross-sectional view of the method for constructing the device structure.

[0046] As is customary, the features and elements described are not drawn to scale, but are drawn to emphasize the features and elements relevant to this disclosure. Detailed Implementation

[0047] It should be understood that, for the sake of simplicity and clarity, the elements shown in the figures are not necessarily drawn to scale. For example, for clarity, the dimensions of some elements are enlarged relative to each other. Furthermore, reference numerals are repeated between figures where deemed appropriate to indicate corresponding elements.

[0048] In the following detailed description, reference is made to the accompanying drawings, which form a part thereof, and specific embodiments in which the invention may be implemented are illustrated by way of illustration. The orientations in the described drawings are referred to using the terms “upper,” “lower,” “vertical,” “horizontal,” “depth,” “height,” “width,” “top,” “bottom,” etc. Because components of embodiments of the invention can be positioned in many different orientations, these terms are used for illustrative purposes and not for limitation.

[0049] The terms "first," "second," etc., used do not indicate any order, but are used to distinguish one element from another. Furthermore, the use of terms "a," "an," etc., does not indicate a limitation on quantity, but rather indicates the presence of at least one of the referenced items.

[0050] As used herein, turning off an electrode means reducing the voltage of that electrode to a level below the common voltage, such as connecting the electrode to ground potential. Alternatively, turning off an electrode may also mean setting the electrode to a floating state. Conversely, turning on an actuator electrode means increasing the voltage of that actuator electrode to a level above the common voltage. The common voltage can be any voltage shared by multiple circuit elements of the EWOD device, such as ground potential.

[0051] As used herein, a droplet is an encapsulated liquid. Droplets can be spherical or non-spherical. A droplet can be divided into multiple very small portions (microdroplets) that are separated from each other and have a substantially uniform size. In this disclosure, the very small portions of a droplet may alternatively be referred to as microdroplets.

[0052] Figure 2A This is a simplified cross-sectional view of a portion of an EWOD device 20A according to one embodiment of this disclosure. (Refer to...) Figure 2A The EWOD device 20A includes a first substrate 22, a dielectric layer 23 on substrate 21, a group of actuating electrodes 24 (e.g., 24a, 24b, 24c) within the dielectric layer 23, and a common electrode 27 attached to a second substrate 28 and facing the actuating electrodes 24. The common electrode 27 may be grounded or have other common voltages. The dielectric layer 23 and the common electrode 27 are spaced apart from each other by spacers 29. (Reference) Figure 2AA droplet 26 is disposed between the actuating electrode 24 and the common electrode 27, and moves laterally across the surface of the dielectric layer 23 by changing or altering the voltage level applied to the actuator electrode relative to the common electrode. In one embodiment, the EWOD device 20A may further include control circuitry (not shown) configured to provide control voltages to the common electrode and the actuating electrode. By switching the voltage applied to the actuating electrode on and off, the control circuitry can cause the droplet 26 to move laterally across the surface of the dielectric layer 23. For example, an electric field is generated by applying a first voltage to the actuating electrode 24a below the droplet 26 and a second voltage to the adjacent actuating electrode 24b, causing the droplet 26 to move toward the actuating electrode 24b. The speed of movement of the droplet 26 can be controlled by the magnitude of the voltage difference between adjacent actuating electrodes. In one embodiment, when the droplet 26 is disposed between the actuating electrode 24 and the common electrode 27, the shape of the droplet 26 can be changed by altering the voltage difference between the actuating electrode 24 and the common electrode 27. It should be understood that the number of actuators in a group of actuator electrodes can be any integer. Figure 2A In the example shown, three actuation electrodes are used in the grouped actuation electrodes. However, it should be understood that this number is arbitrarily chosen to describe the example implementation and should not be limiting.

[0053] Reference Figure 2A Two substrate structures can be formed separately. For example, a first substrate structure can be formed, which includes a substrate 22, a dielectric layer 23, and an actuation electrode 24 within the dielectric layer 23. The substrate 22 can be a thin-film transistor array substrate formed by conventional thin-film transistor (TFT) manufacturing processes. The second substrate structure can include a substrate 28 and a common electrode 27 layer on the substrate 28. Spacers 29 can be formed on either the first or second substrate structure. In some embodiments, the height of the spacers 29 is in the range of several micrometers to several millimeters. Typically, the height of the spacers 29 is smaller than the diameter of the droplet, such that the droplet disposed on the dielectric layer 23 is in physical contact with the second substrate structure. The first and second substrate structures are then combined to form an EWOD device 20A. In other words, the space or air gap between the first and second substrate structures is determined by the height or thickness of the spacers 29. This space or air gap forms a channel for the droplet.

[0054] exist Figure 2A In the illustrated embodiment, the common electrode 27 and the group of actuating electrodes 24 (e.g., 24a, 24b, 24c) are connected via... Figure 1AThe input-output circuit 15 shown is connected to a voltage provided by a control circuit (not shown). In some embodiments, the common electrode may be connected to ground potential or a stable DC voltage. The control circuit applies a time-varying voltage to the group of actuating electrodes via the input-output circuit through a corresponding electronic switch (which may be, for example, a thin-film transistor or MOS circuit outside the substrate or chip) to generate an electric field across the droplet, thereby causing the droplet to move along a path. In some embodiments, the surface of the common electrode 27 is covered with an insulating layer made of a hydrophobic material. In other embodiments, the surface of the dielectric layer 23 is coated with a hydrophobic film having a submicron thickness.

[0055] Figure 2B This is a simplified cross-sectional view of a portion of an EWOD device 20B according to another embodiment of this disclosure. Reference Figure 2B The EWOD device 20B includes a substrate 22b, a dielectric layer 23b on the substrate 21b, a group of actuating electrodes 24 (24a, 24b, 24c) within the dielectric layer 23b, and a group of common electrodes 27 (only one electrode 27b is shown) covering the dielectric layer 23b. The common electrode 27b and the actuating electrodes are spaced apart from each other by a portion of the dielectric layer. Similar to... Figure 2A By applying a first voltage at an actuating electrode (e.g., 24a) below the droplet 26 and a second voltage at an adjacent actuating electrode (e.g., 24b), the droplet 26 can move along a lateral path across the surface of the dielectric layer 23b. The movement and orientation of the droplet 26 are thus controlled by a control circuit (not shown) that applies voltages to certain actuating electrodes via a group of electronic switches (MOS circuitry in substrate 22b, not shown). Figure 2A Unlike the EWOD 20A shown, the EWOD device 20B has a common electrode 27b close to the actuation electrode 24, and the droplet 26 is not sandwiched between the common electrode 27b and the actuation electrode 24. The EWOD device 20B also differs from the EWOD 20A in that it does not have a spacer 29.

[0056] Reference Figure 2B The actuating electrodes 24 and the common electrodes 27 can be two strip electrodes intersecting each other on different planes on the substrate. The actuating electrodes 24 and the common electrodes 27 are operable to move the droplet 26 across the surface of the dielectric layer 23b. In some embodiments, the common electrode 27b has a surface covered by an insulating layer made of a hydrophobic material. In other embodiments, the surface of the dielectric layer 23 is coated with a hydrophobic film having a submicron thickness.

[0057] Figure 2C This is a cross-sectional view of a portion of an EWOD device 20C according to yet another embodiment of this disclosure. Reference Figure 2CThe EWOD device 20C includes a substrate 22c, a dielectric layer 23c on the substrate 22c, a group of actuating electrodes 24 (e.g., 24a, 24b, 24c) within the dielectric layer 23c, and a group of common electrodes (e.g., a common electrode 27c shown) covering the dielectric layer 23c. The common electrode 27c and the actuating electrodes are spaced apart from each other by a portion of the dielectric layer. In some embodiments, the common electrode 27c has a surface covered by an insulating layer made of a hydrophobic material or covered by a submicron hydrophobic coating film on the surface of the dielectric layer 23. The EWOD device 20C may also include a second substrate 28c spaced apart from the substrate 21c by spacers 29c. Similar to Figure 2A The droplet 26 can move along a path within a channel formed by the space or air gap between the surface of the dielectric layer and the second substrate 28c. The movement of the droplet is controlled by a control circuit (not shown) through a voltage applied to the electrode via an electronic switch.

[0058] Figure 2D This is a simplified plan view of an EWOD 20D according to an exemplary embodiment of this disclosure. Reference Figure 2D The actuating electrodes are arranged in an array having routing channels for routing electrical signals from control circuitry 28 to actuating electrode 24 and common electrode 27c. The spacers 29c shown have a circular cross-section; however, the circular cross-sectional shape is not limiting, and any other cross-sectional shape is equally applicable, such as square, rectangular, oval, elliptical, and other shapes. Similarly, the actuating electrodes are shown as having a square shape; however, the square shape is not limiting, and other shapes are equally applicable, such as rectangular, circular, oval, elliptical, and other shapes. In one embodiment, the spacers 29c are spaced apart to leave sufficient space to allow droplet free movement. In other words, the size and spacing of the spacers 29c are set such that they do not impede the movement of droplets across the entire dielectric layer surface. It should be understood that although the routing channels are shown as being coplanar with the electrode array, those skilled in the art will understand that the routing channels and control circuitry can be disposed in the substrate and in different layers of the dielectric layer. It should also be understood that the actuating electrodes 24 and common electrode 27c can have their relative positions transposed, i.e., the common electrode can be disposed below the actuating electrodes.

[0059] In another embodiment, the EWOD device may have a single electrode array. In other words, the common electrode and the actuating electrode are coplanar, meaning they are arranged in the same plane within the dielectric layer. For example, multiple actuating electrodes and multiple common electrodes are arranged alternately adjacent to each other, and the control circuitry can sequentially apply DC or AC voltage and ground potential to the actuating electrodes and common electrodes to control the movement of the droplet. In yet another embodiment, each electrode in the electrode array is individually controlled by the control circuitry via a group of electronic switches, such that each electrode can be an actuator electrode in a first time period and a common electrode in a second time period.

[0060] Figure 2E This is a simplified cross-sectional view illustrating an exemplary electric field 20E generated by electrodes according to one embodiment of the present disclosure. (Reference) Figure 2E The common electrode 27 can be driven by ground potential (gnd), and the actuating electrodes 24a, 24b, and 24c can be driven sequentially by DC or AC voltage (Ve). For example, a DC voltage is first applied to the actuating electrode 24a, and the voltage difference between the actuating electrode 24a and the common electrode 27 generates an electric field E, which causes the droplet 26 to move along the microchannel defined by the electrode 27 and the dielectric layer 23. By applying a voltage at the electrode adjacent to the droplet 26, the droplet 26 can be moved to that electrode in the lateral direction between the dielectric layer 23 and the common electrode 27. This structure is similar to... Figure 2A The EWOD device shown is similar to or the same. In one embodiment, the DC voltage can be controlled by the control circuit. Figure 1A The input-output circuit 15 shown is provided. The main electric field is perpendicular to the surface of the dielectric layer 23.

[0061] Figure 2F This is a simplified cross-sectional view illustrating an exemplary electric field 20F generated by electrodes according to another embodiment of this disclosure. Reference Figure 2F The control circuit (not shown) can apply DC or AC voltages sequentially to the actuation electrodes 24a, 24b, 24c and the common electrode 27b to generate an electric field pattern that controls the movement of the droplet 26. For example, a voltage applied to an electrode adjacent to the droplet will move the droplet onto that electrode. This structure is similar to... Figure 2B Or the EWOD device shown in 2C is the same as or similar to it.

[0062] Figure 2G This is a simplified cross-sectional view showing an electric field 20G generated by electrodes according to another embodiment of this disclosure. (Reference) Figure 2GA control circuit (not shown) can apply DC or AC voltage to the actuation electrodes 24a, 24b, and 24c in a time-sequential manner to generate an electric field pattern that controls the movement of the droplet 26. In this embodiment, the actuation electrodes 24a, 24b, and 24c can alternatively serve as actuator electrodes and a common electrode. A semi-cylindrical field is formed between the actuation electrodes 24a and 24b. (Reference) Figure 2E , 2F And 2G, the electric field E and the generated electric force F are functions of the voltage difference between the electrodes and the electrode size. By changing the voltage difference between adjacent electrodes in a time-sequential manner, an electric field and a resultant force are generated, thereby causing the droplet 26 to be transported along the electric direction. In some embodiments, non-active electrodes that do not contribute to the movement (or transport) of the droplet (e.g., actuation electrode 24c) can remain floating, i.e., not connected. Figure 2G In the example shown, droplet 26 will remain between actuation electrodes 24a and 24b, that is, between voltage Ve and ground.

[0063] Figures 3A to 3C This is a top-sequence view of a droplet moving across the surface of the entire dielectric layer according to an embodiment of this disclosure. Reference Figure 3A Droplet 26 is disposed on an EWOD device, as described above in any of EWOD devices 20A, 20B, and 20C. The EWOD device includes a substrate with a thin-film transistor array or MOS circuitry, a dielectric layer on the substrate, and an array of actuating electrodes (and / or common electrodes) within the dielectric layer. The actuating electrodes and common electrodes are connected to control circuitry via wires in a routing channel and receive control signals from the control circuitry via thin-film transistors. Droplet 26 is disposed on the surface of the dielectric layer above a first actuator electrode 34a. The droplet can move toward the next actuator electrode by closing (or floating) the first actuator electrode below it and opening the next actuator electrode adjacent to it. In one embodiment, a predetermined feature can be used to modify a portion of the surface of the dielectric layer above the actuator electrode array, which has a greater attraction to liquids (e.g., droplets) than a hydrophobic surface. This feature can have dimensions ranging from micrometers to nanometers, corresponding to microliters and nanoliters, respectively. Feature 35 can be precisely fabricated on the dielectric layer thousands or millions of times using currently available submicron semiconductor fabrication processes.

[0064] As used herein, turning off an actuating electrode means reducing the voltage of the actuating electrode to the same level as the common voltage applied to the common electrode. Conversely, turning on an actuating electrode means increasing the voltage of the actuating electrode to a level higher than the common voltage. The EWOD device can operate at DC (DC electrowetting) or AC (AC electrowetting) voltages, provided that the potential between the electrodes is at a DC voltage level to create an electric field for moving the droplet. In some embodiments, when an adjacent electrode is fully or partially turned on, a droplet positioned adjacent to it will be moved to the turned-on electrode and wet a feature placed on the turned-on electrode. As used herein, the term "feature" refers to an area or structure in which a liquid material (e.g., a droplet) is deposited or formed. By moving the droplet to the next turned-on electrode using a time-varying voltage waveform provided by control circuitry, the droplet will move between the electrodes, leaving residual microdroplets (very small or minute drops or microparticles)26a in or on the feature. The volume of residual microdroplets depends entirely on the characteristic size and the contact angle of the liquid droplets on the surface of the environment (e.g., air or oil). Figure 3B This is a top view showing a droplet 26 moving from a first electrode 34a to a second electrode 34b having nine features 35 according to an embodiment of the present disclosure. Figure 3C This is a top view showing a residual droplet 26b moving from the second electrode to the third electrode 34c according to one embodiment of the present disclosure, thereby leaving residual microdroplets (microdroplets) 26a within or on the feature. To prevent the microdroplets from evaporating in the air, they can be surrounded by other immiscible liquids (such as silicone oil). It should be understood that the number of features on the electrode can be any integer. Figures 3A to 3C In the example shown, nine features are used in the second electrode. However, it should be understood that this number is arbitrarily chosen to describe the example implementation and should not be limiting. It should also be understood that each electrode (e.g., the first, second, and third electrodes) may have the same number of features, or they may have different numbers of features. Reference Figures 3A to 3C The feature is shown as having a square shape; however, it should be understood that the shape shown is not limiting, and any other shape is equally applicable, such as circles, rectangles, ovals, ellipses, polygons, and other shapes.

[0065] Note that the electrodes according to embodiments of this disclosure can be arranged in various configurations, and the electrodes can have many shapes. For example, the electrodes can have polygonal shapes (e.g., squares, rectangles, triangles, etc.), circular shapes, oval shapes, etc. The configuration can be a checkerboard configuration or other geometric configurations.

[0066] Patterned features can be implemented in different ways. In one embodiment, the surface of the dielectric layer is selectively divided into hydrophobic and hydrophilic regions. Within a certain range of the surface area ratio between the hydrophobic and hydrophilic regions, and within a certain range of the interfacial tension between the droplet and the environment (oil or air), the droplet can be removed from the hydrophobic regions, leaving residual droplets (microdroplets) in the hydrophilic regions. The volume of the residual droplets (microdroplets) is defined by the size of the hydrophilic feature and the contact angle of the droplet on the surface of the environment (air or oil). Figure 4A This is a cross-sectional view of a portion of an EWOD device 40A according to one embodiment of this disclosure. (Refer to...) Figure 4A The EWOD device 40A includes a substrate structure having a first substrate (not shown) and a second substrate (not shown), a first dielectric layer 43a disposed on the first substrate, and a second dielectric layer 47a disposed on the second substrate (not shown). A droplet 26 is disposed between the first dielectric layer 43a having a first surface 44 and the second dielectric layer 47a 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 48a protruding above the surface of the first dielectric layer 43a. The surface of the first dielectric layer 43a is coated with a hydrophobic film, i.e., the protruding hydrophilic regions 48a are surrounded by interstitial hydrophobic surface regions. The protruding hydrophilic regions 48a can be patterned such that residual small droplets (microdroplets) of the droplet disposed on each hydrophilic region have a desired volume. Each protruding hydrophilic region can have a polygonal shape (e.g., square, rectangular), oval, circular, elliptical, and other shapes. In this disclosure, the term "protruding hydrophilic region" may also be referred to as a "spot" or "island". In some embodiments, the second dielectric layer 47a is made of glass coated with a hydrophobic film. In some embodiments, the first and second dielectric layers are formed separately, and such as in... Figure 2A , 2C Spacers, such as those shown and described in Figures 1 and 2E, are formed on a first dielectric layer or a second dielectric layer. The first and second dielectric layers are then bonded together to form... Figure 4AThe structure shown has a space between the first and second dielectric layers serving as a channel for the droplet 26. In some embodiments, the second dielectric layer 47a is coated with a conductive layer of a metallic material serving as a common electrode (e.g., a ground electrode) and a hydrophobic film on the conductive layer. Protruding hydrophilic regions can be formed on the surface of the first dielectric layer 43a using conventional semiconductor manufacturing techniques. Some of these semiconductor manufacturing techniques will be described in detail in the Examples section below. The EWOD 40A also includes an array of electrodes (not shown) embedded within the first dielectric layer, the second dielectric layer, or both the first and second dielectric layers, which generate a moving electric field in response to a time-varying voltage provided by control circuitry. The droplet 26 moves across the entire surface of the first dielectric layer by the moving electric field, leaving residual small portions (microdroplets) 26' of the droplet 26 on the protruding hydrophilic region 48a.

[0067] Figure 4B This is a cross-sectional view of a portion of an EWOD device 40B according to another embodiment of this disclosure. (Refer to...) Figure 4B EWOD device 40B includes and Figure 4A The substrate structure shown is similar to that of a substrate structure, except that the first dielectric layer 43b includes a plurality of grooves (micropores) 49 of a certain depth instead of protruding areas. In one embodiment, the second surface of the second dielectric layer is hydrophobic, and the first dielectric layer includes a plurality of grooves (micropores) 49 of a certain depth. The terms “groove,” “recess,” and “micropore” are used interchangeably herein. Each groove also has an opening defined by its length and width. In some embodiments, the length and width are less than 1 micrometer. By carefully selecting the shape of the micropores and / or changing the inner surfaces (sidewalls and / or bottom) of the micropores from hydrophobic to hydrophilic through chemical (i.e., by surface treatment) or electrical (i.e., by electrowetting) means, a portion of the water-containing droplets spontaneously infiltrates into the micropores and tends to remain in the micropores even after the electric field is removed. Within a certain range, based on the area ratio between the flat hydrophobic surface of the first dielectric layer and the micropore, and the interfacial tension between the droplet and the environment (oil or air), the droplet can be removed from the micropore, leaving (depositing) residual small droplets (microdroplets) within the micropore. The volume of the residual small droplets (microdroplets) is determined by the predetermined micropore size and the contact angle of the droplet at the micropore opening. Combined with the above... Figure 4ASimilar in structure, the second dielectric layer 47b may be coated with a conductive layer of metallic material serving as a common electrode (e.g., a ground electrode) and a hydrophobic film on the conductive layer. Recessed hydrophilic regions can be formed in the first dielectric layer 43b using conventional semiconductor manufacturing techniques. The EWOD 40B also includes an array of electrodes (not shown) embedded within or in both the first and second dielectric layers, which generate a moving electric field in response to a time-varying voltage provided by control circuitry (not shown). The droplet 26 moves across the entire surface of the first dielectric layer by the moving electric field, leaving small residual portions (microdroplets) 26' of the droplet 26 on the surface of the micropores. In some embodiments, the micropore array can be obtained using photolithographic patterning and etching processes. Some of these semiconductor manufacturing techniques will be described in detail in the Examples section below.

[0068] Figure 4C yes Figure 4A A perspective view of a portion of the EWOD device 40A shown. (Reference) Figure 4C A group of circular cylindrical spots (protruding hydrophilic regions) 48a are arranged in an array. As used herein, the terms "spot," "island," and "protruding region" are used interchangeably. The spots 48a are spaced apart and isolated from each other by interstitial hydrophobic surfaces 44. Note that although the spots 48a are shown as having a circular cylindrical shape in the example shown, those skilled in the art will understand that the spots 48a may also have other shapes, such as rectangular, square, or oval cylindrical shapes.

[0069] Figure 4D yes Figure 4B A perspective view of a portion of the EWOD device 40B shown. (Reference) Figure 4D A group of circular cylindrical grooves (micropores) 49 are arranged in an array. The micropores 49 are filled with a hydrophilic material and spaced apart from each other by interstitial hydrophobic surfaces 46. In some embodiments, the hydrophobic material filling the micropores 49 has an upper surface flush with the upper surface of the interstitial hydrophobic surfaces 46. It should be noted that although the micropores 49 are shown as circular cylindrical pores, depending on the application, the micropores 49 may also be rectangular, square, or oval cylindrical pores.

[0070] Figure 4E This is a perspective view of a portion of an EWOD device according to one embodiment of this disclosure. (Refer to...) Figure 4E The group of hydrophilic surfaces 491 are surrounded (side-connected) by the hydrophobic gap region 461 on the surface of the dielectric layer 43, that is, the hydrophilic surface 491 is flush with the surface of the hydrophobic gap region 461 on the surface of the dielectric layer 43.

[0071] According to this disclosure, a large number of microdroplets of uniform size can be used for droplet digital PCR on a microfluidic chip. Due to the small volume of each sample and the DNA concentration below a certain level required to satisfy a Poisson distribution, each sample (microdroplet) will have one DNA molecule or no DNA molecule. Individual DNA molecules within a target region can be amplified on each sample in an environment (e.g., oil) by thermally cycling the samples (microdroplets) using conventional PCR or incubating them at a specific temperature using isothermal PCR. After reading the DNA concentration of the final droplet by optical detection or pH measurement using an integrated on-chip ion-sensitive field-effect transistor (ISFET) sensor, the absolute amount of target DNA in the sample (microdroplet) array can be quantified, and then the DNA concentration in the host droplet can be calculated using absolute DNA quantification. The terms “sample,” “residual small droplet,” “small portion of droplet,” and “microdroplet” are used interchangeably herein and refer to a small droplet formed from a host droplet according to an embodiment of this disclosure.

[0072] According to this disclosure, droplets containing multiple different DNA targets can be dispensed onto a region of a single microfluidic chip, and then the droplets can be moved to a next region by electrowetting, where a large number of samples (copies of the DNA targets) are generated from the droplets for sample detection or measurement. In some embodiments, the next region to which the droplets are moved may include multiple hydrophilic regions spaced apart by interstitial hydrophobic surfaces. The multiple hydrophilic regions may be combined Figure 4A and 4B Hydrophilic regions are shown and described. Each hydrophilic region may include an ion-sensitive field-effect transistor (ISFET) sensor configured to measure the pH of a sample disposed thereon. That is, a single microfluidic chip may include an array of ISFET sensors, each ISFET sensor associated with one of the samples in the droplet. This arrangement of a single microfluidic chip enables the formation of multiple samples (microdroplets) from the droplet by electrowetting and the measurement of the samples by the array of ISFET devices integrated on the chip. According to this disclosure, the ISFET sensor array embedded in the hydrophilic regions facilitates the simultaneous measurement of different targets from the droplet with high sensitivity and accuracy.

[0073] Figure 5 This is a cross-sectional view of an ISFET device 50 according to one embodiment of the present disclosure. Reference Figure 5The 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 fabricated using standard CMOS manufacturing processes. In the following description, according to an exemplary embodiment of this disclosure, an n-channel MOS transistor (NMOS) is used. However, it should be noted that the choice of NMOS or PMOS depends solely on the chosen process or substrate and is not limiting. Reference Figure 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 also includes a sensing membrane 55 on the floating gate G and below a droplet 56, and a reference electrode 57 that is wholly or partially immersed in the droplet 56 and spaced apart from the sensing membrane 55. The sensing membrane can include any material that provides sensitivity to hydrogen ion concentration (pH), such as silicon nitride, silicon oxynitride, etc. Other sensing membranes sensitive to other ions can be used, as is known to those skilled in the art.

[0074] Still referencing Figure 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, and a voltage source Vds configured to provide a voltage Vds between the source and drain regions. When the voltage Vgs is greater than the threshold voltage Vth of the ISFET device, current will conduct through the channel between the source and drain regions. The amount of current Ids flowing between the source and drain regions represents the concentration or pH value of the droplets 56. In one embodiment, the source region and the substrate have the same potential, such as ground potential.

[0075] exist Figure 5 In the example shown, one ISFET device is used to measure the ion concentration of droplet 56. However, it should be understood that this number is chosen only to describe exemplary embodiments and should not be limiting. In some embodiments, more than one ISFET device may be used to measure the ion concentration of droplet 56. In other words, each spot or microwell may have multiple ISFET devices.

[0076] In some implementation schemes, in the corresponding appendix Figure 4C and 4D The diagram illustrates that each spot in a spot array or each micro-hole in a micro-hole array can have more than one ISFET device to improve measurement sensitivity and accuracy. The number of ISFET devices available per spot or micro-hole depends on the conventional CMOS fabrication process and application requirements. The power supply voltage provided to the ISFET devices via electrical connections can be achieved using conventional CMOS fabrication processes and is not described here for the sake of brevity.

[0077] Figure 6A This is a simplified top view of an integrated on-chip laboratory device 60A according to one embodiment of this disclosure. (Reference) Figure 6A The integrated on-chip laboratory device 60A 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 droplets 26 with one or more reagents 63 to obtain mixed droplets 263; and an EWOD device array configured to split the droplets (mixed or unmixed) into multiple microdroplets and amplify the microdroplets. In one embodiment, the EWOD device array may have a first heating element configured to heat the microdroplets to a first temperature for amplifying the microdroplets and a second heating element for annealing the amplified microdroplets. In one embodiment, the on-chip laboratory device 60 may also include a sensor array, each sensor associated with a sample and configured to measure the concentration or pH of the microdroplets. In one embodiment, the droplet receiving region 61 may have Figure 1A and 1B The device structure is shown. In one embodiment, the reagent receiving area 62 may have... Figure 1A and 1B The device structure is shown. In other words, the integrated on-chip laboratory device 60 can be operable to move one or more droplets and one or more reagents to a mixing region 64, and control the mixing of droplets and reagents according to a user-provided software program. In one embodiment, the EWOD device array may include multiple EWOD devices arranged in a regular pattern, each EWOD device may have... Figures 2A to 2C The devices shown have similar or identical structures. In some implementations, each EWOD device may include multiple ISFETs. An example of an ISFET device is shown in... Figure 5 As shown in the diagram. The integrated on-chip laboratory device 60A may also include a waste (collection) area 66 for collecting residual portions of droplets after they have formed in the EWOD device array and / or for collecting droplets after they have been processed and measured. Figure 6A In the example shown, two electrodes are used in the droplet receiving region 61, eight electrodes are used in the upper part of the reagent receiving region 62, eight electrodes are used in the lower part of the reagent receiving region 62, eight electrodes are used in the mixing region 64, and an array of four EWOD devices is used. However, it should be understood that these numbers are arbitrarily chosen for describing exemplary embodiments and should not be limiting.

[0078] In some embodiments, the integrated on-chip laboratory device 60A may further include control circuitry 67 configured to provide control signals to droplet receiving region 61, reagent receiving region 62, mixing region 64, EWOD device array, and waste region 66 for moving droplets 61, reagents 63, mixed droplets 263, fragmented droplets (i.e., microdroplets), and residual portions of droplets after passing through the EWOD device array. In one embodiment, the integrated on-chip laboratory device 60A may include input / output (IO) ports 68 configured to interface with a host computer. In one embodiment, the host computer may be a separate or external processor configured to provide control signals to the integrated on-chip laboratory device 60. In another embodiment, the host computer may be integrated into the same package as the integrated on-chip laboratory device 60. Many variations, modifications, and alternatives will be recognized by those skilled in the art. Still refer to Figure 6A The control circuit 67 can be located remotely from the integrated on-chip laboratory device 60A and can communicate with the integrated on-chip laboratory device 60A via an input-output port or a serial interface port. In one embodiment, the integrated on-chip laboratory device 60A may further include a first heating block "heater 1" formed within the substrate structure below the surface of the mixing region 64 for maintaining and / or changing the incubation temperature of the mixed droplets 263. In one embodiment, the integrated on-chip laboratory device 60A may further include a second heating block "heater 1" formed within the substrate structure below the surface of the EWOD device array for maintaining and / or changing the incubation temperature of the microdroplets. The first and second heating blocks are formed of metal or polysilicon lines, metal or polysilicon layers, and polysilicon layers that can convert electrical energy from signals received from the control circuit 67 into heat energy.

[0079] Figure 6B This is a simplified top view of an example arrangement of electrodes 60B in an integrated on-chip laboratory device 60A according to one embodiment of the present disclosure. Reference Figure 6B , Figure 6AThe integrated on-chip laboratory device electrode 60B includes a first conductive strip array 611 having strips 611a, 611b, 611c, 611d, 611e, 611f, and 611g arranged parallel to each other, for example, along a first direction from a first region (droplet receiving region) toward a third region (EWOD device array). The first strip array 611 is controlled by control circuitry 67 and configured to generate a moving electric field to transport droplets along the first direction. Electrode 60B also includes a second parallel strip array 612 having strips 612a, 612b, 612c, and 612d arranged parallel to each other, for example, along a second direction perpendicular to the first direction. The second array 612 is configured to generate a moving electric field to transport droplets along the second direction toward a second region (mixing region). The first array intersects with and is spaced apart from the second array by an insulating layer; that is, the first array and the second array are arranged in different layers separated by at least one insulating layer. Figure 6B In the example shown, the second array 612 is arranged in the reagent receiving region for delivering reagents toward the mixing region. However, it should be understood that the second array 612 may also be arranged in the droplet receiving region, in the mixing region, and in the EWOD device array to generate a moving electric field and power to move droplets, mixed droplets, and microdroplets along the second direction. In some other embodiments, electrode 60B may include [missing information - likely related to bonding / contact]. Figure 1A and 1B The electrode array shown and described is similar to the electrode array shown. Those skilled in the art will recognize many variations, modifications, and alternatives.

[0080] Figure 7 This is a simplified flowchart illustrating a method 70 for forming multiple microdroplets from droplets according to one embodiment of the present disclosure. (Refer to...) Figure 7 Method 70 may include, at 701, providing a substrate structure having a lower substrate, a dielectric layer on the lower substrate, and 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 and spaced apart by the hydrophobic surface. In another embodiment, the dielectric layer has a hydrophobic surface and a plurality of micropores (grooves) having substantially the same size in the dielectric layer. Each of the micropores has a hydrophilic bottom and hydrophilic sidewalls (collectively referred to as the hydrophilic surface region). At 703, the method may include dispensing droplets onto the surface region of the dielectric layer. At 705, the method may include applying a time-varying control voltage signal to the electrodes by a control circuit (e.g., a host computer) to move droplets across the hydrophobic surface of the dielectric layer while leaving small residual portions on the hydrophilic surface region to form a plurality of microdroplets having substantially the same size on the hydrophilic surface region.

[0081] Figure 8 This is a simplified flowchart illustrating a method 80 for operating an integrated laboratory-on-a-chip (Lab-on-Chip) device according to one embodiment of the present disclosure. In some embodiments, the integrated Lab-on-Chip device may be the same as or similar to the integrated Lab-on-Chip device 60 shown in FIG. 6. That is, the integrated Lab-on-Chip device includes: a droplet receiving region configured to receive droplets; a reagent receiving region configured to receive one or more reagents; a mixing region configured to mix the droplets with one or more reagents; and an EWOD device array configured to split the received droplets (droplets in the droplet receiving region that may be mixed with one or more reagents or droplets that are not mixed with any reagents) into a plurality of microdroplets, the mixed droplets being incubated at a constant and / or variable temperature controlled by a heater below the surface of the mixing region 64, and the microdroplets also being incubated at a constant and / or variable incubation temperature by the heater below the surface of the EWOD device. In one embodiment, the Lab-on-Chip device may also include a sensor array associated with the microdroplets and configured to measure the ion concentration or pH value of the microdroplets. (See also...) Figure 8 Method 80 may include: at 801, dispensing a droplet onto the surface of a droplet receiving region. At 803, the method may include: moving the droplet across a mixing region toward an EWOD device of an EWOD device array, at which the droplet is fragmented into multiple microdroplets, the pH of which, before and / or 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) portions of the droplet, along with the microdroplets, in a waste area of ​​an integrated on-chip laboratory device. In one embodiment, the method may further include: at 802, dispensing one or more reagent droplets onto the surface of a reagent receiving region. At 802, the method may further include: moving the one or more reagent droplets toward a mixing region to mix the reagent droplets with the droplet under a user-defined software procedure before moving the droplet toward the EWOD device for fragmentation into multiple microdroplets. In one embodiment, the method may further include: at 802', incubating the mixed droplets at a constant or variable temperature for a reaction. In one embodiment, the method may further include: culturing droplets in an EWOD array at a culture temperature at point 804. In one embodiment, the culture temperature in the EWOD array region is maintained at a constant culture temperature. In one embodiment, the culture temperature in the EWOD array region may be variable. In some embodiments, the method may be repeated at step 801 after discarding any remaining portions of the droplets and the droplets that have been measured.

[0082] Figure 9This is a simplified schematic diagram of a mobile computing device 90 that can be used to control an EWOD device according to one embodiment of this disclosure. (Reference) Figure 9 The mobile computing device 90 may include a monitor 910, a computing electronic device 920, a user output device 930, a user input device 940, a communication interface 950, etc.

[0083] The computing electronic device 920 may include one or more processors 960 that communicate with a plurality of peripheral devices via a bus subsystem 990. These peripheral devices may include user output devices 930, user input devices 940, communication interfaces 950, and storage subsystems such as random access memory (RAM) 970 and disk drives 980.

[0084] User input device 940 may include any type of device and interface for inputting information to computer device 920, such as keyboard, keypad, touch screen, mouse, trackball, trackpad, joystick and other types of input devices.

[0085] User output device 930 may include any type of device for outputting information from computing electronics 920, such as a display (e.g., monitor 910).

[0086] Communication interface 950 provides an interface to other communication networks and devices. Communication interface 950 can be used as an interface for receiving data from and sending data to other systems. For example, communication interface 950 may include a USB interface for communicating with EWOD devices or on-chip lab equipment.

[0087] RAM 970 and disk drive 980 are examples of tangible media configured to store data, such as embodiments of this disclosure, including executable computer code, human-readable code, etc. Other types of tangible media include floppy disks, removable hard disks, optical storage media (e.g., CD-ROMs, DVDs, and barcodes), semiconductor memories (e.g., flash memory), non-transitory read-only memory (ROM), battery-supported volatile memory, network storage devices, etc. RAM 970 and disk drive 980 can be configured to store basic programming and data constructs that provide the functionality of this invention.

[0088] The software code modules and instructions that provide the functionality of this disclosure can be stored in RAM 970 and disk drive 980. These software modules can be executed by processor 960.

[0089] Still referencing Figure 9Both the EWOD device 91 and the on-chip laboratory device 92 may include an interface port 94 configured to provide communication with the mobile computing device 90. In some embodiments, the mobile computing device 90 may provide command and control signals via the interface port 94 to control the signal levels of electrodes in the EWOD device 91 or the on-chip laboratory device 92. In some embodiments, the EWOD device 91 may include, for example, Figure 2A-2C The substrate structure described in one of 3A-3C and 4A-4E and Figure 5 One or more of the ISFET devices. The EWOD device 91 is designed to receive droplets and provide a pH value associated with the droplets. In some embodiments, the EWOD device 91 may be a component of an on-chip laboratory device 92.

[0090] Example

[0091] The following embodiments are provided as examples, but do not limit the claimed invention.

[0092] Example 1

[0093] This embodiment discloses the fabrication of patterned features on the surface of a dielectric layer. Figure 4A A cross-sectional view of dielectric layer 43a is shown. Dielectric layer 43a can be deposited on a substrate (e.g., using conventional deposition processes such as chemical vapor deposition). Figure 2A The dielectric layer may be on the substrate 22 shown. The dielectric layer may include silicon oxide, silicon nitride, fluorinated silicate glass (FSG), or organosilicon glass (OSG). In one embodiment, the dielectric layer is coated with a hydrophobic material. A spin coating process can be used to coat the hydrophobic material, wherein 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 for a certain duration. In yet another embodiment, the hydrophobic material can be formed on the dielectric layer by a deposition process (e.g., chemical vapor deposition). The hydrophobic material may include organic or inorganic materials, such as octadecyltrichlorosilane, perfluorodecyltrichlorosilane, other fluorinated layers, such as tetrafluoroethylene, etc. Subsequently, a patterned photoresist layer is formed on the dielectric layer (which is coated with the hydrophobic material) to expose the surface portion of the dielectric layer. The dielectric layer is then subjected to a wet hydrophilicity-enhancing surface treatment (e.g., a spin coating process), wherein the patterned photoresist layer is used as a mask to spray a hydrophilicity-enhancing solution onto the surface of the dielectric layer. In another embodiment, the exposed surface of the dielectric layer is contacted with a hydrophilicity-enhancing solution at a certain temperature for a certain duration. The hydrophilicity-enhancing solution includes a surfactant or wetting agent, comprising one of cationic, anionic, and nonionic surfactants. Subsequently, the patterned photoresist layer is removed to obtain a hydrophilic region 48a on the surface of the dielectric layer, as shown. Figure 4A or Figure 4C As shown in the image.

[0094] In another embodiment, after forming a patterned photoresist layer on the dielectric layer, the patterned photoresist is used as a mask to perform an etching process (wet etching, dry etching, or a combination of wet and dry etching) on ​​the dielectric layer to form multiple recessed grooves within the dielectric layer. Subsequently, a hydrophilicity-enhancing solution is deposited to fill the grooves. The patterned photoresist layer is then removed by a chemical mechanical polishing (CMP) process to obtain the desired result. Figure 4B or Figure 4D The structure shown.

[0095] Example 2

[0096] This embodiment discloses the formation of electrodes within a 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. Subsequently, a second dielectric layer is formed on the metal layer to form an electrode as described above. Figures 2A to 2C The structure shown.

[0097] In another embodiment, a first dielectric layer is formed on a substrate. A metal layer is then deposited on the first dielectric layer. Subsequently, a patterned photoresist layer is formed on the metal layer. An etching 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 a shape as shown in the image. Figures 2A to 2C The structure shown.

[0098] Example 3

[0099] This embodiment discloses a novel apparatus and method for generating large arrays of nanoliters (10⁻⁶) from droplets via electrowetting. -9 liters or nL) and pills (10 -12 Extremely small droplets (microdroplets) within a predetermined volume range (in liters or pL). This novel device can be used with... Figure 4A or Figure 4B The EWOD device shown is similar to or the same as the device. (Refer to...) Figures 3A to 3CThis illustrates a delivery mechanism for dispensed droplets. The droplets move on the surface of a dielectric layer having an array of raised hydrophilic regions (spots) or on the surface of a dielectric layer having an array of micropores, each micropore having a hydrophilic bottom and sidewalls. The raised (protruding) hydrophilic regions (spots) and / or micropores are spaced apart from each other by interstitial hydrophobic surfaces. In one embodiment, the raised hydrophilic regions and / or micropores are circular or oval. In another embodiment, the raised hydrophilic regions and / or micropores are polygonal (rectangular, square, hexagonal) in shape. In one embodiment, the height of the raised hydrophilic regions (spots) and / or micropores is between 1 nanometer and 100 micrometers, preferably between 1 micrometer and 10 micrometers, more preferably below 10 nanometers. In one embodiment, the raised hydrophilic regions and / or micropores are square in shape, with a width or length between 1 nanometer and 100 micrometers, preferably between 1 micrometer and 10 micrometers, more preferably below 10 nanometers. In one embodiment, the raised hydrophilic regions and / or micropores have a circular shape and a diameter between 1 nanometer and 100 micrometers, preferably between 1 micrometer and 10 micrometers, and more preferably below 10 nanometers.

[0100] Example 4

[0101] This embodiment discloses the formation of a device structure having a hydrophobic / hydrophilic interstitial surface with a scale (size) ranging from micrometers to nanometers. Figure 4A and Figure 4C An exemplary implementation is shown in the figure. Figure 10 This is a cross-sectional view of a device structure 1000 having different 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 is disposed on the substrate 1001. The substrate 1001 includes a MOS circuit system (e.g., control circuit 15) and electrodes (e.g., electrodes 14), which are formed by... Figure 1A The control circuit system 15 shown is actuated or controlled.

[0102] A first capping layer 1012 is formed on the top surface of the first thin film region 1011, and a second capping layer 1022 is formed on the top surface of the second thin film region 1021. In some embodiments, a differential surface layer is formed by alternating regions of the first capping layer 1012 and the second capping layer 1022.

[0103] According to some implementation schemes, methods are provided for selecting a first material and a second material to adjust the hydrophobicity of a first capping layer and a second cover layer to form a surface with different hydrophobic / hydrophilic properties.

[0104] In some embodiments, the differentially hydrophobic / hydrophilic surfaces may have alternating nonpolar molecular regions (which repel water) and polar molecular regions (which can form ionic or hydrogen bonds with water molecules). In some embodiments, one method includes first forming alternating inorganic silicon oxide (SiO2) regions and metal oxide material regions, the metal oxide material comprising one or more of various metal oxides, such as anodic aluminum oxide (Al2O3), tantalum oxide (Ta2O5), niobium oxide (Nb2O5), zirconium oxide (ZrO2), and titanium oxide (TiO2), etc. These alternating regions can be formed on a silicon (Si) or glass substrate using standard semiconductor thin film deposition and photolithography processes, as further described below.

[0105] Secondly, metal oxide surfaces can be treated to modify their properties. One approach is to selectively coat the metal oxide surface with polyvinylphosphonic acid (PVPA), a hydrophilic polymer with an inherent pH of 2 (pH=2) from its intramolecular phosphoric acid, at a temperature ranging from 80°C to 100°C. In specific embodiments, this treatment step can be carried out, for example, at 90°C. In some cases, this treatment step can be relatively rapid, for example, less than 2 minutes (min). This step can be followed by a dry annealing step to help form covalent bonds. The dry annealing process can be carried out at a suitable temperature, for example, at 80°C for about 10 minutes. This reaction can be selective, i.e., no reaction occurs on the SiO2 surface.

[0106] Another method involves selectively coating metal oxide regions with self-assembled monolayers (SAMs) based on the adsorption of alkyl phosphate ammonium salts in aqueous solution. Under the same conditions, SAM formation does not occur on SiO2 surfaces. The hydrophobicity of the coated surface can be adjusted by the formulation of this aqueous SAM-forming solution, with water contact angles ranging from 50 to 110 degrees. In some cases, the water contact angle can range from 20 to 130 degrees. Covalent bonds can be formed during the annealing step, and unreacted material can be washed away with deionized (DI) water.

[0107] After PVPA or phosphate ester treatment, the substrate can be dried and then treated with a hydrophobic silane compound (e.g., fluorinated alkyl silane compounds, dialkyl silane compounds, etc.). Alternatively, the substrate can be dried and then treated with a hydrophilic silane compound (e.g., hydroxyalkyl-terminated silane compounds, etc.) in solution or by chemical vapor deposition. These treatments can form stable covalent bonds with the SiO2 surface and make the surface hydrophobic or hydrophilic without affecting the hydrophobicity of the metal oxide surface.

[0108] By using different organic chemicals with varying hydrophobicity to selectively treat inorganic SiO2 and metal oxide surfaces, it is possible to create differentiated hydrophobic / hydrophilic surfaces using semiconductor processes in well-defined patterns.

[0109] Figures 11A to 11F These are cross-sectional views, which illustrate, according to some embodiments of the present disclosure, methods for forming Figure 10 A method for forming a device structure with differentially distributed surface regions. The method includes providing a substrate and forming a surface layer on the substrate having alternating first and second thin-film regions. The surface layer is exposed to a first material to form a first capping 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 capping layer on the second thin-film regions (but not on the first thin-film regions already covered by the first capping layer). The method includes selecting the first and second materials to adjust the hydrophobicity of the first and second capping layers.

[0110] Figure 11A This illustrates the formation of a thin film layer on a substrate. (Reference) Figure 11A A thin film layer 1120 is formed on substrate 1101. Substrate 1101 can be made of any suitable material, such as glass or semiconductor. Semiconductor substrates can include various semiconductor materials, such as silicon, III-V materials on silicon, graphene on silicon, silicon-on-insulator, combinations of the above materials, and the like. Substrate 1101 can be a bare wafer. Substrate 1101 can also include various devices and circuit structures. For example, substrate 1101 can include a CMOS circuit layer (including substrate 12, such as control circuits and other electronic switches) and a dielectric layer (e.g., dielectric layer 13 including electrode layer 14).

[0111] refer to Figure 11A A thin film layer 1120 is formed on substrate 1101. In one embodiment, thin film layer 1120 comprises inorganic silicon oxide, such as SiO2. In some embodiments, thin film layer 1120 may comprise silicon, silicon nitride, metal oxides, or combinations thereof. Thin film layer 1120 may also comprise other silanizable materials. Thin film layer 1120 can be formed on substrate 1101 using any conventional semiconductor thin film deposition technique.

[0112] Figure 11BA second thin film layer 1110 formed on the first thin film layer 1120 is shown. In some embodiments, the second thin film layer 1110 may comprise a metal oxide or a metal. Suitable metal oxides may include, for example, anodic aluminum oxide (Al₂O₃), tantalum oxide (Ta₂O₅), niobium oxide (Nb₂O₅), zirconium oxide (ZrO₂), and titanium oxide (TiO₂), etc. The thin film layer 1110 may also comprise a metallic material, such as tungsten, titanium, titanium nitride, silver, tantalum, tantalum oxide, hafnium, chromium, platinum, tungsten, aluminum, gold, copper, combinations or alloys of the above materials, and the like. Conventional semiconductor thin film deposition techniques may also be used to form the thin film layer 1110.

[0113] Figure 11C A patterned mask layer 1130 formed on a thin film layer 1110 is shown. The mask layer 1130 includes openings that expose areas of the thin film layer 1110. The mask layer 1130 can be applied by any suitable method (e.g., spin coating, dip coating, and / or similar methods). The mask layer 1130 can also be made of any suitable material, such as a photoresist. Figure 11C As shown, the mask layer 1130 is patterned to have openings according to conventional semiconductor photolithography techniques. In some embodiments, the mask layer 1130 may be a hard mask, which is a patterned layer of suitable thin film material with suitable etch selectivity to serve as an etch mask.

[0114] After the patterned mask layer 1130 is formed, an etching process can be performed to remove the exposed portions 1117 in the thin film layer 1110 to obtain the thin film region 1111. This etching process can be performed using conventional semiconductor process technology. Subsequently, the patterned mask layer 1130 is removed using conventional semiconductor process technology. Figure 11D The resulting device structure is shown in the figure.

[0115] Figure 11E A capping layer 1112 selectively formed on the thin film region 1111 is shown. This selective capping layer formation process can be performed by exposing the device structure to a suitable first material, such that capping layer 1112 is formed on the top surface of thin film region 1111 but not on the top surface of thin film region 1121. Various materials can be used depending on the materials of thin film regions 1111 and 1121, as detailed below. After material treatment, an annealing process can be performed to selectively form capping layer 1112 on thin film region 1111. This annealing process can be performed at a temperature in the range of 70°C to 90°C for 5 to 15 minutes. For example, a dry annealing process can be performed at a temperature of 80°C for 10 minutes. A rinsing process can be performed in deionized (DI) water to remove unreacted material.

[0116] Figure 11F A second capping layer 1122 selectively formed on the top surface of the second thin film region 1121 is shown. This selective capping layer is formed by exposing the device structure to a suitable second material, such that the capping layer 1122 is formed on the top surface of the thin film region 1121, but not on the top surface of the thin film region 1111 having the capping layer 1112. Various materials can be used depending on the materials of the thin film regions 1111 and 1121, as detailed below. After treatment with this second material, Figure 11F A cross-sectional view of the device structure 1100 is shown according to some embodiments of the present invention, which is similar to Figure 10 The device structure 1000 has a surface layer 1102, which includes differential surface regions 1112 and surface regions 1122. As shown in the figure, the device structure 1100 includes a substrate 1101. The surface layer 1102, which includes a plurality of first thin film regions 1111 and a plurality of second thin film regions 1121, is disposed on the substrate 1101.

[0117] A first capping layer 1112 is formed on the top surface of the first thin film region 1111, while a second capping layer 1122 is formed on the top surface of the second thin film region 1121. In this embodiment, a differential surface layer is formed by alternating regions of the first capping layer 1112 and the second capping layer 1122.

[0118] In some embodiments, the differential surface regions may include alternating hydrophilic and hydrophobic surfaces. In some embodiments, the differential surface regions may include alternating positively charged and negatively charged surfaces. In the following description, film region 1111 is referred to as the first film region, and film region 1121 is referred to as the second film region. Capping layer 1112 is referred to as the first capping layer, which is formed by a reaction between the first film region and a first material. Capping layer 1122 is referred to as the second capping layer, which is formed by a reaction between the second film region and a second material.

[0119] In some embodiments, the first film region may include a metal oxide film or a metal film as described above. Subsequently, the metal oxide or metal may be treated and exposed to a phosphonic acid compound, such as PVPA (polyvinylphosphonic acid). In some embodiments, this treatment may be performed at a temperature in the range of 80°C to 100°C for 1 to 3 minutes. For example, the treatment may be performed at 90°C for 2 minutes. This treatment can form a hydrophilic coating.

[0120] In some embodiments, metal oxides or metals may be exposed to phosphates during a SAM (self-assembled monolayer) process. For example, using ammonium hydroxydodecyl phosphate (OH-DDPO4(NH4)2) in a SAM process can form a hydrophobic capping layer with a contact angle of about 110 degrees. In another example, using 12-hydroxydodecyl phosphate (OH-DDPO4) in a SAM process can form a hydrophilic capping layer with a contact angle of about 50 degrees. In still other examples, using a mixture of different phosphate ester compounds in a SAM process can form capping layers with different hydrophobicities ranging from 50 degrees to 110 degrees. Furthermore, suitable combinations of different phosphate esters can be used to form capping layers with different hydrophobicities, with contact angles ranging, for example, from 20 degrees to 130 degrees.

[0121] After forming a first capping layer on a metal oxide film or a metal film, a second capping layer may be selectively formed on a region of a second film region, such as inorganic silicon oxide. For example, a hydrophobic capping layer can be formed by treating the device in a hydrophobic silane (e.g., fluorinated alkylsilanes, dialkylsilanes, etc.). Alternatively, a hydrophilic capping layer can be formed by treating the device in a hydrophilic silane (e.g., silanes with hydroxyalkyl terminals, etc.). By appropriately selecting the silane compound, the second capping layer can be formed only on the second film region of, for example, inorganic silicon oxide, and not on the first capping layer already formed on the first film material. In addition to inorganic silicon oxide, the second film region may also contain materials such as silicon, silicon nitride, metal oxides, or combinations of these materials.

[0122] In the above process, alternating first thin film regions 1110 and second thin film layers 1120 are formed sequentially, such that the first thin film region 1110 is formed on the second thin film region 1120. In some other embodiments, the second thin film region 1120 may be formed on the first thin film region 1110, as referenced below. Figures 12A to 12C As shown in the image.

[0123] Figures 12A to 12C This is a cross-sectional view, which, according to an alternative embodiment of the present disclosure, illustrates the method for forming... Figure 10 A method for constructing a device structure with different surface regions.

[0124] Figure 12A A cross-sectional view of a device structure with alternating surface regions of thin film layer 1210 and thin film layer 1220 is shown. (Reference) Figure 12A A surface layer comprising a plurality of first thin film regions 1211 and a plurality of second thin film regions 1221 is formed on a substrate 1201.

[0125] Figure 12A The equipment structure in it is similar to Figure 11D In the device structure, the first thin film layer 1210 corresponds to Figure 11D The first thin film layer 1110, and the second thin film layer 1220 corresponding to Figure 11D The second thin film layer 1120 in the middle. Figure 11D Structure and Figure 12A One difference between the structures is that the first thin film layer 1210 is below the second thin film layer 1220. It can be used as follows: Figures 11A to 11C The similar process described herein forms the thin film layers in the reverse order. Figure 12A The equipment structure within.

[0126] Figure 12B A capping layer 1212 selectively formed on thin film region 1211 is shown. This selective capping layer is formed by exposing the device structure to a suitable first material such that capping layer 1212 is formed on the top surface of thin film region 1210, but not on the top surface of thin film region 1220. This processing and annealing process are similar to those used in [the following context, possibly related to a specific process or process]. Figure 11E The aforementioned processing technology and annealing process, wherein the covering layer 1212 corresponds to Figure 11E The overlay layer 1112 in the middle.

[0127] Figure 12C A second capping layer 1222 is shown selectively formed on the top surface of the second thin film region 1221. This selective capping layer is formed by exposing the device structure to a suitable second material such that the capping layer 1222 is formed on the top surface of the thin film region 1221, but not on the top surface of the thin film region 1211 having the capping layer 1212. This process is similar to bonding. Figure 11F The aforementioned processing technology. For example... Figure 12C As shown, the device structure 1200 includes a substrate 1201. A surface layer 1202 comprising a plurality of first thin film regions and a plurality of second thin film regions is disposed on the substrate 1201.

[0128] A first capping layer 1212 is formed on the top surface of the first thin film region 1211, while a second capping layer 1222 is formed on the top surface of the second thin film region 1221. In this embodiment, a differential surface layer is formed by alternating regions of the first capping layer 1212 and the second capping layer 1222.

[0129] In the alternative implementation plan, Figures 11A to 11F as well as Figures 12A to 12C The process shown can also be modified. For example, the order of surface layer formation can be reversed by appropriately selecting the thin film material and the compound used for surface treatment. In some embodiments, in Figures 11D to 11FIn the middle, you can first in Figure 11D A capping layer 1122 is formed on the thin film region 1121 in the structure, and subsequently a capping layer 1112 is formed on the thin film region 1111. Similarly, in Figures 12A to 12C In the middle, you can first in Figure 12A A capping layer 1222 is formed on the thin film region 1221 in the structure, and a capping layer 1212 is subsequently formed on the thin film region 1211.

[0130] Although the process described herein is based on a specific number of steps performed in a particular order, it is contemplated that additional steps not explicitly shown and / or described may be included. Furthermore, it is contemplated that fewer steps than 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). Additionally, it is contemplated that the steps described herein may be performed in a different order than that described.

[0131] For example, the first and second thin film regions can be one of metal, metal oxide, or silicon oxide. Although in the above examples, a capping layer formed on the metal oxide by means of phosphonic acid or phosphate ester is formed first, followed by a capping layer formed on the silicon oxide by means of a silane compound. In some embodiments, a first capping layer may be formed on the silicon oxide first with a silane, followed by a second capping layer formed on the metal oxide with phosphonic acid or phosphate ester. In some embodiments, the metal oxide is treated with phosphonic acid or phosphate ester followed by an annealing process as described above.

[0132] Example 5

[0133] This embodiment discloses a novel apparatus and method for mixing droplets with one or more reagents and delivering the mixed droplets to an EWOD array to obtain multiple microdroplets. In one embodiment, the microdroplets produced by the novel apparatus and method have a uniform size. The novel apparatus may be an integrated laboratory-on-a-chip device similar to 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 droplets, a reagent discharge region for receiving reagents, a mixing region for mixing droplets with reagents, and an array of IWOD devices for generating multiple samples of uniform size. In one embodiment, the apparatus may also include a temperature regulating element for performing conventional PCR or incubating the microdroplets at a predetermined temperature (isothermal PCR). In one embodiment, each IWOD region may have an array of raised (protruding) hydrophilic surface regions or spots (e.g., Figure 4A and 4C (as shown) or arrays of recessed hydrophilic surface regions or micropores (such as... Figure 4B and Figure 4D(As shown). In some embodiments, each spot or microwell includes one or more ISFETs for measuring the ion concentration or pH of the associated microdroplet. In one embodiment, the device may include an interface port configured to communicate with an external host that collects the measured pH of the sample and calculates the DNA concentration of the droplets.

[0134] It should be understood that the embodiments and implementations described herein are for illustrative purposes only, and various modifications or changes made thereto will be suggested by those skilled in the art and will be included within the spirit and scope of this application and the appended claims. All publications, patents, and patent applications cited herein are incorporated herein by reference in their entirety for all purposes.

[0135] It should be understood that the above description is intended to illustrate rather than limit. Many embodiments will be apparent to those skilled in the art upon reading the above description. Therefore, the scope of the invention should not be determined by reference to the above description, but rather by reference to the full scope of the appended claims and their equivalents.

[0136] While the foregoing disclosure illustrates illustrative aspects of this disclosure, it should be noted that various changes and modifications may be made herein without departing from the scope of this disclosure as defined by the appended claims. The functions, steps, and / or actions of the method claims according to aspects of this disclosure described herein do not need to be performed in any particular order. Furthermore, although elements of this disclosure may be described or claimed in the singular, plural forms are conceivable unless a limitation on the singular is expressly stated.

[0137] For all the flowcharts in this article, it should be understood that many steps can be combined, executed in parallel, or executed in different orders without affecting the functionality implemented.

Claims

1. An apparatus for forming a plurality of microdroplets from a droplet, the apparatus comprising: A first substrate having a first surface; A dielectric layer is located on the first surface of the first substrate and has a plurality of hydrophilic surface regions separated from each other by hydrophobic surfaces; Multiple electrodes, located within the dielectric layer, are configured to generate an electric field across the droplet in response to a voltage provided by a control circuit, thereby moving the droplet laterally across the upper surface of the dielectric layer while leaving portions of the droplet on the hydrophilic surface region to form the plurality of microdroplets on the hydrophilic surface region. Each of the plurality of microdroplets is disposed above the upper surface of the dielectric layer and protrudes into the channel.

2. The apparatus of claim 1, wherein the plurality of hydrophilic surface regions are arranged in an array of uniformly sized structures.

3. The apparatus of claim 2, wherein each of the structures protrudes above the upper surface of the dielectric layer.

4. The device according to claim 2, wherein the structure has a polygonal, circular or oval shape.

5. The apparatus of claim 2, wherein each of the structures is a groove below the upper surface of the dielectric layer, wherein each groove comprises a hydrophilic material having an upper surface flush with the upper surface of the dielectric layer.

6. The apparatus of claim 1, wherein the hydrophilic surface is flush with the surface of the hydrophobic gap region on the surface of the dielectric layer.

7. The apparatus of claim 1, further comprising a plurality of sensors associated with a counterpart in the microdroplet, wherein each sensor comprises: An ion-sensitive field-effect transistor includes an ion-sensing membrane configured to be exposed to microdroplets and to provide a signal associated with the concentration level of the microdroplets. as well as A reference electrode is configured to be fully or partially immersed in the microdroplet and spaced apart from the sensing membrane to provide a reference voltage to the microdroplet.

8. The apparatus of claim 1, further comprising a second substrate having a second surface facing the first surface of the first substrate and spaced apart from the first substrate by a spacer having a height, wherein the height of the spacer defines a channel for the droplet.

9. The apparatus of claim 8, further comprising a common electrode on the second surface of the second substrate, wherein the common electrode has a common voltage less than the voltage applied to one or more of the plurality of electrodes.

10. The apparatus of claim 9, wherein the voltage applied to one or more of the plurality of electrodes is a direct current (DC) voltage.

11. A method for forming a plurality of microdroplets from droplets on a substrate structure, comprising: A substrate structure is provided, comprising: a first substrate; a dielectric layer having a hydrophobic surface on the first substrate; control circuitry; a plurality of conductive wirings; and a plurality of electrodes communicating with the control circuitry via the conductive wirings and configured to form an electric field in response to a voltage supplied by the control circuitry; and The dielectric layer is configured to include: The first region is used to receive droplets; A second region, configured to receive one or more reagents, is connected to the first region and configured to mix the droplet with the one or more reagents to obtain a mixed droplet. A third region, which communicates with the second region and includes a plurality of hydrophilic surface regions spaced apart from each other by the hydrophobic surface, wherein, as the droplet moves above the hydrophilic surface regions, a portion of the droplet forms a plurality of microdroplets on the hydrophilic surface regions. Each of the plurality of microdroplets is disposed above the upper surface of the dielectric layer and protrudes into the channel.

12. The method of claim 11, further comprising configuring the plurality of electrodes to move the microdroplets above the upper surface of the dielectric layer.

13. The method of claim 12, further comprising arranging the plurality of hydrophilic surface regions in an array of uniformly sized structures, wherein each structure protrudes above the hydrophobic surface of the dielectric layer.

14. The method of claim 13, wherein the structure has a polygonal, circular, or oval shape.

15. The method of claim 13, wherein each of the structures is a groove below the upper surface of the dielectric layer, wherein each groove comprises a hydrophilic material having an upper surface flush with the upper surface of the dielectric layer.

16. The method of claim 11, wherein the hydrophilic surface is flush with the surface of the hydrophobic gap region on the surface of the dielectric layer.

17. The method of claim 11, wherein the substrate structure further comprises a second substrate and a conductive layer located on the second substrate and facing the hydrophobic surface of the dielectric layer, and the space between the conductive layer and the dielectric layer forms a channel for the droplet.

18. The method of claim 17, wherein the conductive layer operates as a common electrode, and the plurality of electrodes comprises a first parallel strip array along a first direction from the first region to the third region and a second parallel strip array located in the second region and along a second direction perpendicular to the first direction.

19. The method of claim 11, wherein the third region further comprises one or more sensors associated with a counterpart of one of the plurality of hydrophilic surface regions, wherein the sensors comprise: An ion-sensitive field-effect transistor includes an ion-sensing membrane configured to be exposed to microdroplets and to provide a signal associated with the concentration level of the microdroplets. as well as A reference voltage electrode is configured to be fully or partially immersed in the microdroplet and spaced apart from the sensing membrane to apply a reference voltage to the microdroplet.

20. The method of claim 11, further comprising configuring the dielectric layer to include a fourth region connected to the third region and configured to collect the remaining portion of the droplet after the remaining portion of the droplet has moved across the third region.

21. A method for forming a plurality of microdroplets having substantially uniform size from a droplet, the method comprising: A substrate structure is provided, the substrate structure comprising: a substrate; A dielectric layer located on the substrate; Multiple electrodes are located in the dielectric layer, wherein the dielectric layer has a hydrophobic surface and multiple hydrophilic surface regions spaced apart from each other by the hydrophobic surface; The droplets are discharged onto the dielectric layer; A time-varying voltage waveform is applied to the electrode to move the droplet across the hydrophilic surface region of the dielectric layer to form the plurality of microdroplets on the hydrophilic surface region. Each of the plurality of microdroplets is disposed above the upper surface of the dielectric layer and protrudes into the channel.

22. The method of claim 21, further comprising: The droplet is mixed with one or more reagents to obtain a mixed droplet, and then the mixed droplet is moved across the hydrophilic surface region of the dielectric layer.

23. The method of claim 21, further comprising: Provide one or more sensors associated with one of the hydrophilic surface regions, wherein the sensors include: An ion-sensitive field-effect transistor includes an ion-sensing membrane and a reference electrode, the ion-sensing membrane and the reference electrode being configured to be immersed in a sample and provide a signal correlated with the concentration level of the sample; A first voltage source applies voltage to the reference electrode; and A second voltage source is applied to the source and drain of the ion-sensitive field-effect transistor.

24. The method of claim 21, wherein each of the hydrophilic surface regions protrudes above the upper surface of the dielectric layer.

25. The method of claim 21, wherein the hydrophilic surface region is a groove, each groove comprising a hydrophilic material having an upper surface flush with the upper surface of the dielectric layer.

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