Splitting droplets on electrowetting devices on dielectrics

Abstract

A method of operating a set of electrodes on an on-dielectric electrowetting device is described. The on-dielectric electrowetting device includes: a central electrode (75), a first electrode (77) adjacent to the central electrode, a second electrode (78) adjacent to the central electrode, a third electrode (85) adjacent to the first electrode, the third electrode having an area larger than the central electrode, and a fourth electrode (86) adjacent to the second electrode, the fourth electrode having an area larger than the central electrode. The method includes activating the central electrode, the first, second, third, and fourth electrodes to cause a first droplet (41) to remain above the central electrode, the first, second, third, and fourth electrodes, wherein the first droplet is confined above the central electrode. The method also includes deactivating the first and second electrodes to cause the first droplet to divide into second, third, and fourth droplets (42, 43, 3) that are respectively retained above the third, fourth, and central electrodes.

Description

Technical Field

[0001] The present invention relates to a method for dividing liquid droplets on an on-dielectric electrowetting device. Background Art

[0002] Electrowetting on dielectric (EWOD) is a unique technology in digital microfluidics for lab-on-a-chip (LoC) systems that has the potential to manipulate small volumes of fluid samples, also known as droplets. EWOD devices can serve as a precursor to complex sample processing workflows for other LoCs.

[0003] An introduction to electrowetting is given by F. Mugele and J.C. Baret: “Electrowetting: from basics to applications”, Journal of Physics: Condensed Matter, Vol. 17, p. R705 (2005), and a review of electrowetting devices on dielectrics is given by Nelson & C-J Kim: “Droplet Actuation by Electrowetting-on-Dielectric (EWOD): A Review”, Journal of Adhesion Science and Technology, Vol. 26, pp. 1747-1771 (2012), which are incorporated herein by reference.

[0004] Single-cell whole-genome amplification (scWGA) can be applied to nucleic acids in single cells before single-cell whole-genome sequencing (scWGS) can be performed. scWGS is used in a variety of applications where it is desirable to assess genetic variation between cells. For example, copy number variations, alterations, and single nucleotide variants (SNVs) can be assessed within a single cell.

[0005] However, scWGA is challenging due in part to the low copy number of nucleic acids present in each cell and the rarity of genomic variation within a cell population. Existing methods for scWGA have low throughput and require complex and expensive tracking and identification procedures, such as individually coding tubes.

[0006] Cells intended for scWGA and / or scWGS are typically prepared in a suspension containing a plurality of cells. This suspension is typically injected or introduced into an on-medium electrowetting device, thereby forming droplets within the device. Droplets containing the cell suspension may have a volume that is difficult to manipulate on an on-medium electrowetting device. Furthermore, identifying and tracking droplets at the required number of cells can be challenging, requiring expensive barcodes to be attached to the sample. Furthermore, the barcode may be damaged during processing, potentially rendering the sample useless. Summary of the Invention

[0007] Disclosed herein is a method for operating an electrowetting device (2) on a dielectric, the electrowetting device comprising a central electrode (75); a first conical electrode (77) adjacent to the central electrode; a second conical electrode (78) adjacent to the central electrode; a first side electrode (85) adjacent to the first conical electrode, the first side electrode having a larger area than the central electrode; and a second side electrode (86) adjacent to the second conical electrode, the second side electrode having a larger area than the central electrode. The method comprises: activating the central electrode, the first conical electrode, the second conical electrode, the first side electrode, and the second side electrode so as to cause a first droplet (41) to be held on the central electrode, the first conical electrode, the second side electrode, and the second side electrode. a second conical electrode, the first side electrode, and the second side electrode, wherein the first droplet is confined above the central electrode; deactivating the first conical electrode and the second conical electrode to cause the first droplet to divide into a second droplet (42), a third droplet (43), and a sample droplet (3) held above the first side electrode, the second side electrode, and the central electrode, respectively; the volumes of the second conical electrode, the first side electrode, and the second side electrode are related to the areas of the first side electrode, the second side electrode, and the central electrode, respectively; the volume of the sample droplet is smaller than the volume of the second droplet; the volume of the sample droplet is smaller than the volume of the third droplet; the first side electrode and the second side electrode may have an area five times larger than the central electrode. The first side electrode and the second side electrode may have an area one hundred times larger than the central electrode. The first conical electrode and the second conical electrode may have an area between the area size of the central electrode and the area size of one of the first side electrode or the second side electrode; the electrodes in the device may be coplanar; the first conical electrode and the second conical electrode may be arranged relative to each other. The first conical electrode and the second conical electrode can be arranged opposite to each other in the first direction; the first side electrode and the second side electrode can be arranged opposite to each other; the first side electrode and the second side electrode can be arranged opposite to each other in the first direction; the central electrode, the first conical electrode, the second conical electrode, the first side electrode and the second side electrode can be arranged in a row in the first direction; that is, the electrodes are arranged in a row in the first direction in the order of the first side electrode, the first conical electrode, the central electrode, the second conical electrode and then the second side electrode; the arrangement of the electrodes can be symmetrical using a symmetry line passing through the central electrode.

[0008] In one embodiment, the device further comprises: a first convex electrode (70) adjacent to the central electrode; a second convex electrode (71) adjacent to the central electrode; wherein the method further comprises: activating the central electrode, the first conical electrode, the second conical electrode, the first side electrode and the second side electrode so as to cause a first droplet (41) to be held above the activated central electrode, the activated first conical electrode, the activated second conical electrode, the activated first side electrode and the activated second side electrode; activating the central electrode, the first conical electrode, the second conical electrode, the first convex electrode and the second convex electrode so as to cause a droplet (4) to be held above the central electrode, the first conical electrode, the second conical electrode, the first convex electrode and the second convex electrode; deactivating the first convex electrode and the second convex electrode; and activating the first side electrode and the second side electrode; the first convex electrode and the second convex electrode may be arranged relative to each other; the first convex electrode and the second convex electrode may be arranged relative to each other in a second direction that is not parallel to the first direction; the second direction may be perpendicular to the first direction.

[0009] In one embodiment, the method further comprises: determining the number of particles (21) in the sample droplet held above the central electrode using a sensor; in response to the number of particles in the sample droplet being outside a predefined range: activating at least the first conical electrode and the second conical electrode to cause the second droplet, the third droplet, and the sample droplet to merge into a single droplet (4); the on-dielectric electrowetting device may further comprise a seventh electrode line (83), wherein at least one seventh electrode is adjacent to the central electrode; the method further comprises: in response to the number of particles in the sample droplet being within a predefined range: activating and deactivating electrodes in the seventh electrode line to move the sample droplet to a further processing portion; the at least one seventh electrode adjacent to the central electrode may be at one end of the line. The method further comprises activating electrodes to cause a first process on the sample droplet to be moved to the first processing portion, wherein the first process is a thermally regulated process; the first process may be single cell nucleic acid amplification; the first process may be cell culture.

[0010] In one embodiment, the electrowetting device on a dielectric comprises a plurality of central electrodes, the method further comprising: wherein deactivating the first conical electrode and the second conical electrode to cause the first droplet to divide into a second droplet (42), a third droplet (43) and a sample droplet (3) comprises: for each given central electrode of the plurality of central electrodes: determining, using a sensor, the number of particles (21) in the first droplet held above the given central electrode; marking the given electrode in response to the number of particles (21) in the first droplet held above the given central electrode being within a predefined range; unmarking any central electrode that is marked and is adjacent to another marked central electrode; and simultaneously deactivating the first conical electrode and the second conical electrode upon deactivating the unmarked central electrode, causing the first droplet to divide into second droplets (42) held above the first side electrode and the second side electrode. ) and a third droplet (43), and a sample droplet (3) held above each electrode in the marked central electrode; the electrowetting on the medium may also include a seventh electrode line (83), wherein at least one seventh electrode is adjacent to the central electrode; the method may also include: in response to the number of particles in the sample droplet being within a predefined range: activating and deactivating the electrodes in the seventh electrode line to move the sample droplet to a further processing portion; the seventh electrode line may divide the first convex electrode and / or the second convex electrode into multiple parts; the seventh electrode line may be used to transfer one or more droplets or samples out of the arrangement of the droplet dividing electrodes; such an arrangement may allow droplets or samples to be moved, for example, to multiple further processing portions (for example, to be circulated) or to an extraction electrode; the further processing portion may be on the same electrowetting on the medium device or it may be on a different device.

[0011] In one embodiment, wherein the number of particles in the droplet is determined by capturing an image of the droplet held above an electrode using a camera (22) and analyzing the image using machine vision in a processor; the particles may be biological cells; this may allow droplets containing individual biological cells to be identified. In one embodiment, wherein the electrowetting on dielectric device has a region of tessellated electrodes (89), wherein at least one of the electrodes in the electrowetting on dielectric device comprises a group of tessellated electrodes within the tessellated electrodes, wherein the groups of electrodes are activated or deactivated simultaneously; this may enable the electrowetting on dielectric device comprising an array of connected electrodes, which may also be a tessellated array of electrodes, to perform methods that would otherwise require a device comprising electrodes having a specifically designed and manufactured shape; the electrodes in the tessellated array of electrodes may be square, rectangular, triangular, hexagonal, or octagonal.

[0012] A computer program (16) is disclosed which, when executed by at least one processor, causes the at least one processor to perform a method of operating an on-medium electrowetting device.

[0013] A computer program product is disclosed, comprising a computer-readable medium storing the computer program according to the invention.

[0014] Disclosed is an apparatus comprising: an electrowetting device on a dielectric (2); and a controller (8); the electrowetting device on a dielectric comprises a central electrode (75); a first conical electrode (77) adjacent to the central electrode; a second conical electrode (78) adjacent to the central electrode; a first side electrode (85) adjacent to the first conical electrode, the first side electrode having a larger area than the central electrode; a second side electrode (86) adjacent to the second conical electrode, the second side electrode having a larger area than the central electrode; the controller is configured to activate the central electrode, the second conical electrode, the second side electrode, the first side electrode, the second ... a first conical electrode, a second conical electrode, a first side electrode and a second side electrode so as to cause a first droplet (41) to be held above the central electrode, the first conical electrode, the second conical electrode, the first side electrode and the second side electrode, wherein the first droplet is confined above the central electrode; and deactivating the first conical electrode and the second conical electrode so as to cause the first droplet to be divided into a second droplet (42), a third droplet (43) and a sample droplet (3) which are respectively held above the first side electrode, the second side electrode and the central electrode.

[0015] In one embodiment, the on-dielectric electrowetting device further comprises: a first convex electrode (70) adjacent to the central electrode; a second convex electrode (71) adjacent to the central electrode; the controller is further configured to: wherein, activate the central electrode, the first conical electrode, the second conical electrode, the first side electrode and the second side electrode so as to cause the first droplet (41) to be held above the activated central electrode, the activated first conical electrode, the activated second conical electrode, the activated first side electrode and the activated second side electrode; activate the central electrode, the first conical electrode, the second conical electrode, the first convex electrode and the second convex electrode so as to cause the droplet (4) to be held above the central electrode, the first conical electrode, the second conical electrode, the first convex electrode and the second convex electrode; deactivate the first convex electrode and the second convex electrode; and activate the first side electrode and the second side electrode.

[0016] In one embodiment, the apparatus further comprises: a sensor configured to determine a number of particles (21) within a droplet; and the controller, in response to the number of particles in the sample droplet being outside a predefined range, configured to: activate at least the first conical electrode and the second conical electrode to cause the second droplet, the third droplet, and the sample droplet to merge into a single droplet (4).

[0017] In one embodiment, the apparatus further comprises: a sensor configured to determine the number of particles within the droplet; wherein the on-dielectric electrowetting device further comprises a plurality of central electrodes; the controller further configured to: wherein deactivating the first conical electrode and the second conical electrode to cause the first droplet to separate into a second droplet (42), a third droplet (43) and a sample droplet (3) comprises: for each given central electrode of the plurality of central electrodes: determining the number of particles (21) in the first droplet held above the given central electrode using the sensor; marking the given electrode in response to the number of particles (21) in the first droplet held above the given central electrode being within a predefined range; unmarking any central electrode that is marked and adjacent to another marked central electrode; and deactivating the first conical electrode and the second conical electrode simultaneously with deactivating the unmarked central electrode, causing the first droplet to separate into a second droplet (42) and a third droplet (43) held above the first side electrode and the second side electrode, and a sample droplet (3) held above each of the marked central electrodes.

[0018] In one embodiment, the apparatus further comprises: a processor; wherein the sensor is a camera (22), and wherein the number of particles in the droplet is determined by capturing an image of the droplet held above an electrode using a camera and analyzing the image using machine vision in the processor.

[0019] In one embodiment, the electrowetting device on the dielectric has a region of tessellated electrodes (89), wherein at least one of the electrodes in the electrowetting device on the dielectric comprises a group of tessellated electrodes within the tessellated electrodes; and the controller is further configured to simultaneously activate or deactivate the group of electrodes.

[0020] A system is disclosed, comprising the apparatus described in the preceding embodiments; and a computer system (11) operably connected to the controller, the computer system being configured to execute the method of any one of claims 1 to 8 and record the position of at least one droplet. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Specific embodiments of the present invention will now be described, by way of example, with reference to the accompanying drawings, in which:

[0022] Figure 1 is a schematic block diagram of a microfluidic system including an on-die electrowetting droplet manipulation device;

[0023] Figure 2 is a schematic block diagram of a sample inspection system including a camera and a lens;

[0024] Figure 3 is a schematic block diagram of a thermal control system including an electrowetting droplet manipulation device on a dielectric;

[0025] Figure 4 is a cross-sectional view of an electrowetting device on a dielectric;

[0026] Figure 5 Droplet actuation is schematically shown;

[0027] Figure 6 A plan view of the first set of droplet partitioning electrodes for sample preparation and single-cell amplification on an on-dielectric electrowetting device.

[0028] Figure 7 is a plan view of a second set of droplet partitioning electrodes on an electrowetting-on-dielectric device for sample preparation and single-cell amplification;

[0029] Figure 8 A plan view of the third set of droplet partitioning electrodes on an electrowetting-on-dielectric device for sample preparation and single-cell amplification.

[0030] Figure 9 Plan view of the fourth set of droplet partitioning electrodes on an electrowetting-on-dielectric device for sample preparation and single-cell expansion.

[0031] Figure 10 is a process flow diagram of a first method of droplet partitioning and recycling;

[0032] Figure 11 Shown using Figure 6 The first set of droplet dividing electrodes shown in FIG. Figure 10 The droplet division stage of the first method is shown in FIG;

[0033] Figure 12A shows the use of the fifth set of droplet partitioning electrodes and Figure 10 The first stage of droplet division of the first method is shown;

[0034] Figure 12B shows the use of the fifth set of droplet partitioning electrodes and Figure 10 The second stage of droplet division of the first method shown;

[0035] Figure 13A shows the use of the sixth set of droplet partitioning electrodes and Figure 10 The first stage of droplet division of the first method is shown;

[0036] Figure 13B shows the use of the sixth set of droplet partitioning electrodes and Figure 10 The second stage of droplet division of the first method shown;

[0037] Figure 14 is a process flow diagram of a second method of droplet partitioning and recycling;

[0038] Figure 15 Shown using Figure 6 The first set of droplet dividing electrodes shown in FIG. Figure 14 The droplet division stage of the second method shown in FIG;

[0039] Figure 16 Shown using Figure 8 The third set of droplet dividing electrodes shown in FIG. Figure 14 The droplet division stage of the second method shown in FIG;

[0040] Figure 17 Shown using Figure 9 The fourth set of droplet dividing electrodes shown in FIG. Figure 14 The droplet division stage of the second method shown in FIG;

[0041] Figure 18 is a plan view of the seventh set of droplet partitioning electrodes on an electrowetting-on-dielectric device for sample preparation and single-cell amplification;

[0042] Figure 19 is a plan view of the eighth set of droplet partitioning electrodes on an electrowetting-on-dielectric device for sample preparation and single-cell amplification;

[0043] Figure 20 is a process flow diagram of a third method of droplet partitioning and recycling;

[0044] Figure 21 Shown using Figure 18 The seventh set of droplet dividing electrodes shown in FIG. Figure 20 The droplet division stage of the third method shown in FIG;

[0045] Figure 22 Shown using Figure 19 The eighth set of droplet dividing electrodes shown in FIG. Figure 20 The droplet division stage of the third method shown in FIG;

[0046] Figure 23 Shown using Figure 19The eighth set of droplet dividing electrodes shown in FIG. Figure 20 The droplet division stage of the second method shown in FIG;

[0047] Figure 24 is a plan view of a first on-medium electrowetting device for preparing a sample for nucleic acid amplification;

[0048] Figure 25 is a plan view of a second on-medium electrowetting device for preparing a sample for nucleic acid amplification;

[0049] Figure 26 It is a process flow chart of the method for single cell expansion;

[0050] Figure 27A shows the use of the ninth set of droplet partitioning electrodes and Figure 10 The first stage of droplet division of the first method is shown;

[0051] Figure 27B shows the use of the ninth set of droplet partitioning electrodes and Figure 10 The second stage of droplet partitioning of the first method is shown. DETAILED DESCRIPTION

[0052] Microfluidic systems

[0053] refer to Figure 1 A microfluidic system 1 for preparing a sample is shown. The microfluidic system 1 comprises an electrowetting on medium (EWOD) device 2 (also referred to as a "droplet manipulation device", or simply "device") for preparing sample droplets 3 from droplets 4 and moving the sample droplets 3 to a further processing portion 6. The electrowetting on medium device 2 comprises electrodes 7. The microfluidic system 1 may also comprise a fluid handling system (not shown) for delivering a fluid to the electrowetting on medium device 2. The microfluidic system 1 further comprises a controller 8, for example in the form of a microcontroller or a single board computer, for controlling the fluid handling system (not shown) and at least one electrode driver 9. The one or more electrode drivers 9 are connected to the electrodes 7 in the electrowetting on medium device 2, and the one or more electrode drivers 9 are arranged to control the application of or the elimination of deviations from one or more specific electrodes 7.

[0054] System 1 includes a computer system 11 that can be used to control controller 8. Computer system 11 includes at least one processor 12 and memory 13 that stores a droplet control program 15 and a computer program 16. Each of droplet control program 15 and computer program 16 can take the form of one or more scripts (not shown) and / or a series of tables (not shown), one table storing a list of active electrodes 7 for each frame. In some cases, controller 8 can store droplet control program 15 and computer program 16. Thus, computer system 11 can be omitted, at least during manipulation. Further details of how to control electrode movement and manipulate sample droplets 3 and 4 are detailed in British Patent No. 2,559,216 B, particularly on page 4, line 20 to page 7, line 24.

[0055] Sample inspection system

[0056] Additional references Figure 2 The microfluidic system 1 may further include a sample inspection system 20. The sample droplets 3 and / or 4 may include or contain multiple fluids, such as, for example, saline buffer, biological samples (such as DNA or protein), or body fluids (such as blood or urine). The sample droplets 3 and / or 4 may include or contain more than one type of fluid. The sample droplets 3 and / or 4 may contain one or more particles 21. The particles 21 may be, for example, artificial cells or biological cells, such as eukaryotic cells or prokaryotic cells, cell nuclei, or organelles. The sample inspection system 20 may be used to determine the number of particles 21 within the sample droplet 3.

[0057] The volume of the droplet 4 and / or sample droplet 3 can vary from, for example, 10 μl to 1 ml. The volume of the droplet 4 introduced into the electrowetting device 2 on the medium depends on the number and volume of sample droplets 3 to be prepared by the device. Typically, the first, or initial, droplet 41 introduced into the electrowetting device 2 on the medium will be 1000 times larger than the desired volume of the sample droplet 3, enabling approximately 1000 sample droplets 3 to be prepared from one droplet 4. However, the ratio between the volume of the droplet 4 and the volume of the sample droplets 3 can be larger or smaller, depending on the desired number and volume of the sample droplets 3. For example, if the desired volume of the sample droplet 3 is 25 nanoliters, the volume of the droplet 4 will be greater than 25 microliters. The diameter of the droplet 4 or sample droplet 3 will depend on the volume of the droplet 4 or sample droplet 3, respectively. The diameter of the droplet 4 or sample droplet 3 can typically be between 50 and 1000 micrometers, or more typically between 100 and 500 micrometers.

[0058] The sample inspection system 20 includes a camera 22, a lens 23, an illumination source 24, a processor 25 for processing the output from the camera 22, a memory 27 for storing images or output data, and image analysis / particle counting software 28 for analyzing images and / or counting the number of particles 21 within the sample droplets 3 or 4. The illumination source 24 can be integrated into the camera 22 or arranged separately from the camera 22 and the rest of the system 20. The sample inspection system 20 can also include at least one output device 30. Alternatively, the camera 22 can be operably connected to the computer system 11, and the image analysis / particle counting software 28 stored in the computer system memory 13. The memory 13 can also store images 31 and output data from the image analysis performed. The software 28 can be a set of instructions that can take the form of a script (not shown).

[0059] A lens 23 is used to focus the camera 22 on at least a portion of a sample droplet 3. An illumination source 24 is used to illuminate at least a portion of the sample droplet 3. The camera 22 then captures an image 31 of at least a portion of the sample. The image 31 is then transferred to a memory 13, 27. The image 31 is then analyzed in a processor using software 28. The software 28 determines the number of particles 21 in at least a portion of the sample droplet 3. Machine vision methods are used to isolate and count the individual particles 21 in at least a portion of the sample droplet 3. Various machine vision methods can be used, such as thresholding, edge detection, color analysis, blob detection, and pattern recognition. Neural network processing, deep learning, and / or machine learning can also be used in conjunction with the machine vision methods to identify and count the number of particles 21 in at least a portion of a sample droplet 3.

[0060] The sample inspection system 20 may also include a device (not shown), such as a motorized X, Y, Z camera stage, to move the camera 22 and lens 23 to focus on different portions of the sample droplet 3. The camera 22 and lens 23 are moved to focus on different portions of the sample droplet 3 until an entire sample droplet 3 has been captured and analyzed. Alternatively, the camera 22 and lens 23 are positioned to focus on a single, intact sample droplet 3 or multiple, intact sample droplets 3.

[0061] After the sample inspection system 20 has determined the number of particles 21 within the sample droplet 3, the system 20 will output at least a binary response indicating a positive or negative evaluation of the sample droplet 3 based on the user input. For example, if the user wishes to isolate a sample droplet 3 containing only a single particle 21, the system 20 will output a positive response if the sample droplet 3 contains one particle 21, and a negative response if the sample droplet 3 contains fewer or more than one particle 21. The user control and / or stored droplet control program 15 can use the positive or negative output to determine the next step. For example, depending on the output, the sample can be moved to further processing section 6. Alternatively, the sample inspection system 20 can output the number of particles 21 identified within the sample droplet 3. The number of particles 21 can then be used to determine how to further process the sample droplet 3. For example, if the number of particles 21 identified within the sample droplet 3 is greater than one, the sample droplet 3 can be recombined with a larger droplet 4. If no particles 21 are present in the sample droplet 3, the sample droplet 3 can be moved to waste. Regardless of the number of particles 21 identified within each sample droplet 3, all sample droplets 3 can be passed to the further processing section 6. The number of particles 21 in each sample droplet 3 will be recorded together with the position of the sample droplet 3 on the on-medium electrowetting device 2. The number of particles 21 within each sample droplet 3 can be used to determine the action of subsequent steps, for example, whether to extract the nucleic acid sequence of the droplet or whether to discard it to waste.

[0062] The sample inspection system 20 can also be used to assess the condition of the sample droplet 3 during or after further processing in the further processing section 6. For example, the camera 22 can be sensitive to fluorescence emitted from the sample droplet 3. The sample inspection system 20 can then assess the stage of the reaction or process occurring within the sample droplet 3. For example, the sample droplet 3 may include nucleic acid containing a non-specific fluorescent dye embedded in double-stranded DNA, and the sample droplet 3 may be subjected to a thermal cycling quantitative polymerase chain reaction (qPCR). An image 31 of the sample droplet 3 undergoing the qPCR can be processed in a processor using sample analysis software 32 to determine the level of fluorescence emitted from the nucleic acid within the sample droplet 3. The fluorescence level measured from the image 31 can be used to assess the amount of nucleic acid that has been amplified in the sample droplet 3. The level of fluorescence can determine the stage of the reaction and, therefore, how long, or more, how many thermal cycles, the sample droplet 3 needs to complete before the reaction is complete.

[0063] The sample inspection system 20 used to evaluate the sample condition during or after further processing can be the same sample inspection system 20 that evaluated the sample droplet 3 before further processing, or it can be a second, different sample inspection system. Each sample inspection system 20 can have more than one camera 22, lens 23, and illumination source 24.

[0064] Thermal control system

[0065] refer to Figure 3 The microfluidic system 1 may further include a thermal control system 34. The thermal control system 34 includes a thermal controller 35 that controls the temperature of one or more thermally controlled areas 36 in the electrowetting-on-dielectric device 2. The thermally controlled areas 36 are temperature-regulated in a suitable manner (e.g., by using heating or cooling pads, microwaves, or chemical heating). The thermally controlled areas also include temperature sensors (not shown) that can feed temperature information back to the thermal controller 35. The thermal controller 35 may be operably connected to the computer system 11. Thermal control software 37 may also be included in the memory 13.

[0066] The thermally controlled zones 36 may include one or more electrodes 7. Each thermally controlled zone 36 may be individually controlled to a temperature required for a particular process, for example, a thermally controlled zone 36 may be set and adjusted to a temperature that facilitates a portion of a thermal cycling reaction, such as a polymerase chain reaction (PCR).

[0067] Electrowetting on dielectric devices

[0068] refer to Figure 4 The electrowetting-on-dielectric device 2 comprises a substrate 40 having first and second sides 41, 42 and a perimeter 43. The substrate 40 is generally in the form of a sheet and is longer in first and second perpendicular principal axes than in a third axis perpendicular to the plane of the first and second axes.

[0069] An array of electrodes 7 is embedded on a first side 41 of the substrate. Each electrode 7 has a front face 45 and a rear face 46. The electrode front face 45 is typically flat. Each electrode front face 45 may be flush with the first side 41 of the substrate. The electrodes 7 may be coplanar, or the electrodes 7 may be on more than one plane. The electrodes 7 are connected to the electrode driver 9 by electrode connectors, and electrode connectors 47 are attached to the electrode rear faces 46 of the electrodes 7. The electrode connectors 47 pass from the electrode rear faces 46 of the electrodes 7 through the substrate 40 to the second side 42 of the substrate 40. Each electrode connector 47 may be flush with the second side 42 of the substrate 40. Each electrode 7 connected to the electrode connector 47 and the electrode driver 9 herein may also be referred to as a "pixel."

[0070] A dielectric layer 50 having first and second sides 51, 52 is disposed on the first side 41 of the substrate and the front face 45 of the electrode 7. The first side 51 of the dielectric layer 50 is positioned adjacent to the first side 41 of the substrate 40 and the front face 45 of the electrode 7. The second side 52 of the dielectric layer 50 has a hydrophobic coating 53, thereby providing a surface for removal of sample droplets 3 and / or droplets 4. A microfluidic structure 55 is positioned adjacent to the perimeter 43 of the substrate 40 from the second side 42 of the substrate 40 and extends beyond the dielectric layer 50 and the hydrophobic coating 53. The microfluidic structure 55 has first and second sides 56, 57. A lid or cover 58 having first and second sides 59, 60 is disposed on the microfluidic structure 55 so that the second side 60 of the lid 58 faces the first side 56 of the microfluidic structure 55.

[0071] The cover 58 comprises a material that can be used as a substrate in the field of microconductors. For example, the cover 58 can comprise any one of poly(methyl methacrylate) (PMMA), polyethylene terephthalate (PET), polyethylene naphthalate (PEN), polystyrene (PS), or polyimide (PI), or any combination thereof. The cover 58 can comprise a glass material. The second side 60 of the cover 58 can be coated with a conductive material 62 and a hydrophobic material 63. The conductive material 62 can also be hydrophobic. The coated conductive material 62 and / or the coated hydrophobic material 63 can take the form of a thin film.

[0072] Conductive material 62 can be opaque or transparent. Conductive material 62 can be a transparent conductive oxide (TCO). Conductive material 62 can be cadmium tin oxide (CTO). Conductive material can be indium tin oxide (ITO). Hydrophobic material 63 coating second side 60 of cover 58 can be a fluorite material. Hydrophobic material 63 can be an amorphous fluoropolymer, such as polytetrafluoroethylene (PTFE or Teflon™) or CYTOP™.

[0073] The second side 60 of the cover 58 is placed on the first side 59 of the microfluidic structure 55, leaving a space 64 for the droplet 4 and / or sample droplet 3 to move in. The space 64 is typically one-tenth the width of the narrowest electrode 7, but can be between one-third and one-twentieth. The electrode driver 9 and the conductive coating of the cover 58 are connected to ground 65.

[0074] Each electrode 7 is arranged adjacent to at least one other electrode 7. The electrodes 7 can be arranged to form a continuous electrode group. As will be described in more detail later, the continuous electrode group can be an array of checkerboard electrodes. The continuous electrode group can be arranged and controlled in a manner that allows them to work as if they were a single electrode 7. Each electrode 7 in a continuous electrode 7 group can have a boundary, and the conductive parts of the electrodes 7 in a group of electrodes can not be adjacent to each other, that is, there can be a gap (e.g., <1 mm, or typically between 2 microns and 50 microns) between the conductive parts of adjacent electrodes 7.

[0075] Droplet actuation

[0076] refer to Figure 5 , the process of actuating a sample droplet 3 and / or a droplet 4 from the first electrode 71 to the fifth electrode 75 across the three center electrodes 72, 73, and 74 in a row of five electrodes 7 will now be described. The droplet 4 and / or sample droplet 3 can be moved (or "actuated") along a transportable path, in other words, along a path whose route can be selectively set and changed. As will be described in more detail later, a route can be defined as a sequence of coordinates, such as Cartesian coordinates (x, y, z) or three-axis coordinates (a, b, c), a vector, or other route-defining parameters stored in a table, script, or other suitable computer-readable data structure. Manipulation of the sample droplet 3 can also take the form of two or more droplets 4 being merged into a single droplet 4, or conversely, a single droplet 4 being split into two or more droplets 4. A series of droplet manipulations can be performed. The position of each droplet 4 on the on-media electrowetting device 2 can be recorded as it moves over the droplet 4.

[0077] The electrodes 7 are individually controllable and can be in an activated state (e.g., an activated state in which a positive bias is applied) or in an inactivated state (e.g., a state in which the electrodes 7 are grounded or ungrounded). Activating the electrodes 7 causes the droplet 4 or sample droplet 3 to be held in position above the electrodes 7. The hydrophobic coating 53 of the electrowetting device 2 on the dielectric means that the droplet 4 does not substantially cover the inactivated electrodes 7. In step S1, the electrode driver 9 (under the control of the controller 8) deactivates the first, third, fourth, and fifth electrodes 71, 73, 74, 75 and activates the second electrode 72. In step S2, the electrode driver 9 activates the third electrode 73 and deactivates the second electrode 72, causing the droplet 4 to move from above the second electrode 72 to above the third electrode 73. Finally, in step S3, the electrode driver 9 activates the fourth electrode 74 and deactivates the third electrode 73, causing the droplet 4 to move from above the third electrode 73 to above the fourth electrode 74. When the electrode driver 9 activates an electrode 7 or a continuous group of electrodes 7, they may remain activated, causing the droplet 4 to be held above the electrode 7. The electrode 7 remains in this state until the electrode driver 9 deactivates the electrode 7. Likewise, the electrode 7 will remain in an inactive state until the electrode driver 9 deactivates the electrode 7. The electrode 7 may also be activated for a short period of time (e.g., when rapidly actuating a droplet 4 and / or a sample droplet 3 across the hydrophobic coating 53 of the electrowetting device 2 on the medium). Instructions for moving one or more droplets 4 along a path or route may be stored in a stored droplet control program 15.

[0078] The array of electrodes 7 can be controlled in this manner to actuate sample droplets 3 and / or droplets 4 on the surface of the hydrophobic coating 53 in the electrowetting-on-media device 2. The state of each electrode 7 can be controlled manually (i.e., by a user controlling the activation and deactivation of the electrode in real time) or programmed to a predefined path that will control the movement of the electrode above the hydrophobic coating 53. The control of the electrodes 7 can be integrated with other systems (such as sensors, cameras, and processors that can process data from the electrowetting-on-media device 2 before feeding back into a program that determines the next movement of one or more sample droplets 3 and / or droplets 4).

[0079] Droplet partitioning electrode

[0080] refer to Figure 6, a first set of droplet-splitting electrodes 691 on a dielectric electrowetting device for preparing sample droplets 3 includes first and second convex electrodes 70, 71 having a first convex edge 73 and a second convex edge 74 facing each other and spaced apart at a certain distance along a first direction 72. In the first set of droplet-splitting electrodes 691, the first and second convex electrodes 70, 71 are semicircular in shape. The curved edges of the semicircular shape form the first convex edge 73 and the second convex edge 74. This arrangement forms a waist-shaped (also referred to as a "waisted") area between the first and second convex electrodes 70, 71. The first convex edge 73 and the second convex edge 74 of the first and second convex electrodes 70, 71 may include a plurality of continuous edges forming the first convex edge 73 and the second convex edge 74.

[0081] A square central electrode 75 is disposed in the waist-shaped region between the first and second convex electrodes 70 and 71. There may be more than one central electrode 75. If there is more than one central electrode 75, the central electrodes 75 may be arranged in a line, a grid, or a cluster. Multiple central electrodes 75 may be activated and deactivated simultaneously or independently. The central electrode 75 may be any suitable shape for retaining the sample droplet 3 or 4 above it. For example, the central electrode 75 may be triangular, rectangular, hexagonal, or octagonal.

[0082] The waisted region may be wider at the two opposite ends than in the center, forming a dumbbell or hourglass shape, or a shape defined by a hyperbolic function (e.g., x2-y2=1). The waisted region may gradually narrow from the wider two opposite ends to a narrower portion near the central electrode.

[0083] The first and second tapered electrodes 77 and 78 are arranged along a second direction 79 transverse to the first direction 72, on either side of the at least one central electrode 75, and between the first and second raised electrodes 70 and 71. The second direction 79 may be perpendicular to the first direction 72. Thus, the first and second tapered electrodes fill the waist-shaped region between the first and second raised electrodes 70 and 71 (narrowing near the central electrode 75 and widening as the distance from the central electrode 75 increases). The first and second tapered electrodes 77 and 78 may only fill some of the space between the first and second raised electrodes 70 and 71.

[0084] The first convex electrode 70 is divided or segmented into first and second portions 81, 82 by a row of lead-out seventh electrode lines 83 leading from a central electrode 75 to beyond the first convex electrode 70. The electrodes 7 in the lead-out electrode lines 83 are square and arranged adjacent to each other, however, they can be of any suitable shape for holding the sample droplet 3 or droplet 4 above them and actuating the sample droplet 3 or droplet 4, for example, the lead-out electrode lines 83 can be triangular, rectangular, hexagonal or octagonal.

[0085] First and second side electrodes 85 and 86 are arranged along the second direction 79 adjacent to the first and second tapered electrodes 77 and 78, respectively. The first and second side electrodes 85 and 86 are arranged adjacent to the ends of the first convex edge 73 and the second convex edge 74 of the first and second convex electrodes 70 and 71. The first and second side electrodes 85 and 86 in the first set of droplet-splitting electrodes 691 are square, but they can be of different suitable shapes. They can have a mixture of curved and straight edges to improve the manipulation of the droplets 4, for example, they can have one or more concave or convex edges.

[0086] The arrangement of the electrodes 7 in the droplet-splitting electrode 69 can form a rectangle with an aspect ratio of 3:1, with one-third of the area at each end of the group 69 being filled with the first and second side electrodes 85, 86. The central electrode 75 can have a length lcentral typically between 125 and 350 microns and / or a width wcentral typically between 125 and 350 microns. However, the dimensions of the central electrode 75 can be smaller or larger, depending on the device application. The first and second side electrodes 85, 86 can have an area at least five times greater than that of the central electrode 75, but the area of ​​the first and second side electrodes 85, 86 can be 100 times greater than that of the central electrode 75. The area of ​​the first and second side electrodes 85, 86 can be greater than 100 times the area of ​​the central electrode 75. The first and second side electrodes 85, 86 typically have similar dimensions to each other and can have a length lflanking between 280 and 3500 microns and a width wflanking between 280 and 3500 microns. However, the dimensions of the first and second side electrodes 85, 86 may be smaller or larger, depending on the application of the device.

[0087] The first or second tapered electrodes 77, 78 typically have an area between the area of ​​the central electrode 75 and the area of ​​the first and second side electrodes 85, 86. The area of ​​the first and second tapered electrodes 77, 78 can be between one-half and one-hundredth the area of ​​the first and second side electrodes 85, 86. The first and second tapered electrodes 77, 78 typically have a length of between 280 microns and 3500 microns and / or a width of between 125 microns and 1750 microns. The first and second tapered electrodes 77, 78 typically have similar dimensions to each other.

[0088] The first or second convex electrode 70, 71 typically has an area between the area of ​​the central electrode 75 and the area of ​​the first and second side electrodes 85, 86. The area of ​​the first and second tapered electrodes 77, 78 can be between one-half and one-hundredth the size of the area of ​​the first and second side electrodes 85, 86. The first and second convex electrodes 70, 71 typically have similar dimensions to each other and have a length lconvex between 280 microns and 3500 microns and / or a width wconvex between 125 microns and 1750 microns.

[0089] Each electrode 7 in the extraction electrode line 83 is typically of similar size to the central electrode 75 and has a length (extrude) between 125 microns and 350 microns and / or a width (extrude) between 125 microns and 350 microns. Each electrode 7 in the extraction electrode line 83 is typically of similar size to the central electrode 75.

[0090] Each electrode 7 in the first set of drop-splitting electrodes 691 is connected to an electrode driver 9 controlled by a controller 8. Each electrode 7 may be individually controllable, but may also be controlled in pairs of electrodes 7, or in larger groups or sets of electrodes 7.

[0091] refer to Figure 7 The second set of droplet-splitting electrodes 692 on the on-dielectric electrowetting device for preparing sample droplets 3 has the same layout as the first set 691. The on-dielectric electrowetting device can be made of an array of electrodes 89 in a checkerboard pattern. Each electrode 7 in the array of electrodes 89 can have an area smaller than that of the central electrode. For example, the area of ​​each electrode 7 in the array of electrodes 89 can be between one-fifth and one-fiftieth of the area of ​​the central electrode. One or more of the central electrode 75, the lead-out electrode lines 83, the first and second convex electrodes 70, 71, the first and second conical electrodes 77, 78, or the first and second side electrodes 85, 86 can be formed by a set of checkerboard electrodes on the on-dielectric electrowetting device. For example, in the second set of droplet-splitting electrodes 692, the second convex electrode 71 is not formed as a single electrode 7, but rather is formed by a set of electrodes 7 within the array of electrodes 89 in a continuous pattern.

[0092] In the second group 692, the array of continuous electrodes, a tessellated grid of electrodes 89, is made of hexagonal electrodes 7; however, the array of tessellated electrodes 89 can be made of electrodes 7 having any suitable shape. For example, the shape of the electrodes 7 can be triangular, square, rectangular, or octagonal. The shape of the electrodes 7 in the array of tessellated electrodes 89 can be regular or irregular. A mixture of different electrode 7 shapes and sizes can be present in the array of tessellated electrodes 89.

[0093] Each electrode 7 in the continuous array of electrodes 89 has an identifier (not shown) that allows the controller 8 and / or computer system 11 to identify its position in the array of electrodes 89. A group of adjacent electrodes 7 in the array of electrodes 89 that form the desired electrode 7 shape in the droplet-splitting electrode 69 are then simultaneously activated or deactivated by the electrode driver 9 under the control of the controller 8. Simultaneous control of the electrode group can be achieved through software or, alternatively, can be hard-wired with each electrode 7 in the electrode group connected to a common connector from the electrode driver 9. Simultaneous control of a group of electrodes 7 in the shape of one of the first and second convex electrodes 70, 71, the central electrode 75, the first and second tapered electrodes 77, 78, the extractor electrode wire 83, and / or the first and second side electrodes 85, 86 allows the second group of droplet-splitting electrodes 692 to be controlled in a manner similar to the first group of droplet-splitting electrodes 691. As will be described in more detail later, the second group of droplet-splitting electrodes 692 can be used to split droplets 4 in a manner similar to the first group 691.

[0094] An enlarged portion 91 of a portion of the second set of droplet-splitting electrodes 692 shows portions of the first and second convex electrodes 70, 71, first and second tapered electrodes 77, 78, the central electrode 75, portions of the extraction electrode line 83, and portions of the first side electrode 85 formed on the continuous tessellated array of electrodes 89 of the hexagonal electrode 7. The density of the electrodes 7 in each of the shaped electrodes can vary, for example, there can be 1 to 1000 electrodes within the convex electrode length lconvex. There can be more than 1000 electrodes within the convex electrode length.

[0095] refer to Figure 8 The third set of droplet-splitting electrodes 693 is similar to the arrangement of the first set of droplet-splitting electrodes 691. The third set of droplet-splitting electrodes 693 includes diamond-shaped first and second convex electrodes 70, 71, trapezoidal first and second conical electrodes 77, 78, and hexagonal first and second side electrodes 85, 86. The first convex edge 73 and the second convex edge 74 are straight.

[0096] refer to Figure 9, the fourth set of droplet-splitting electrodes 694 is also arranged similarly to the third set of droplet-splitting electrodes 693. The fourth set of droplet-splitting electrodes 694 comprises a tessellated array of equilateral triangular electrodes. A square central electrode 75 is arranged at the points of the six surrounding triangular electrodes 7. This has the effect of changing the shape of some of these electrodes 7. For example, the first and second conical electrodes 77, 78 are trapezoidal. The second convex electrode 71 is formed by first and second equilateral triangular portions 93, 94. The corners of each portion nearest the central electrode 75 have been removed and replaced by two sides of the square central electrode 75, which makes the first and second portions pentagonal. The first and second side electrodes 85, 86 are each formed by three tessellated equilateral triangular portions 851, 852, 853, 861, 862, 863.

[0097] The array of checkerboard electrodes 89 of the second set of drop-splitting electrodes 692 can be used to make the electrode shape and arrangement of any of the first, third and fourth sets of drop-splitting electrodes 691, 693, 694, or combine shapes from these sets.

[0098] The dimensions of the electrodes 7 in the second, third and fourth groups of drop-splitting electrodes 69 are similar to the dimensions of the electrodes in the first group of drop-splitting electrodes 691 .

[0099] Sample preparation method

[0100] refer to Figure 10 to Figure 1 3, the first process of preparing the sample droplet 3 from the droplet 4 will now be described. In step S11, the central electrode 75, the first and second conical electrodes 77, 78, and the first and second side electrodes 85, 86 are activated, and the first droplet 41 is held above all the activated electrodes and confined above the waist-shaped region and the central electrode 75. In step S12, the first and second conical electrodes 77, 78 are deactivated, which causes the first droplet 41 to split into second and third droplets 42, 43, which are respectively held above the first and second side electrodes 85, 86, and causes the sample droplet 3 to be held above the central electrode 75.

[0101] The method can allow for the manipulation of large droplets and for the division of the large droplets into at least one smaller droplet or sample, which can then be moved to another portion or region of an on-medium electrowetting device. It can be difficult to manipulate large droplets on an on-medium electrowetting device. Dividing the droplets into droplets having smaller volumes can make manipulation easier and can allow droplets containing single particles to be separated more easily. These smaller droplets can be more easily controlled and can be more easily further divided or divided into smaller droplets or samples of an appropriate size for further processing. For example, small droplets containing single particles (e.g., biological cells) can be moved to a portion of an on-medium electrowetting device for cultivation and nucleic acid amplification.

[0102] In step S11, the shape of the first liquid droplet 41 confined above the activation electrode 7 may be hourglass-shaped. For example, the shape of the first liquid droplet 41 may be approximately described by the following formula:

[0103] x4-4(8x2-y2+0.25)=0

[0104] The sample droplet 3 can then be inspected by the sample inspection system 20 to determine the number of particles 21 within the sample droplet 3. Depending on the number of particles 21 present in the sample droplet 3, the sample droplet 3 can be moved to the further processing portion 6 using the extraction electrode line 83, or the first and second conical electrodes 77, 78 can be activated again and the second and third droplets 42, 43 and the sample droplet 3 will recombine into one droplet 4.

[0105] The method can allow for the inspection of droplets or samples before they are moved for further processing to determine the number of particles retained within the droplet or sample. Droplets or samples that do not contain the desired number of particles or cells can be discarded before further processing. This can increase the throughput of, for example, single-cell whole genome amplification and can also reduce waste in the form of reagents and sample identification. The resulting product can then be further processed (e.g., by sequencing). The manipulation of the droplets and the decision on how to process the droplets based on the number of particles within the droplets can be automated.

[0106] With particular reference to Figures 12 and 13, the droplet-splitting electrodes 69 can take different forms. For example, in Figure 12, in the fifth set of droplet-splitting electrodes 695, the first and second tapered electrodes 77, 78 may not be directly opposite each other in a straight line (i.e., 180°), but may be opposite each other at different angles (e.g., approximately 120°). With particular reference to Figure 13, the sixth set of droplet-splitting electrodes 696 may have first and second tapered electrodes 77, 78 surrounding the central electrode 75 at approximately 90° angles to each other. The first and second tapered electrodes 77, 78 and the central electrode 75 form a waist shape and, when activated, the first droplet 41 is confined above the central electrode 75.

[0107] refer to Figures 14 to 17 , a second process for preparing a sample droplet 3 from a droplet 4 using the first, second, third, or fourth set of droplet-splitting electrodes 69 will now be described. In step S21, the electrode driver 9 actuates the first and second convex electrodes 70, 71, the first and second conical electrodes 77, 78, at least one central electrode 75, and the extraction electrode wire 83, which results in the first droplet 41 being held above these activated electrodes. The first and second side electrodes 85, 86 are inactive at this step, and due to the hydrophobic coating 53 above each electrode, the droplet is not held above them. In step S22, the electrode driver 9 deactivates the first and second convex electrodes 70, 71 and the extraction electrode wire 83, and simultaneously activates the first and second side electrodes 85, 86. This results in the first droplet 41 forming a waist shape above the first and second conical electrodes 77, 78 and the central electrode 75. In step S23 , the electrode driver 9 deactivates the first and second conical electrodes 77 , 78 , which causes the first droplet 41 to split into second and third droplets 42 , 43 held above the first and second side electrodes 85 , 86 , respectively, and causes the sample droplet 3 to be held above the central electrode 75 .

[0108] In step S24, the sample inspection system 20 determines the number of particles 21 within the sample droplet 3 held above the central electrode 75. The number of particles 21 is sent to the computer system 11. Based on the results of this step, the sample is moved to the further processing section 6 (step S25), recombined with the second and third droplets 42 and 43 (step S26), or the sample is moved to a waste area (not shown) (step S27). If the droplet-splitting electrode 69 has multiple central electrodes 75, at least one central electrode 75 can be activated and deactivated to separate sample droplets 3 containing only one particle 21 (step S28). In this case, the sample inspection system 20 can inspect the sample droplet 3 twice, or the system 20 can be run in real time, thereby sending information about the number of particles 21 in each sample droplet 3 held above any one of the central electrodes 75 to the computer system 11 in real time. Once the sample droplet 3 containing a single particle is separated, the process can move to step S25, and the sample droplet 3 can be moved to the further processing section 6.

[0109] The method of partitioning the droplet 4 is stored in the computer program 16, which can determine which step is required next based on information received from, for example, the sample inspection system 20. The number of particles 21 is analyzed by the droplet control program 15 and / or the computer program 16 and / or the user. For example, if the user is performing single-cell whole genome amplification (scWGA), if a single cell is identified in the sample droplet 3, the sample will be moved for further processing. If the sample droplet 3 contains zero or more than one cell, the sample droplet 3 can be recombined with the second and third droplets 42, 43 or moved to a waste area. Alternatively, if the user desires a high-throughput system for processing the sample droplets 3, the number of particles 21 identified in each sample droplet 3, recorded in the memory 13, along with the location and unique identifier of each sample droplet 3 on the on-media electrowetting device 2 (not shown), can be used to move all sample droplets 3 to the further processing section 6. This may allow the user to process large numbers (eg thousands) or sample droplets 3 at once, and only after a further processing stage make a decision as to whether to retain or discard the sample droplets 3 or their products (not shown).

[0110] The process of preparing sample droplets 3 from the droplet 4 continues until the desired number of sample droplets 3 has been prepared or the droplets 4 have been exhausted. Typically, 1000 sample droplets 3 will be prepared from a single droplet 4, but the number of sample droplets 3 prepared will depend on the capacity and / or size of the further processing section 6, as well as the requirements of the user.

[0111] Droplet partitioning electrode

[0112] refer to Figure 18The seventh set of droplet-splitting electrodes 697 is similar to the first set of droplet-splitting electrodes 691. The seventh set of droplet-splitting electrodes 697 has five central electrodes 751 ... 755 arranged in a row along the second direction 79. Each central electrode 75 is square and has the same size. Each central electrode 75 can be controlled independently of the other electrodes 7 or simultaneously with the other electrodes 7. A set of droplet-splitting electrodes 69 can have more than five central electrodes 75, for example, the droplet-splitting electrodes 69 can have 10 to 100 central electrodes 75. As will be described in more detail later, multiple central electrodes can make it easier to separate the sample droplet 3 from the droplet 4, or to separate more than one sample droplet 3 from the droplet 4.

[0113] refer to Figure 19 The eighth set of droplet-splitting electrodes 698 is similar to the seventh set of droplet-splitting electrodes 697, with the addition of an extraction electrode line 83 extending from each central electrode 75. The eighth set of droplet-splitting electrodes 698 has first, third, and fifth extraction electrode lines 831, 833, 835 extending from the first, third, and fifth central electrodes 751, 753, and 755 along the first direction 72 beyond the flat edge of the semicircular first convex electrode 70. The second and fourth extraction electrode lines 832, 834 extend from the second and fourth central electrodes 752, 754 beyond the flat edge of the second convex electrode 71 in a direction opposite to the first direction 72. The number of extraction electrode lines 83 can be between one and several central electrodes 75. The extraction electrode lines 83 can branch into lines of multiple electrodes 7 (not shown). As will be described in more detail later, multiple central electrodes can make it easier to separate a sample droplet 3 from a droplet 4, or to separate more than one sample droplet 3 from a droplet 4. As will be described in more detail later, the addition of multiple extraction electrode lines 83 can allow multiple sample droplets 3 to be moved to different further processing parts 6 and / or allow sample droplets 3 to be extracted to one or more further processing parts 6 simultaneously.

[0114] Sample preparation method

[0115] refer to Figures 20 to 23The third process for preparing a sample droplet 3 from a droplet 4 using the seventh or eighth set of droplet-splitting electrodes 697 and 698 will now be described. In step S31, the electrode driver 9 activates the first and second convex electrodes 70 and 71, the first and second tapered electrodes 77 and 78, the plurality of central electrodes, and the extraction electrode wire 83, which results in the first droplet 41 being held above these activated electrodes 7. The first and second side electrodes 85 and 86 are inactive at this stage, and due to the hydrophobic coating 53 above each electrode, the droplet is not held above the first and second side electrodes 85 and 86. In step S32, the electrode driver 9 deactivates the first and second convex electrodes 70 and 71 and the extraction electrode wire 83, and simultaneously activates the first and second side electrodes 85 and 86. This results in the first droplet 41 forming a waist shape above the first and second tapered electrodes 77 and 78 and the central electrode 75.

[0116] In step S33, the sample inspection system 20 determines the number of particles 21 within the portion of the first droplet 41 held above the plurality of central electrodes 75, and if particle counting is not performed on the computer system 11, the number of particles 21 is sent to the computer system 11. A given central electrode 75 having a particle held above it will be marked. A central electrode 75 that does not have a particle held above it will be unmarked. If a marked central electrode 75 is adjacent to one or more other marked central electrodes 75, the marked central electrode 75 is unmarked. In step S34, based on the number of particles 21 held above each central electrode 75, the microfluidic system 1 instructs the electrode driver 9 to deactivate the first and second conical electrodes 77, 78, as well as the unmarked central electrodes 75 that do not have a single particle 21 held above them. This can allow for the separation of more than one particle 21 in the sample droplet 3 at a time.

[0117] For example, special reference Figure 21 In step S33, the sample inspection system 20 identifies a single particle 21 in the first droplet 41 held above the fourth central electrode 754. In step S34, the first and second conical electrodes 77, 78 and the first, second, third and fifth central electrodes 751, 752, 753, 755 are simultaneously deactivated. This leaves the sample droplet 3 containing the single particle 21 held above the fourth central electrode 754. With particular reference to Figure 23 In step S33, the sample inspection system 20 identifies a single particle 21 in the first droplet 41 held above the first and fourth central electrodes 751, 754. In step S34, the first and second conical electrodes 77, 78 and the second, third, and fifth central electrodes 752, 753, 755 are simultaneously deactivated. This leaves the sample droplet 3 containing the single particle 21 held above the first and fourth central electrodes 751, 754.

[0118] If there is no particle 21 held above any of the multiple central electrodes 75, or more than one particle 21 is held above a single central electrode 75, the first and second convex electrodes 70, 71 can be activated and the first and second side electrodes 85, 86 are deactivated and the process returns to step S31.

[0119] In step S35, one or more sample droplets 3 are moved from the plurality of central electrodes 75 to the further processing portion 6 along one of the extraction electrode lines 83. For example, with particular reference to Figure 21 , the sample droplets 3 containing the single particles 21 are moved along the extraction electrode line 83 to the further processing section 6. The droplet splitting electrode 69 can be arranged adjacent to more than one further processing section 6. The droplet splitting electrode 69 can also be arranged adjacent to a processing section 97, which can facilitate the recycling of sample droplets 3 and / or droplets 4 for further use (e.g., by merging large droplets 4). For example, with particular reference to Figure 23 , the first sample droplet 31 is moved to the further processing section 6 and the second sample droplet 32 ​​is moved to the processing section 97. There may be a number of reasons why a particular sample droplet 3 is moved to the processing section 97. For example, the further processing section 6 may already have the required number of sample droplets 3, additional particles 21 may have been detected in the sample after the splitting, or the user may simply want to reduce the volume of fluid in the electrowetting-on-dielectric device 2.

[0120] The process of preparing sample droplets 3 from the droplets 4 continues until the desired number of sample droplets 3 has been prepared or the droplets 4 have been exhausted. Typically, 1000 sample droplets 3 will be prepared from a single droplet 4, but the number of sample droplets 3 prepared will depend on the capacity and / or size of the further processing section 6, as well as the requirements of the user.

[0121] This method can allow droplets and / or samples containing only a desired number of particles to be separated. Assessing the number of particles held in a droplet held above a central electrode before separating individual droplets and / or samples can be more efficient and produce fewer wasted droplets and / or samples.

[0122] Nucleic acid amplification equipment

[0123] refer to Figures 24 to 26 , the preparation of sample droplets 3 and the amplification of nucleic acids in the on-medium electrowetting device 2 will now be described. With particular reference to Figure 24In the first electrowetting device on dielectric 21, the second side electrode 86 of the first set of droplet-splitting electrodes 691 is arranged adjacent to a rectangular reservoir electrode line 98. Each reservoir electrode has a length lreservoir that is the same as the side electrode length lflanking. Each reservoir electrode has a width wreservoir that is one-half to one-twelfth of the reservoir length lreservoir. The reservoir electrode line 98 is led out from the second side electrode 86 and is arranged adjacent to the inlet electrode 100. The inlet electrode 100 is used to introduce droplets 4 into the electrowetting device on dielectric 2. The inlet electrode 100 is hexagonal, but can be any suitable shape. The inlet electrode 100 typically has a length le and a width we between 100 microns and 300 microns. The reservoir electrode line 98 can hold large droplets 4 before moving the droplets 4 to the droplet-splitting electrode 69.

[0124] Arranging the extraction electrode wire 83 adjacent to the further processing section 6 can allow sample droplets 3 to be moved from the droplet-splitting electrode 69 into the further processing section 6. The further processing section 6 includes at least one set of parallel rows 102 (also referred to as "amplification rows") of electrodes 7. The electrodes 7 can have appropriate dimensions to allow the on-dielectric electrowetting device 2 to hold the droplets 4 or sample droplets 3 above the activation electrodes 7. Typically, the electrodes 7 in the further processing section 6 can have a width we between 100 microns and 300 microns and a length le between 100 microns and 300 microns.

[0125] The electrodes 7 in the further processing section 6 can be thermally insulated and thermally regulated using the previously described thermal control system 34. Typically, the number of rows 102 will be between 5 and 100 rows; however, the number of rows 102 will depend on the application for which the microfluidic system 1 is being used. For example, if the microfluidic system 1 is being used for experimental or research and development purposes, there may be 5 to 50 rows 102, and if the microfluidic system 1 is being used for industrial processing of sample droplets 3, there may be 100 to 1000 rows 102. The rows 102 are separated from each other by dielectric regions 103 and / or by electrodes 7 in an inactive state. Two adjacent rows 102 are typically connected to each other at two locations, one near a first end 104 of the row 102 and another near a second end 105 of the row 102, by a row 106 of connecting electrodes 7.

[0126] The sample outlet portion 108 generally includes a series of electrodes 7 adjacent one of the one or more amplification rows 102 and / or connection rows 106 and also adjacent an outlet electrode 109. The outlet electrode 109 may allow a user to remove a sample droplet 3 for further processing, e.g., for DNA sequencing.

[0127] The further processing section 6 typically includes a cell lysis zone 110 for performing cell lysis on biological cells. The cell lysis zone 110 typically spans a series of amplification rows 102 near the first end 104 of the row 102 in the further processing section 6. The cell lysis zone 110 is wide enough for at least one sample droplet 3 to be held above the electrode 7 in each amplification row 102. Typically, one to five sample droplets 3 will be held in the amplification row 102 in the cell lysis zone 110, however, the cell lysis zone 110 may be wide enough to allow more sample droplets 3 (e.g., 10 sample droplets 3 or 50 sample droplets 3) to be held in each row. The cell lysis zone 110 may be capable of performing different suitable cell lysis methods, such as temperature treatment (thawing and heat treatment), osmotic lysis, and / or chemical lysis. Additional reagents can be added to the sample droplets 3 at this stage via the connection electrodes 7 (not shown) arranged adjacent to the amplification row 102. If the cell lysis method is temperature treatment, the thermal control system 34 controls the temperature of the cell lysis zone 110, or otherwise thermal regulation is required. External devices (e.g. devices delivering ultrasonic homogenization, pressure homogenization, not shown) can also be directed to the sample droplet 3 in the cell lysis section. Different cell lysis techniques may be available, such as heat treatment and addition of chemicals to the sample droplet 3.

[0128] The further processing section 6 typically includes three to ten thermally controlled zones 36 adjacent to the cell lysis zone 110. A thermal control system 34 controls the temperature of the thermally controlled zones 36. Similar to the cell lysis zone 110, each thermally controlled zone 36 spans the amplification rows 102. Each thermally controlled zone 36 is wide enough to hold at least one sample droplet 3 above the electrodes 7 in each amplification row 102. Typically, one to five sample droplets 3 are held in the processing section 97 within the thermally controlled zones 36; however, the thermally controlled zones 36 may be wide enough to hold more sample droplets 3 (e.g., ten or fifty sample droplets 3) per row. Thermal control of the electrodes 7 in these zones 36 allows for chemical or biological processing of sample droplets 3 containing necessary molecular biological reagents and nucleic acids. For example, thermally controlled electrodes 7 can allow for nucleic acid amplification. The zones 36 can be thermally controlled to appropriate temperatures and thermally isolated from each other. The zones 36 include at least one electrode 7. There may be more than 10 thermally controlled zones 36 in the on-medium electrowetting device 2. The number of thermally controlled zones 36 will depend on the chemical or microbiological process being performed, and the throughput of the microfluidic system 1. For example, sample droplets 3 may be moved in only one row along the row 102 in the on-medium electrowetting device 2 to reduce contamination. Performing 30 cycles of a polymerase chain reaction using this method would require at least 90 thermally controlled zones 36.

[0129] The thermally controlled regions 36 can be arranged and thermally controlled to promote nucleic acid amplification and / or the incubation of nucleic acids or products of nucleic acid amplification reactions (e.g., polymerase chain reactions). For example, a first thermally controlled region 361, arranged adjacent to the cell lysis zone 110, can be controlled to a temperature that denatures nucleic acids in the sample droplet 3 held above the region 361. A second thermally controlled region 362, arranged adjacent to the first thermally controlled region 361, can be thermally controlled to a temperature suitable for the annealing step of the polymerase chain reaction. A third thermally controlled region 363, arranged adjacent to the second thermally controlled region 362, can be thermally controlled to a temperature suitable for the elongation / extension step of the polymerase chain reaction. Additional thermally controlled regions 36 can be arranged and thermally controlled to appropriate temperatures that allow additional stages of nucleic acid amplification to be performed in the sample droplet 3 held thereon. For example, additional regions 36 can be controlled to temperatures that allow initialization, final extension, or other molecular biology processes. The sample droplet 3 can be moved or actuated between thermally controlled regions 36 to perform thermal cycling in, for example, the following reactions: polymerase chain reaction (PCR), quantitative polymerase chain reaction (qPCR), degenerate oligonucleotide primer polymerase chain reaction (DOP-PCR), multiple annealing amplification loops (MALBAC), or ligase chain reaction (LCR). Alternatively, there can be only one thermally controlled region 36 that can cycle between temperatures required to promote a particular reaction.

[0130] There may be only one thermally controlled region 36 that can be controlled to a temperature that allows an isothermal nucleic acid amplification reaction to occur in the sample droplet 3. Examples of isothermal nucleic acid amplification reactions include, for example, multiple displacement amplification (MDA), loop-mediated isothermal amplification (LAMP), self-sustaining sequence replication (3SR) or nucleic acid sequence-dependent amplification (NASBA), strand displacement (SDA), and rolling circle amplification (RCA).

[0131] A waste container 111 is arranged adjacent to at least one electrode 7 on the connection row 106 of electrodes 7. Unwanted and / or discarded droplets 4 and / or sample droplets 3 are moved to the waste container 111 and are not further processed. The waste container 111 may be an electrode 7 and may be activated to assist in removing waste from the on-medium electrowetting device 2.

[0132] Special References Figure 25 In the electrowetting device 2 on the second medium, there may be additional reservoir electrode lines 98 and inlet electrodes 100 arranged adjacent to other electrodes 7 in the droplet-splitting electrode 69. For example, the reservoir electrode line 98 of the reservoir electrode may be arranged adjacent to the first side electrode 85. The processing portion 97 may include one or more rows of electrodes 7 connecting the extraction electrode line 83 to the reservoir electrode line 98. One or more reservoir connection electrodes 112 may connect two reservoir electrode lines 98.

[0133] Once each sample droplet 3 has been separated from the droplet 4, the sample droplets 3 are individually actuated to electrodes 7 in a row in the processing portion 97 of electrodes 7 in the cell lysis zone 110. The sample droplets 3 can be held at these electrodes 7 until the desired number of sample droplets 3 have been moved to the cell lysis zone 110. If desired, a row in the amplification rows 102 of electrodes 7 can be used to move sample droplets 3 between rows 97.

[0134] If desired, a cell lysis process as previously described can then occur in the cell lysis zone 110 to break down the cell walls of the biological cells in the sample droplet 3 (step S41). Cell lysis can be performed outside the on-medium electrowetting device 2, and the products from the process are input into the on-medium electrowetting device 2 for further processing, e.g., purification and amplification. The sample droplet 3 may already contain nucleic acids prepared for amplification, e.g., the nucleic acids may have already been purified. After cell lysis, the components of the sample droplet 3 can then be mixed with a buffer and / or subjected to a purification step (step S42) to prepare the nucleic acids for amplification.

[0135] In step S33, when the components of the sample droplet 3 are ready for amplification, the sample is moved to a first thermally controlled area 361 within the further processing section 6. In this area 361, the sample droplet 3 begins the amplification process (step S44). If the amplification process (such as MDA, LAMP, 3SR, NASBA, SDA, or RCA) is isothermal, the sample droplet 3 can remain in the first thermally controlled area 361 until the amplification process is complete.

[0136] If the amplification process (such as PCR, qPCR, and LCR) is based on thermal cycling, the sample droplet 3 can be moved through thermally controlled zones 36 to facilitate nucleic acid amplification. For example, for the denaturation step, a first thermally controlled zone 361, regulated between 94°C and 98°C, denatures the nucleic acids. The sample droplet 3 can then be actuated to a second thermally controlled zone 362, regulated between 50°C and 65°C, for annealing. The sample droplet 3 can then be actuated to a third thermally controlled zone 363, regulated between 75°C and 80°C, for elongation and extension. Typically, the sample droplet 3 is actuated between these zones in approximately thirty cycles; however, the number of cycles can be more or less than thirty. As previously described, the number of temperature-controlled zones 36 will depend on the process being performed in the further processing section 6. The temperature-controlled zones 36 are maintained at temperatures as appropriate to facilitate the desired portion of the amplification reaction. The sample droplet 3 can be moved between the temperature-controlled zones 36 as many times as necessary for the amplification reaction to occur and can be maintained at each temperature for the time required for the reaction to occur. There may be multiple sample droplets 3 at different stages of amplification on the on-medium electrowetting device 2. Different forms of amplification may occur simultaneously on the on-medium electrowetting device 2.

[0137] In step S45, the sample droplet 3 can be monitored by the sample inspection system 20 at intervals or continuously to assess the status of the amplification cycle and quantify the total amount of deoxyribonucleic acid (DNA). For example, the nucleic acid amplification can be quantitative PCR (qPCR, also known as "real-time PCR"). For example, the fluorescence of the sample droplet 3 can be measured to assess the amount of nucleic acid that has been amplified in the reaction. The fluorescence of a non-specific fluorescent dye embedded in double-stranded deoxyribonucleic acid can be detected and measured. The level of fluorescence or another marker used to indicate the amount of nucleic acid present in the reaction can be recorded, processed, and analyzed using the sample inspection system 20 to determine the amount of nucleic acid in the sample droplet 3. When the sample emits a certain level of fluorescence indicating that the desired amplification has been achieved for the sample droplet 3, the amplification cycle will stop. Some sample droplets 3 may complete the cycle before others.

[0138] Once the amplification reaction has been completed, the sample droplet 3 can be moved to the electrodes 7 which are maintained at a temperature between 4° C. and 15° C. The amplified nucleic acids can be temporarily stored at these temperatures.

[0139] The sample droplet 3 that has undergone the amplification cycle or other processing is then evaluated to determine the total amount of nucleic acid product present (e.g., by using the fluorescence measurement described above). The measured fluorescence level, along with other information about the sample droplet 3, can be fed back into the storage droplet control program 15 or computer program 16 or forwarded to the user to allow the program or user to decide to move the sample to the exit electrode 109. In step S46, based on the evaluation results, the sample is moved to the exit electrode 109 for further analysis or to a waste container 111, where the sample droplet 3 can be removed from the on-medium electrowetting device 2. In step 47, the sample is extracted for further analysis, such as DNA sequencing.

[0140] In a high-throughput process for single-cell whole genome amplification, sample droplets 3 with no cells, single cells, or multiple cells separated from a large droplet 4 can undergo lysis, amplification, and incubation processes, but then be discarded to a waste container 111 without extraction, and samples identified as having single cells are moved to the outlet electrode 109. Thus, the on-medium electrowetting device 2 can automatically move sample droplets 3 that do not have single cells or in which the amplification process failed to occur to the waste container 111.

[0141] Using such a microfluidic system 1 and on-medium electrowetting device 2, it is possible to easily store the locations of all sample droplets 3 along with associated metadata, such as the number and type of cells or particles 21 present in each sample droplet 3 before the cell lysis stage. Such a microfluidic system 1 can eliminate the need for tracking identifiers (such as barcodes attached to tubes, etc.). In addition, the on-medium electrowetting device 2 can be easily adjusted, which allows a large number of sample droplets 3 to be amplified simultaneously, and the number of products will be automatically evaluated and selected for further processing.

[0142] Cell culture and cultivation

[0143] The on-medium electrowetting device 2 can be used to culture cells. For example, a single cell or a cell group within the sample droplet 3 can be moved to a further processing section 6. Here the cells can be immobilized by active (e.g., modifying the lid to activate physical containment within the porous matrix) or passive cell theft deterrents (not shown). The further processing section 6 can be thermally regulated by a thermal control system 34 to a temperature that promotes cell culture, which allows the cells to grow and divide to form larger cell groups. For example, the temperature of the further processing section 6 can be controlled to between 37°C and 38°C, which temperature range will depend on the requirements of the one or more cells in the sample droplet 3. Depending on the evaluation by the sample inspection system 20, these cell groups can be moved to the extraction electrode or outlet electrode 109 for further processing or they can be moved to waste.

[0144] Additional reagents used in the cultivation, growth, and processing of cells, such as cell release solutions and cell nutrient broth, can be added to the sample droplets 3 before, during, and after cultivation to assist in the movement, cultivation, inspection, and evaluation of the cell population. Additional reagents can be added to the sample droplets 3 containing one or more cells via connection electrodes (not shown) arranged adjacent to the connection row 106 and / or the cultivation and expansion row 102. Additional reagents can be added to the sample droplets 3 via mixing electrodes (not shown) before the sample droplets 3 are moved to the further processing section 6. It is possible to remove cellular waste products from the sample by partitioning the sample droplets 3 in the further processing section 6 using a method similar to the second stage of droplet 4 partitioning previously described. The sample droplets 3 containing cells can then have additional buffer, nutrient broth, or other reagents added to the sample droplets 3 to dilute the concentration of waste products in the sample droplets 3.

[0145] Droplet partitioning electrode

[0146] 27 , a ninth set of droplet-splitting electrodes 699 is similar to the fifth set 695 . The ninth set 699 has an equilateral hexagonal central electrode 75 . First, second, and third tapered electrodes 77 , 78 , 113 are arranged on alternating sides of the central electrode 75 (each arranged at 120° to each other). First, second, and third side electrodes 85 , 86 , 114 are arranged adjacent to the first, second, and third tapered electrodes 77 , 78 , 113 , respectively. The ninth set 699 may further include first, second, and third convex electrodes (not shown) each arranged between the three tapered electrodes 77 , 78 , 113 and the extraction electrode line 83 .

[0147] Sample preparation method

[0148] Special References Figure 27A In step S11, the central electrode 75, the first, second and third conical electrodes 77, 78, 113 and the first, second and third side electrodes 85, 86, 114 are activated so that the first droplet 41 is confined above the central electrode 75 and forms three lobes above the first, second and third conical electrodes and the side electrodes, respectively. Figure 27BIn step S12, the first, second, and third conical electrodes are deactivated, which causes the first droplet 41 to divide into the second, third, and fourth droplets 42, 43, 44 and the sample droplet 3. If present, the first, second, and third convex electrodes can allow the first droplet to be held above the first, second, and third convex electrodes, and the first, second, and third conical electrodes 77, 78, 113 and the center electrode assist in the formation of the trilobal shape of the first droplet 41 before being deactivated while activating the first, second, and third side electrodes 85, 86, 114. As in the previous example, the first side electrode 85 can be inspected by the sample inspection system 20 and used to move the sample to further processing 6 or to reassemble the sample.

[0149] Such an arrangement may allow for easier manipulation of larger droplets 4 on the electrowetting-on-dielectric device 2 .

[0150] Revise

[0151] It will be appreciated that various modifications may be made to the embodiments described above. Such modifications may include equivalent and other features known in the design, manufacture, and use of on-dielectric electrowetting devices or digital microfluidic devices and component parts thereof, and which may be used instead of or in addition to features already described herein. Features of one embodiment may be substituted for or supplemented by features of another embodiment.

Claims

1. A method of operating an electrowetting-on-dielectric device, the electrowetting-on-dielectric device comprising a central electrode (75); a first conical electrode (77) adjacent to the central electrode; a second conical electrode (78) adjacent to the central electrode; a first side electrode (85) adjacent to the first conical electrode, the first side electrode having a larger area than the central electrode; a second side electrode (86) adjacent to the second conical electrode, the second side electrode having a larger area than the central electrode; The method comprises: activating the central electrode, the first conical electrode, the second conical electrode, the first side electrode, and the second side electrode to cause a first droplet (41) to be held above the central electrode, the first conical electrode, the second conical electrode, the first side electrode, and the second side electrode, wherein the first droplet is confined above the central electrode; deactivating the first and second conical electrodes to cause the first droplet to divide into a second droplet (42), a third droplet (43), and a sample droplet (3) held above the first side electrode, the second side electrode, and the center electrode, respectively; The volumes of the second conical electrode, the first side electrode, and the second side electrode are respectively related to the areas of the first side electrode, the second side electrode, and the central electrode; the volume of the sample droplet is smaller than the volume of the second droplet; and the volume of the sample droplet is smaller than the volume of the third droplet; The first side electrode and the second side electrode have an area five times larger than the central electrode, the first side electrode and the second side electrode have an area one hundred times larger than the central electrode, and the first tapered electrode and the second tapered electrode have an area between the area size of the central electrode and the area size of one of the first side electrode or the second side electrode; The electrodes in the device are coplanar; The first conical electrode and the second conical electrode are arranged opposite to each other, and the first conical electrode and the second conical electrode are arranged opposite to each other in the first direction; the first side electrode and the second side electrode are arranged opposite to each other; the first side electrode and the second side electrode are arranged opposite to each other in the first direction; the central electrode, the first conical electrode, the second conical electrode, the first side electrode and the second side electrode are arranged in a row in the first direction; the electrodes are arranged in a row in the first direction in the order of the first side electrode, the first conical electrode, the central electrode, the second conical electrode and then the second side electrode; the arrangement of the electrodes is symmetrical using a symmetry line passing through the central electrode.

2. The method according to claim 1, wherein The device further comprises: a first convex electrode (70) adjacent to the central electrode; a second convex electrode (71) adjacent to the central electrode; The method further comprises: wherein the central electrode, the first conical electrode, the second conical electrode, the first side electrode, and the second side electrode are activated to cause a first droplet (41) to be held above the activated central electrode, the activated first conical electrode, the activated second conical electrode, the activated first side electrode, and the activated second side electrode; activating the central electrode, the first conical electrode, the second conical electrode, the first convex electrode, and the second convex electrode to cause a droplet (4) to be held above the central electrode, the first conical electrode, the second conical electrode, the first convex electrode, and the second convex electrode; deactivating the first convex electrode and the second convex electrode; and activating the first side electrode and the second side electrode; The first convex electrode and the second convex electrode are arranged opposite to each other; the first convex electrode and the second convex electrode are arranged opposite to each other in a second direction which is not parallel to the first direction; and the second direction is perpendicular to the first direction.

3. The method according to claim 2, further comprising: determining, using a sensor, a number of particles (21) in the sample droplet held above the central electrode; In response to the number of particles in the sample droplet being outside a predefined range: activating at least the first conical electrode and the second conical electrode to cause the second droplet, the third droplet, and the sample droplet to merge into a single droplet (4); The on-dielectric electrowetting device comprises a seventh electrode line (83), wherein at least one seventh electrode is adjacent to the central electrode; The method includes: in response to the number of particles in the sample droplet being within a predefined range: activating and deactivating electrodes in the seventh electrode line to move the sample droplet to a processing portion; The at least one seventh electrode adjacent to the central electrode is at one end of the line, the method comprising activating the electrodes to cause a first treatment of the sample droplet to be moved to a first treatment portion, wherein the first treatment is a thermally regulated treatment; The first treatment is single-cell nucleic acid amplification; the first treatment is cell culture.

4. The method according to claim 2, wherein the electrowetting device on a dielectric comprises a plurality of central electrodes, wherein the method further comprises: include: in, Deactivating the first conical electrode and the second conical electrode to cause the first droplet to divide into a second droplet (42), a third droplet (43), and a sample droplet (3), comprising: For each given central electrode in the plurality of said central electrodes: determining, using a sensor, the number of particles (21) in the first droplet held above the given central electrode; marking a given central electrode in response to the number of particles (21) in the first droplet being held above the given central electrode being within a predefined range; Any central electrode that is labeled and adjacent to another labeled central electrode is not labeled; deactivating the first and second conical electrodes simultaneously with deactivating the unlabeled central electrode, causing the first droplet to divide into a second droplet (42) and a third droplet (43) held above the first and second side electrodes, and a sample droplet (3) held above each of the labeled central electrodes; The electrowetting on the dielectric includes a seventh electrode line (83), wherein at least one seventh electrode is adjacent to the central electrode; The method includes: in response to the number of particles in the sample droplet being within a predefined range: activating and deactivating electrodes in the seventh electrode line to move the sample droplet to a processing portion; The seventh electrode line divides the first convex electrode and / or the second convex electrode into multiple parts; the seventh electrode line is used to transfer one or more droplets or samples from the arrangement of the droplet dividing electrodes; such an arrangement allows droplets or samples to be moved to multiple further processing parts or to extraction electrodes; the further processing parts are on the same dielectric electrowetting device or on different devices.

5. The method according to claim 3, wherein: determining the number of particles in the droplet by capturing an image of the droplet held above an electrode using a camera (22) and analyzing the image using machine vision in a processor; The particles are biological cells; This allows droplets containing single biological cells to be identified.

6. The method according to any one of claims 1 to 5, wherein The electrowetting-on-dielectric device has a region of tessellated electrodes (89), wherein at least one of the electrodes in the electrowetting-on-dielectric device comprises a group of tessellated electrodes within the region of the tessellated electrodes, wherein the group of electrodes is activated or deactivated simultaneously; This enables an on-dielectric electrowetting device comprising an array of connected electrodes to perform the method of a device comprising electrodes having a specifically designed and manufactured shape, wherein the array of connected electrodes is a tessellated electrode array; the electrodes in the tessellated electrode array are square, rectangular, triangular, hexagonal or octagonal.

7. A computer program product, which, when executed by at least one processor, causes the at least one processor to perform the method of any one of claims 1 to 5.

8. A computer program product comprising a computer readable medium storing the computer program product according to claim 7.

9. A microfluidic manipulation device comprising: A dielectric electrowetting device (2) and a controller (8); The dielectric electrowetting device includes a central electrode (75); a first conical electrode (77) adjacent to the central electrode; a second conical electrode (78) adjacent to the central electrode; a first side electrode (85) adjacent to the first conical electrode, the first side electrode having a larger area than the central electrode; a second side electrode (86) adjacent to the second conical electrode, the second side electrode having a larger area than the central electrode; The controller is configured to activate the central electrode, the first conical electrode, the second conical electrode, the first side electrode, and the second side electrode to cause a first droplet (41) to be held above the central electrode, the first conical electrode, the second conical electrode, the first side electrode, and the second side electrode, wherein the first droplet is confined above the central electrode; deactivating the first conical electrode and the second conical electrode to cause the first droplet to divide into a second droplet (42), a third droplet (43), and a sample droplet (3) held above the first side electrode, the second side electrode, and the center electrode, respectively; The dielectric electrowetting device further comprises: a first convex electrode (70) adjacent to the central electrode; a second convex electrode (71) adjacent to the central electrode; The controller is further configured to: wherein the central electrode, the first conical electrode, the second conical electrode, the first side electrode, and the second side electrode are activated to cause a first droplet (41) to be held above the activated central electrode, the activated first conical electrode, the activated second conical electrode, the activated first side electrode, and the activated second side electrode; activating the central electrode, the first conical electrode, the second conical electrode, the first convex electrode and the second convex electrode so as to cause the droplet (4) to be held above the central electrode, the first conical electrode, the second conical electrode, the first convex electrode and the second convex electrode; deactivating the first convex electrode and the second convex electrode; and activating the first side electrode and the second side electrode.

10. The apparatus according to claim 9, further comprising: a sensor configured to determine a number of particles within the droplet; The controller, in response to the number of particles in the sample droplet being outside a predefined range, is configured to: At least the first conical electrode and the second conical electrode are activated to cause the second droplet, the third droplet, and the sample droplet to merge into a single droplet (4).

11. The apparatus according to claim 9, further comprising: a sensor configured to determine a number of particles (21) within the droplet; Wherein, the electrowetting device on the dielectric further comprises a plurality of central electrodes; The controller is further configured to: wherein deactivating the first conical electrode and the second conical electrode to cause the first droplet to separate into a second droplet (42), a third droplet (43), and a sample droplet (3) comprises: For each given central electrode of the plurality of central electrodes: determining, using the sensor, a number of particles (21) in the first droplet held above the given central electrode; marking a given electrode in response to a number of particles (21) within the droplet in the first droplet held above the given central electrode being within a predefined range; Any central electrode that was labeled and adjacent to another labeled central electrode was not labeled; Deactivating the first and second conical electrodes while deactivating the unlabeled central electrode causes the first droplet to divide into a second droplet (42) and a third droplet (43) held above the first and second side electrodes, and a sample droplet (3) held above each of the labeled central electrodes.

12. The apparatus according to claim 11, further comprising: processor; wherein the sensor is a camera (22), and wherein the number of particles in the droplet is determined by capturing an image of the droplet held above an electrode using a camera and analyzing the image using machine vision in the processor.

13. The device according to any one of claims 9 to 12, wherein: The electrowetting-on-dielectric device has a tessellated electrode (89), wherein at least one of the electrodes in the electrowetting-on-dielectric device comprises a set of tessellated electrodes within the region of the tessellated electrode; The controller is further configured to simultaneously activate or deactivate groups of electrodes.

14. A microfluidic manipulation system comprising: The device according to any one of claims 9 to 13; and a computer system (11) operatively connected to the controller, the computer system being configured to perform the method of any one of claims 1 to 6 and to record the position of at least one droplet.

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