CN113203607A - Preparation of samples on electrowetting-on-dielectric devices - Google Patents

Abstract

The invention discloses a method for preparing a sample on a dielectric electrowetting device. The method comprises activating a first, a second and a third set of consecutive electrodes (7) in the electrowetting-on-dielectric device (2), the third set of electrodes being interposed between the first and second set of electrodes to form a path (48) between the first and second set of electrodes such that the first droplet (4) is caused to drop₁) Held above the first, second and third sets of electrodes. The method further comprises deactivating at least one electrode of the third set of electrodes so as to break the path between the first set of electrodes and the second set of electrodes and to divide the first droplet into a second and a third droplet (4) held above the first set and the second set of electrodes, respectively₂，4₃)。

Description

Preparation of samples on electrowetting-on-dielectric devices

Technical Field

The present invention relates to a method of preparing a sample on a electrowetting device on a medium.

Background

Electrowetting-on-dielectric (EWOD) is a useful technique employed in digital microfluidic technology for lab-on-a-chip (LoC) systems to manipulate small volumes of a liquid sample (commonly referred to as "droplets"). The EWOD device can be used as a front-end platform to perform complex sample processing procedures for other locs.

Mugel and j.c. barret, journal of physics: condensed matter,volume 17, page R705 (2005): "electrowetting: an introduction to electrowetting is given from basic to application "and w.nelson and CJ Kim in journal of adhesion science and technology, volume 26, pages 1747-: an overview of electrowetting on dielectric devices is given in the review ", which is incorporated herein by reference.

Single cell whole genome amplification (scWGA) can be applied to nucleic acids in a single cell before single cell whole genome sequencing (scWGS) can be performed. scWGS is used in a variety of applications where assessment of intercellular genomic variation is required. For example, the number of replications, alterations and Single Nucleotide Variations (SNVs) occurring within a single cell are assessed.

However, scWGA is challenging, in part because of the low number of nucleic acid replications present in each cell and the rare variation of the genome in cell populations. Current scWGA methods have low throughput and require complex and expensive tracking and identification procedures, e.g., individual barcode tubes.

Cells intended to undergo scWGA and/or scWGS are typically prepared in a suspension containing a plurality of cells. Such suspensions are typically injected or inserted into EWOD devices, forming droplets within the devices. The droplets containing the cell suspension may have a large volume, which is difficult to manipulate on electrowetting devices on the medium. Furthermore, identifying and tracking or recording the location of droplets with the desired number of cells can be challenging, requiring expensive bar codes to be affixed to the sample. In addition, the bar code may be damaged during handling, possibly rendering the sample unusable.

Disclosure of Invention

According to a first aspect of the invention, a method for splitting droplets on an electrowetting device on a medium is provided. The method includes activating first, second, and third sets of successive electrodes in the electrowetting-on-dielectric device, the third set of electrodes interposed between the first and second sets of electrodes to form a path between the first and second sets of electrodes such that the first droplet is retained over the first, second, and third sets of electrodes. The method also includes thereafter deactivating at least one electrode in the third set of electrodes to break the path between the first and second sets of electrodes and to cause the first droplet to break into second and third droplets that remain above the first and second sets of electrodes, respectively. The method further includes, thereafter, activating a fourth set of continuous electrodes including at least one electrode of the second set of electrodes and forming a row of continuous electrodes having first and second opposing ends to elongate the third droplet. The method further includes thereafter activating a fifth set of consecutive electrodes adjacent to the first end of the fourth set of electrodes, and deactivating at least one electrode of the fourth set of electrodes between the first end and the second end, and activating at least one electrode of the fourth set of electrodes at or adjacent to the second end to cause the third droplet to separate into a fourth droplet and a fifth droplet.

This may allow a large droplet to be introduced into the device and then split or divided into smaller droplets (referred to herein as "sample droplet(s)") comprising one or more droplets for further processing. When split or separated, larger droplets are more difficult to control. These smaller droplets are more easily controlled and can be more easily broken up or separated into smaller sample droplets of a size suitable for further processing. Further processing may include recovery of droplets, chemical treatment of droplets (e.g., addition of reagents), biochemical treatment of droplets, physical treatment of droplets (e.g., exposure of droplets to electromagnetic radiation, thermal cycling of droplets, etc.), analysis of droplets (e.g., optical, electrical, etc.), or other forms of processing.

The third set of consecutive electrodes may have a smaller surface area than either the first or second set of electrodes, resulting in a narrower path between the first and second set of electrodes. When the three sets of consecutive electrodes are activated, this may result in the droplets forming an hourglass or dumbbell shape.

The fifth set of electrodes may have a smaller area than the fourth set of electrodes.

The method may further include determining a number of particles within the fifth droplet using the sensor. Thereafter, the fifth droplet is moved to the first processing section or the fifth droplet is moved to the second processing section via a recovery path including a recovery electrode according to the number of particles in the fifth droplet.

The method may allow for recovery of the droplet and the sample droplet when the number of particles in the droplet or sample droplet is not a desired number. For example, the number of particles may be 0, 1, or more than 1. For example, if the particles are biological cells that require further processing for whole genome amplification and sequencing (scWGA and scWGS), sample droplets with 0 or more than 1 cell can be recovered and combined with droplets stored in a reservoir.

The method may further comprise activating a sixth set of continuous electrodes and a seventh set of continuous electrodes so as to maintain a sixth and seventh droplet, respectively, above each set of electrodes, wherein each set of electrodes is arranged with a space between them and is also arranged adjacent to the deactivated set of continuous mixed electrodes. The method may further comprise, thereafter, activating the set of mixing electrodes and deactivating at least one of the sixth or seventh sets of continuous electrodes such that droplets held above the sixth and seventh sets of continuous electrodes coalesce into a single droplet held above the at least one set of continuous electrodes and/or the set of mixing electrodes. The sixth and seventh sets of continuous electrodes may include any one of the first through fifth sets of continuous electrodes.

The number of particles in the droplet may be determined by capturing an image of the droplet held over the electrode using a camera and lens and analyzing the image using machine vision in a processor.

The particle may be a cell. The cell may be an artificial cell or a biological cell, such as a eukaryotic cell or a prokaryotic cell.

The sensor may be an optical sensor. The optical sensor may be a Charge Coupled Device (CCD) or a Complementary Metal Oxide Semiconductor (CMOS). The optical sensor may include a camera and a lens.

If the particles are biological cells, the sensor for determining the number of biological cells in the droplet may be an electromechanical sensor, an electrical sensor or a flow cytometer. The electrical sensor may measure resistance using a coulter counter or a CASY cell counter.

The method may further comprise activating the electrodes to perform a first process on the sample droplet moved to the first processing portion, wherein the first process is a thermal conditioning process.

The first process may be single cell nucleic acid amplification. The first process may be cell culture.

Nucleic acid amplification may be a thermal cycling method or an isothermal method. The nucleic acid amplification may be Polymerase Chain Reaction (PCR), quantitative polymerase chain reaction (qPCR), or loop-mediated isothermal amplification (LAMP).

The method may further comprise recording the position of at least one droplet.

The method may be implemented in software, for example in a (programmable) computer system comprising a memory and at least one processor. The method may be implemented at least partially in hardware. For example, an Application Specific Integrated Circuit (ASIC) or a Field Programmable Gate Array (FPGA) may be used to perform at least some of the steps of the method.

According to a second aspect of the invention, there is provided a computer program which, when executed by a computer system, causes the computer to perform the method.

According to a third aspect of the invention, a computer readable medium storing a computer program is provided, which may be a non-transitory computer readable medium.

According to a fourth aspect of the present invention, there is provided an apparatus comprising an electrowetting on dielectric device and a controller. The controller is configured to activate a first, a second, and a third set of consecutive electrodes in the electrowetting-on-dielectric device, the third set of electrodes being interposed between the first and second set of electrodes to form a path between the first and second set of electrodes such that the first droplet is retained over the first, second, and third set of electrodes. Thereafter, the controller is further configured to deactivate at least one electrode of the third set of electrodes so as to break the path between the first set of electrodes and the second set of electrodes and cause the first droplet to break into a second droplet and a third droplet held above the first set of electrodes and the second set of electrodes, respectively. Thereafter, the controller is further configured to activate a fourth set of consecutive electrodes, including at least one electrode of the second set of electrodes, and form a row of consecutive electrodes having first and second opposite ends to elongate the third droplet. Thereafter, the controller is further configured to activate a fifth set of consecutive electrodes adjacent to the first end of the fourth set of electrodes, and deactivate at least one electrode of the fourth set of electrodes between the first end and the second end, and activate at least one electrode of the fourth set of electrodes at or adjacent to the second end, thereby causing the third droplet to separate into a fourth droplet and a fifth droplet.

This may allow automatic splitting, movement and tracking of the sample.

The device may further include: a sensor for determining the number of particles in the droplet; and a controller configured to cause activation and deactivation of the electrodes to move the fifth droplets to the first processing section, or activation and deactivation of a path of the continuous recovery electrode to move the fifth droplets to the second processing section, depending on the number of particles in the fifth droplets.

This may allow for automatic splitting, movement and tracking of samples containing known quantities of particles.

The device may further comprise a first processing section adjacent to the fourth set of electrodes comprising a set of sequential thermally regulated electrodes to facilitate nucleic acid amplification. The controller may also be configured to cause activation and deactivation of the electrodes so as to cause a first process to be performed on the sample droplet moved to the first processing section, wherein the first process is single-cell nucleic acid amplification.

The arrangement of the recycling lanes may allow a droplet or sample droplet to move from the second set of electrodes to the first set of electrodes while bypassing the second and/or third and/or fifth set of electrodes.

Thus, the device may allow manipulation and tracking of samples containing a known number of cells, which then undergo whole genome amplification. The droplets or sample droplets that do not contain the desired number of particles or cells may be discarded before further processing. This greatly improves the throughput of single cell whole genome amplification and also greatly reduces waste in the form of reagents and sample markers. The resulting product may then continue to be further processed, for example, sequenced. Manipulation of the droplets and the decision of how to treat the droplets based on the number of particles within the droplets may be automated.

Nucleic acid amplification may be thermocycling or isothermal. The nucleic acid amplification may be Polymerase Chain Reaction (PCR), quantitative polymerase chain reaction (qPCR), loop-mediated isothermal amplification (LAMP).

The device may comprise a bulb of successively separated electrodes to allow separation of the droplet into two or more droplets or sample droplets.

The device may comprise a continuous row of separation electrodes and at least one separation electrode arranged transversely to the row of separation electrodes to allow a second separation of the droplets.

The recovery path may bypass an electrode arranged for droplet separation.

The electrodes or groups of consecutive electrodes may be of shapes other than quadrilateral shapes.

The controller may be further configured to activate a sixth set of continuous electrodes and a seventh set of continuous electrodes such that a sixth and seventh droplet, respectively, remains over each set of electrodes, wherein each set of electrodes is arranged with a space disposed therebetween, and is further arranged adjacent to the deactivated set of continuous mixed electrodes. Thereafter, the controller may be further configured to activate the set of mixing electrodes and deactivate at least one of the sixth or seventh sets of continuous electrodes, thereby causing the droplets held above the sixth and seventh sets of continuous electrodes to coalesce into a single droplet held above the at least one set of continuous electrodes and/or the set of mixing electrodes. The sixth and seventh sets of continuous electrodes may include electrodes of any one of the first through fifth sets of continuous electrodes.

The nucleic acid amplification may be single cell whole genome nucleic acid amplification (scWGA).

The first processing section may include a plurality of electrodes or groups of consecutive electrodes that are thermally regulated and thermally isolated from each other and configured to allow droplets to move within and between them.

Thus, the device may allow for automatic isolation of droplets with a known number of cells within each droplet, and then the droplet containing the desired number of cells may be moved to a processing section where cell lysis may be performed, followed by movement of the droplet between thermally regulated electrodes for a thermal cycling procedure such as PCR.

There may be a plurality of electrodes forming at least one row of successive electrodes, the row of electrodes being arranged and controlled to allow actuation of a droplet along the row.

There may be at least one row of consecutive electrodes, wherein at least a portion of the row comprises a plurality of adjacent electrodes that are thermally conditioned together and are thermally isolated from at least one other portion of the row.

There may be any number of adjacent thermally regulated and thermally isolated sets of contiguous electrodes. There may be two, three, four, five or six adjacent regions.

There may be at least first and second rows of continuous electrodes and a third row of continuous electrodes arranged and controlled to allow a droplet to move between the first and second rows of electrodes.

The device may include an optical sensor configured to determine an amount of nucleic acid within the at least one droplet.

The optical sensor may be a fluorescence sensor configured to measure fluorescence of the at least one droplet.

The optical sensor may be a CCD or CMOS adapted to sense the wavelength emitted by the product in the sample droplet. For example, the CCD or CMOS may be in a camera attached to a lens, wherein the camera is suitable for fluorescence detection.

The device may further comprise at least one droplet outlet to allow removal of droplets from the device. The device may further comprise a droplet inlet to allow introduction of a droplet to at least one electrode on the device.

According to a fifth aspect of the present invention there is provided a computer system operatively connected to the controller of the fourth aspect of the present invention. The computer system is configured to perform the method steps of the first aspect of the invention. The computer system is configured to record a position of at least one droplet.

The computer system may be configured to perform the second aspect of the invention. The computer system may be configured to read the computer readable medium of the third aspect of the invention.

Drawings

Certain embodiments of the invention will now be described, by way of example, with reference to the accompanying drawings, in which:

FIG. 1 is a schematic block diagram of a microfluidic system including an electrowetting on-dielectric liquid droplet manipulation device;

FIG. 2 is a schematic block diagram of a specimen inspection system including a camera and lens;

FIG. 3 is a schematic block diagram of a thermal control system including an electrowetting droplet on dielectric manipulation device;

FIG. 4 is a plan view of an electrowetting device on a first medium for sample preparation and single cell amplification;

FIG. 5 is a plan view of an electrowetting device on a second medium for sample preparation and single cell amplification;

FIG. 6 is a cross-sectional view of an electrowetting device on a dielectric;

FIG. 7 schematically illustrates droplet actuation;

FIG. 8 is a process flow diagram of a single cell amplification method;

FIG. 9 is a process flow diagram of a droplet separation and recirculation method;

FIG. 10 is a process flow diagram of a droplet separation method;

FIG. 11 is a plan view of the device of FIG. 1 including a droplet containing biological cells;

FIG. 12 shows a stage of droplet separation using the device shown in FIG. 1;

FIG. 13 shows a stage of droplet separation using the device shown in FIG. 1;

FIG. 14 shows a stage of droplet separation using the device shown in FIG. 1;

FIG. 15 shows a stage of sample droplet isolation and evaluation using the device shown in FIG. 1;

FIG. 16 is a plan view of an electrowetting device on a medium for sample preparation and single cell amplification;

FIG. 17 is a plan view of an electrowetting device on a medium for sample preparation and single cell amplification;

FIG. 18 is a process flow diagram of a single cell amplification method;

FIG. 19 shows a cell lysis stage using the device of FIG. 1;

FIG. 20 shows nucleic acid amplification using the device of FIG. 1;

FIG. 21 shows a sample droplet evaluation using the device of FIG. 1;

FIG. 22 illustrates sample collection and sample disposal using the device of FIG. 1;

Detailed Description

Microfluidic system

Referring to fig. 1, amicrofluidic system 1 for preparing a sample is shown. Themicrofluidic system 1 comprises an electrowetting on dielectric (EWOD) device 2 (also referred to as "droplet manipulation device", or simply "device") for preparing asample 3 fromdroplets 4 and moving thesample 3 to afurther processing section 6. The electrowetting ondielectric device 2 comprises anelectrode 7. Thesystem 1 may further comprise a fluid handling system (not shown) for delivering fluid to the electrowetting device onmedia 2. Thesystem 1 further comprises acontroller 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 oneelectrode driver 9. One ormore electrode drivers 9 are connected to theelectrodes 7 in thedroplet manipulation device 2 and are arranged to control the application or removal of a bias voltage to one or moreparticular electrodes 7.

Thesystem 1 includes acomputer system 11 that may be used to control thecontroller 8. Thecomputer system 11 includes at least oneprocessor 12 andmemory 13,memory 13 storing adroplet control program 15 anddroplet manipulation instructions 16, both of which may take the form of one or more scripts (not shown) and/or a series of tables (not shown), one per frame, each table storing a list ofactive electrodes 7. In some cases,controller 8 may store acontrol program 15 anddroplet operations instructions 16. Thus, at least during operation, thecomputer system 11 may be omitted. More details of how the electrodes are controlled to move and manipulate thesample 3 and thedroplet 4 are described in detail in british patent GB 2,559,216B, particularly onpage 4,line 20 topage 7, line 24.

Specimen inspection system

Referring also to fig. 2, themicrofluidic system 1 may further comprise asample inspection system 20. Thesample 3 and/or thedroplets 4 may comprise or comprise a variety of liquids, such as saline buffer, a biological sample (e.g. DNA or protein) or a body fluid (e.g. blood or urine). Thesample 3 and/or thedroplet 4 may contain or comprise more than one type of liquid. Thesample 3 and/or thedroplet 4 may contain one ormore particles 21. Theparticles 21 may be, for example, artificial or biological cells, such as eukaryotic or prokaryotic cells, cell nuclei or organelles. Thesample inspection system 20 may be used to determine the number ofparticles 21 in thesample 3.

Thesample inspection system 20 comprises acamera 22, alens 23, an illumination source 24, a processor 25 for processing the output of thecamera 22, amemory 27 for storing images or output data, and image analysis/particle counting software 28 for analyzing the images and/or counting the number ofparticles 21 within thesample 3 ordroplet 4. Illumination source 24 may be integrated intocamera 22 or arranged separately fromcamera 22 and the rest ofsystem 20. Thespecimen inspection system 20 may further include at least oneoutput device 30. Alternatively, thecamera 22 may be operably connected to thecomputer system 11 and the image analysis/particle counting software 28 stored in thecomputer system memory 13. Thememory 13 may also store theimage 31 and output data from the performed image analysis. Thesoftware 28 may be a set of instructions that may take the form of a script (not shown).

Thelens 23 is used to focus thecamera 22 on at least a part of onesample 3. At least a portion of thesample 3 is illuminated using an illumination source 24. Thecamera 22 then acquires animage 31 of at least a portion of the sample. Theimage 31 is then transferred to thememory 13, 27. Theimage 31 is then analyzed in theprocessor 12, 25 using thesoftware 28. Thesoftware 28 determines the number ofparticles 21 in at least a part of thesample 3. The machine vision method is used to isolate and countindividual particles 21 in at least a portion of thesample 3. Various machine vision methods may be used, such as thresholding, edge detection, color analysis, blob detection, and pattern recognition. Neural network processing, deep learning and/or machine learning may also be used in combination with machine vision methods to identify and count the number ofparticles 21 in at least a portion of onesample 3.

Thesample inspection system 20 may also include a device (not shown), such as a motorized X, Y, Z camera stage, to move thecamera 22 andlens 23 to focus different portions of thesample 3. Thecamera 22 andlens 23 are moved to focus different parts of thesample 3 until theentire sample 3 is captured and analysed. Optionally, thecamera 22 andlens 23 are positioned to focus on a singlecomplete sample 3 or multiplecomplete samples 3.

After thesample inspection system 20 determines the number ofparticles 21 within thesample 3, thesystem 20 will output at least a two-state response indicating a positive or negative evaluation of thesample 3 based on the user-entered condition. For example, if the user wants to isolatesample 3 containing only asingle particle 21,system 20 will output a positive response ifsample 3 contains oneparticle 21; and if thesample 3 contains less or more than oneparticle 21, thesystem 20 will output a negative response. The user and/or dropcontrol 15 can use the positive or negative output to determine the next step. For example, depending on the output, the sample may be moved to thefurther processing section 6. Alternatively, thesample inspection system 20 may output the number ofparticles 21 identified within thesample 3. The number ofparticles 21 can then be used to determine how thesample 3 is to be further processed. For example, if the number ofparticles 21 identified in thesample 3 is greater than 1, thesample 3 may recombine withlarger droplets 4. If noparticles 21 are present in thesample 3, thesample 3 may be moved to waste. Regardless of the number ofparticles 21 identified in eachsample 3, allsamples 3 may be transferred to thefurther processing section 6. The number ofparticles 21 in eachsample 3 will be recorded together with the position of thesample 3 on thedevice 2. The number ofparticles 21 in eachsample 3 can be used to determine the action of a later step, e.g. whether to extract droplets for nucleic acid sequencing or discard to waste.

Thesample inspection system 20 may also be used to assess the condition of thesample 3 during or after further processing in thefurther processing section 6. For example, thecamera 22 may be sensitive to fluorescence emitted by thesample 3. This may allow thesample inspection system 20 to assess the stage of the reaction or process occurring within thesample 3. For example,sample 3 may contain nucleic acids comprising non-specific fluorescent dyes that intercalate into double stranded DNA, andsample 3 may be subjected to thermal cycling quantitative polymerase chain reaction (qPCR). Theimage 31 of thesample 3 subjected to the quantitative polymerase chain reaction may be processed in theprocessor 12, 25 usingsample analysis software 32 to determine the level of fluorescence emitted from nucleic acids within thesample 3. The level of fluorescence measured fromimage 31 can be used to assess the amount of amplified nucleic acid insample 3. The level of fluorescence can determine the stage of the reaction and thus how long or how manythermal cycles sample 3 needs to be completed before the reaction is completed.

Thesample inspection system 20 used to evaluate the condition of the sample during or after further processing may be the samesample inspection system 20 that evaluated thesample 3 prior to further processing, or it may be a second, different sample inspection system. Eachsample inspection system 20 may have more than onecamera 22,lens 23, and illumination source 24.

Temperature control system

Referring to fig. 3, themicrofluidic system 1 may also include athermal control system 34. Thethermal control system 34 includes athermal controller 35 that controls the temperature of one or morethermal control zones 36 in the electrowetting device on themedium 2. The thermally controlledzone 36 may be temperature regulated in a suitable manner by, for example, using a heating or cooling pad, microwave, or chemical heating. The thermal control zone also includes a temperature sensor (not shown) that can feed temperature information back to thethermal controller 35.Thermal controller 35 may be operatively connected tocomputer system 11.Thermal control software 37 may also be included inmemory 13.

The thermally controlledregion 36 may include one ormore electrodes 7. Eachthermal control region 36 can be individually controlled to a temperature required for a particular process, for example, thethermal control region 36 can be set and adjusted to a temperature that facilitates a portion of a thermal cycling reaction, such as a Polymerase Chain Reaction (PCR).

Electrowetting on dielectric device

Referring to fig. 3 and 4, electrowetting devices 2 (hereinafter also referred to as "devices") on a first and a second medium for preparing asample 3 for further processing are shown. Further processing of thesample 3 may include single cell amplification. Thedroplet 4 can be freely manipulated in two or three dimensions over thesurface 40 using an array ofelectrodes 7. Thedevice 2 may be a passive or activedielectrically electrowetting device 2. Eachelectrode 7 may be arranged adjacent to at least oneother electrode 7. Theelectrodes 7 may be arranged to form a continuous set of electrodes. The set of consecutive electrodes may be a checkerboard array. The set of consecutive electrodes may be arranged and controlled in a manner that allows them to function as asingle electrode 7. Eachelectrode 7 of the set ofconsecutive electrodes 7 may have a boundary and the conductive portions of theelectrodes 7 of the set may not abut each other, that is, there may be a gap (e.g., <1mm, or typically between 2 μm and 50 μm) between the conductive portions ofadjacent electrodes 7. Theelectrode 7 in thedevice 2 may be coplanar with at least oneother electrode 7 in thedevice 2.

Referring also to fig. 6, eachelectrode 7 may be individually controllable in an activated or deactivated/non-activated state, as will be explained in more detail later. As will be explained in more detail later, thesurface 40 may be provided by adielectric layer 41 having a hydrophobic coating (not shown) which may be used to help isolate thedroplet 4 from theunderlying electrode 7. Thedroplets 4 may comprise or contain a variety of liquids, for example a saline buffer, a biological sample (such as DNA or protein) or a body fluid (such as blood or urine). Thedroplets 4 may contain or comprise more than one liquid. Thedroplet 4 may contain one ormore particles 21. Theparticles 21 may be, for example, artificial or biological cells, such as eukaryotic or prokaryotic cells, cell nuclei or organelles.

The volume of thedroplet 4 and/or thesample 3 may vary from e.g. 10pl to 1 ml. The volume of thedroplets 4 introduced into thedevice 2 may depend on the number and volume ofsamples 3 to be prepared by the device. Typically, the volume of theinitial droplet 4 introduced into thedevice 2 will be more than 1000 times the size of the volume of the desiredsample 3, so that approximately 1000samples 3 can be prepared from onedroplet 4. However, the ratio between the volume of thedroplets 4 and thesample 3 may be larger or smaller, depending on the number and volume of thesamples 3 required. For example, if the desiredsample 3 volume is 25nl, the volume ofdroplet 4 will be greater than 25 μ l. The diameter of thedroplet 4 or thesample 3 will depend on the volume of thedroplet 4 or thesample 3, respectively. The diameter of thedroplet 4 or thesample 3 may typically be between 50 μm and 1000 μm, or more typically between 100 μm and 500 μm.

Droplet 4 manipulation may take different forms.

Thedroplets 4 may move (or "actuate") along a routable path, in other words, a path along which the route may be selectively set and changed. As will be explained in more detail later, a route may be defined as a sequence of coordinates, such as cartesian (x, y, z) or three-axis (a, b, c) coordinates, vectors, or other route defining parameters stored in a table, script, or other suitable computer-readable data structure. The manipulation of thedroplet 3 may also take the form of two ormore droplets 4 merging into asingle droplet 4, or conversely, the splitting of asingle droplet 4 into two ormore droplets 4. A series of droplet manipulations can be performed. The position of eachdroplet 4 on theelectrowetting device 2 on the medium can be recorded as thedroplet 4 moves over thesurface 4.

Thedevice 2 comprises asample inlet electrode 42 for delivering thedroplets 3 into thedevice 2. In these examples, theentrance electrode 42 is hexagonal, however, theentrance electrode 42 need not be hexagonal. Theentrance electrode 42 may be polygonal, such as a regular polygon, or other shape. Theentrance electrode 42 typically has a size between 100 μm and 300 μm. Theinlet electrode 42 may be adjacent a first end of arow 43 of reservoir electrodes which together constitute asample reservoir 44. Thesample reservoir 44 may allowdroplets 4 of a size larger than the desired size of thedroplets 4 to be processed to be inserted (or "introduced") into thedevice 2. Thereservoir electrode 43 is generally rectangular in shape and typically has a length similar to the inlet electrode and awidth 5 to 10 times greater than its length. Thereservoir electrodes 43 are arranged with their long sides abutting each other. This arrangement forms a larger surface than in other areas of thedevice 2. Thedroplets 4 in thereservoir 44 tend to be larger than the droplets elsewhere in thedevice 2.

Thedevice 2 comprises adroplet separation section 46, thedroplet separation section 46 comprising an array ofseparation electrodes 47, at least one of the array ofseparation electrodes 47 abutting thereservoir electrode 43 at the opposite end of a row of reservoir electrodes adjacent to theinlet electrode 42. Thedroplet separation section 46 generally comprises a narrowingpath 48 from thesample reservoir 44 for theelectrodes 7, 47 and then widening into abulb 49 of the checkerboardcontinuous electrodes 7, 47. A row ofseparation electrodes 7, 47 connects thedroplet separation section 46 to the further processing section 6 (hereinafter also referred to as "incubation and amplification section") of thedevice 2. The incubation andamplification part 6 comprises at least one set of parallel rows 50 (also called "amplification rows") ofelectrodes 7. Theelectrodes 7 in the incubation andamplification section 6 may be thermally isolated and thermally regulated using thethermal control system 34 described above. Typically, the number of rows will be between five and one hundred, however, the number ofrows 50 will depend on the application for which thesystem 1 is used. For example, there may be 5 to 50rows 50 if thesystem 1 is used for experimental or research and development purposes, and 100 to 1000rows 50 if thesystem 1 is used for industrial processing of asample 3. Therows 50 are separated from each other bydielectric regions 51 and/orelectrodes 7 in an inactive state. Twoadjacent rows 50 are typically interconnected by arow 54 of connectingelectrodes 7 at two locations, one near afirst end 52 of therow 50 and the other near asecond end 53 of therow 50.

Thesample outlet portion 56 typically comprises a series ofelectrodes 7 adjacent to one of the one ormore amplification rows 50 and/orconnection rows 54, and also adjacent to anoutlet electrode 57. Theexit electrode 57 may allow a user to remove thesample 3 for further processing, e.g. for DNA sequencing.

Thedevice 2 generally includes acell lysis zone 58 for cell lysis of biological cells (not shown). Thecell lysis zone 58 typically spans a series ofexpansion rows 50 in the incubation andexpansion portion 6 near thefirst end 52 of therows 50. Thecell lysis zone 58 may be wide enough to hold at least onesample 3 above theelectrodes 7 in eachamplification row 50. Typically, between one and fivesamples 3 will be held in eachrow 50 in thecell lysis zone 58, however, thecell lysis zone 58 may be wide enough to allowmore samples 3 to be held in each row, for example tensamples 3 or fiftysamples 3. Thecell lysis zone 58 may be capable of performing various suitable cell lysis, for example, temperature treatment (freeze-thaw and heat treatment), osmosis, and/or chemical lysis. At this stage, additional reagent may be added to thesample 3 through connecting electrodes (not shown) arranged adjacent to the connectingrows 54. If the cell lysis method is temperature treatment, or requires thermal regulation,thermal control system 34 controls the temperature ofcell lysis zone 58. It is also possible to guide an external device (not shown) to thesample 3 in the cell lysis section, for example, a device for performing ultrasonic homogenization treatment, pressure homogenization treatment. Different cell lysis techniques may be available, such as heat treatment and addition of chemicals to thesample 3.

The incubation andamplification portion 6 typically includes three to tenthermal control regions 36 adjacent to thecell lysis zone 58. Thethermal control system 34 controls the temperature of thethermal control zone 36. Similar to thecell lysis zone 58, eachthermal control zone 36 spans theamplification row 50. Eachthermal control region 36 may be wide enough to hold at least onesample 3 over theelectrodes 7 in eachamplification row 50. Typically, one to fivesamples 3 will be held in eachrow 50 in thethermal control zone 36, however, thethermal control zone 36 may be wide enough to allowmore samples 3 to be held in each row, for example tensamples 3 or fiftysamples 3. The thermal control of theelectrodes 7 in thesezones 36 may allow thesample 3 containing the necessary molecular biological agents and nucleic acids to undergo chemical or biochemical processes. For example, the thermally controlledelectrode 7 may allow nucleic acid amplification. Thezones 36 may be thermally controlled to a suitable temperature and thermally isolated from each other. Saidzone 36 comprises at least oneelectrode 7. There may be more than tenthermal control regions 36 in thedevice 2. The number ofthermal control zones 36 will depend on the chemical or microbiological process being performed and the throughput of thesystem 1. For example, thesample 3 may be moved in only one pass along therows 50 in thedevice 2 to reduce contamination. Thirty cycles of a polymerase chain reaction using this method would require at least ninetythermal control regions 36.

Thethermal control region 36 can be arranged and thermally controlled to facilitate nucleic acid amplification and/or incubation of nucleic acids or products of a nucleic acid amplification reaction (e.g., polymerase chain reaction). Example (b)For example, the firstthermal control region 36 can be disposed adjacent thecell lysis region 58₁Is controlled to remain in thiszone 36₁Temperature of nucleic acid denaturation insample 3 above. May be adjacent to the first thermal control region 36₁A secondthermal control zone 36 arranged₂The heat is controlled to a temperature suitable for the polymerase chain reaction annealing step to occur. May be adjacent to the second thermal control region 36₂A thirdthermal control zone 36 arranged₃The heat is controlled to a temperature suitable for the extension/elongation step of the polymerase chain reaction to occur. The additional thermally controlledzone 36 may be arranged and thermally controlled to a suitable temperature that allows for an additional stage of nucleic acid amplification to be performed in thesample 3 held on that zone. For example, theadditional region 36 may be controlled to a temperature that allows for initiation, ultimate elongation, or other molecular biological processes.Sample 3 may be moved or actuated betweenthermal control regions 36 to perform thermal cycling, for example, in a Polymerase Chain Reaction (PCR), a quantitative polymerase chain reaction (qPCR), a degenerate oligonucleotide primer polymerase chain reaction ((DOP-PCR), multiple annealing, and loop-based amplification cycles (MALBAC), or a Ligase Chain Reaction (LCR). alternatively, there may be only onethermal control region 36 that cycles between temperatures required to facilitate a particular reaction.

There may be only onethermal control region 36 that can be controlled to a temperature that allows an isothermal nucleic acid amplification reaction to occur in thesample 3. Examples of isothermal nucleic acid amplification reactions include, for example, Multiple Displacement Amplification (MDA), loop-mediated isothermal amplification (LAMP), self-sustained sequence replication (3SR) or nucleic acid sequence-based amplification (NASBA), Strand Displacement Amplification (SDA), and Rolling Circle Amplification (RCA).

Thewaste container 61 is arranged adjacent to at least oneelectrode 7 on the connectingrow 54 ofelectrodes 7. The unwanted and/orwaste droplets 4 and/orsamples 3 are moved into thewaste container 61 and are not further processed. Thewaste container 61 may be anelectrode 7 and may be activated to assist in removing waste from thedevice 2.

Thedevice 2 may comprise one ormore sample reservoirs 44, aseparation section 46, an incubation andamplification section 6, and asample outlet section 56. Theelectrodes 7 may have any suitable shape for holding and moving droplets of the size of thesample 3. Typically, theelectrodes 7 will be rectangular, square, hexagonal or octagonal. The hexagon is closer to the shape of thedroplet 4 than the square and may allow improved actuation and storage. Furthermore, for a given pitch, the hexagonal array may pack theelectrodes 7 more closely than thesquare electrodes 7. A hexagonal array may more easily divide adroplet 4 into a plurality ofdroplets 4 and/orsamples 3. In addition, a hexagonal array may provide a better design of theentrance electrode 42. Thedevice 2 may comprise primarily or entirelynon-quadrilateral electrodes 7, for example thedevice 2 may comprise a mixture ofhexagonal electrodes 7 andoctagonal electrodes 7. Theelectrodes 7 may also be triangular. The shaped side of anelectrode 7 may have a plurality of edges in order to allow anotherelectrode 7 adjacent to it to abut it as closely as possible.

Theelectrodes 7 may be of suitable size to allow thedevice 2 to hold thedroplet 4 orsample 3 above theactive electrodes 7. Typically, the width w of theseparation electrode 7, 47_seAnd length l_seMay be between 100 μm and 300 μm. Typically, the width w of thereservoir electrode 7, 43_reBetween 500 μm and 15mm and a length l_reBetween 100 μm and 300 μm. The area of the droplet reservoir may be more than 1000 times the area of thesample 3 on the device, allowing approximately 1000samples 3 to be prepared from thedroplets 4. Typically, theelectrodes 7 in the amplification andincubation portion 6 may have a width w between 100 μm and 300 μm_eAnd a length l between 100 μm and 300 μm_e。

Referring to fig. 5, a second example of an electrowetting-on-dielectric device 2 comprises a combination of hexagonal andoctagonal electrodes 7 in the incubation andamplification section 6 and thesample outlet section 56. Hexagonal andoctagonal electrodes 7 are advantageous in some cases because theelectrodes 7 have a shape that is closer to thedroplet 4 orsample 3 held above them. Furthermore, the tessellation ofhexagonal electrodes 7 may allow actuation of thesample 3 along three linear paths in different directions. Similarly, theoctagonal electrodes 7 may be arranged to allow actuation of thedroplets 4 and/or thesamples 3 along four linear paths in different directions. This may reduce the time required to move a droplet over thesurface 40 of thedevice 2. A combination of differently shapedelectrodes 7 may be used to obtain theoptimum sample 3 actuation characteristics required for a particular application.

Fig. 6 is a cross-sectional view of the electrowetting device on a medium 2 capable of moving adroplet 4 and/or asample 3 as shown in fig. 4 and 5. Thedevice 2 comprises asubstrate 62 having afirst side 63 and asecond side 64 and aperimeter 65. Thesubstrate 62 is typically in the form of a sheet, and thesubstrate 62 may be longer on first and second orthogonal principal axes than on a third axis that is perpendicular to the plane of the first and second axes.

An array ofelectrodes 7 is embedded on thefirst side 63 of thesubstrate 62. Eachelectrode 7 has afront portion 66 and arear portion 67. Thefront 66 of each electrode may be flush with thefirst side 63 of thesubstrate 62. Theelectrode 7 is connected to thedrive electronics 9 by aconnector 69, theconnector 69 being connected to the rear 67 of theelectrode 7. Theconnector 69 passes through thesubstrate 62 from the rear 67 of theelectrode 7 to thesecond side 64 of the substrate. Each connection may be flush with thesecond side 64 of the substrate. Eachelectrode 7 may be connected to a correspondingconnector 69, and thedrive electronics 9 are also referred to herein as "pixels".

Adielectric layer 41 having afirst side 70 and asecond side 71 may be disposed on thefirst side 63 of thesubstrate 62 and thefront portions 66 of theelectrodes 7. Thefirst side 71 of thedielectric layer 41 may be arranged adjacent to thefirst side 63 of thesubstrate 62 and thefront portion 66 of theelectrode 7. The second side of thedielectric layer 41 has ahydrophobic coating 72. Themicrofluidic structure 73 may be positioned adjacent theperimeter 65 of thesubstrate 62 from thesecond side 64 of the substrate and extend through thedielectric layer 41 and thehydrophobic coating 72.Microfluidic structure 73 has afirst side 75 and asecond side 76. A cover or cover 77 having afirst side 78 and asecond side 79 may be disposed onmicrofluidic structure 73 such thatsecond side 79 ofcover 77 may be adjacent tofirst side 75 ofmicrofluidic structure 73.

Thecover 77 comprises a material that can be used as a substrate in the semiconductor field. For example, thecover 77 may include any one or any combination of poly (methyl methacrylate) (PMMA), polyethylene terephthalate (PET), polyethylene naphthalate (PEN), Polystyrene (PS), Polyimide (PI), or the like. Thecover 77 may comprise a glass material. Thesecond side 79 of thecover 77 may be coated with a conductive material and a hydrophobic material (not shown). The conductive material may also be hydrophobic.

The conductive material may be opaque or transparent. The conductive material may be a Transparent Conductive Oxide (TCO). The conductive material can be Cadmium Tin Oxide (CTO). The conductive material may be Indium Tin Oxide (ITO). The hydrophobic material coating thesecond side 79 of thecap 77 may be a fluorine material. The hydrophobic material may be an amorphous fluoropolymer, such as polytetrafluoroethylene (PTFE or Teflon)^OTM) Or CYTOP^OTM。

Asecond side 79 of thecover 77 may be placed on thefirst side 75 of themicrofluidic structure 73, leaving aspace 80 for thedroplet 4 and/or thesample 3 to move into. Thedrive electronics 9 and the conductive coating of thecover 77 are connected to ground 81.

Droplet actuation

With reference to fig. 7, it will now be described that thesample 3 and/or thedroplet 4 are transferred from thefirst electrode 7 in a row of fiveelectrodes 7₁Through threecentral electrodes 7₂、7₃And 7₄Actuated to thefifth electrode 7₅The process of (1). Theelectrode 7 may be in an active state, e.g. a positive bias may be applied; or in an inactive state, for example theelectrode 7 may be grounded or floating. Activating theelectrodes 7 keeps thedroplet 4 orsample 3 in position above theelectrodes 7. Thehydrophobic layer 72 of thedevice 2 means that thedroplets 4 do not substantially cover thenon-active electrodes 7. In step S1, the electrode driver 9 (under the control of the controller 8) deactivates the first, third, fourth andfifth electrodes 7₁、7₃、7₄、7₅And activating thesecond electrode 7₂. In step S2, theelectrode driver 9 activates thethird electrode 7₃And deactivating thesecond electrode 7₂Resulting in adroplet 4 from thesecond electrode 7₂Is moved upwards to thethird electrode 7₃And (4) upward. Finally, in step S3, theelectrode driver 9 activates thefourth electrode 7₄And deactivating thethird electrode 7₃Resulting in adroplet 4 from thethird electrode 7₃Is moved upward to thefourth electrode 7₄And (4) upward. When theelectrode driver 9 activates theelectrodes 7 or a consecutive group of electrodes, they may remain activated, resulting in theliquid droplet 4 remaining above theelectrodes 5. Theelectrode 7 remains in this state until theelectrode driver 9 deactivates theelectrode 7. Also, the same appliestoThe electrode 5 will remain in the inactive state until theelectrode driver 9 activates theelectrode 7. Theelectrodes 7 may also be activated for a very short time, for example when thedroplet 4 and/or thesample 3 are actuated rapidly across thesurface 40 of thedevice 2.

The array ofelectrodes 7 may be controlled in such a way as to actuate thedroplets 4 on thesurface 40. The state of eachelectrode 7 may be controlled manually (i.e. by the user controlling the activation and deactivation of the electrodes in real time), or a predetermined path may be programmed which will control the movement of a droplet on thesurface 40 of thedevice 2. The control of theelectrodes 7 may be integrated with other systems, such as sensors, cameras and processors, which may process the data from thedevice 2 before feeding the data from thedevice 2 back to the program determining the next movement of one ormore droplets 4.

Sample preparation

Referring to fig. 8 to 22, a sample preparation process for amplification will now be described. Referring specifically to fig. 8 and 9,droplet 4 may be inserted intodevice 2 at insertion electrode 42 (step S11). Thedroplet 4 may contain zero, one ormore particles 21. Theparticles 21 may be suspended within thedroplets 4. Theparticles 21 may be cells, organelles, or a collection of different molecules, such as nucleic acids or nuclei, that are visible using light microscopy. The cell may be an artificial cell or a biological cell, such as a eukaryotic cell or a prokaryotic cell.Droplet 4 may be actuated and held indroplet reservoir 44 using methods similar to those described previously (step S21). For example, two or moreadjacent reservoir electrodes 43 are activated simultaneously, and adroplet 4 may remain over all activatedelectrodes 43, with no orlittle droplet 4 volume over thenon-activated electrodes 43. Then, thedroplet 4 may be actuated toward the droplet-separating portion 46 (step S22).Droplet 4 may contain all necessary reagents for nucleic acid amplification. Alternatively, the necessary reagents for nucleic acid amplification may be added to thedroplets 4 at a later stage, which will be explained in more detail later.

Reagents may include primers and a DNA polymerase, such as Taq polymerase. The reagents may also include buffers, non-specific fluorescent dyes capable of intercalating any double-stranded DNA, or sequence-specific DNA probes consisting of oligonucleotides labeled with fluorescent reporters.

With particular reference to fig. 9 to 15, the preparation ofsample 3 is generally carried out in two stages. First stagefirst droplet 4₁Is divided intosecond droplets 4₂And athird droplet 4₃. Second stagethird droplet 4₃Is divided intofourth droplets 4₄Andfifth droplet 4₅Also referred to as "sample droplet", or simply "sample" 3.

Thedroplet separation section 46 comprises a series ofelectrodes 47, theseelectrodes 47 being adjacent to thereservoir electrode 43 at the end of the row ofreservoir electrodes 43 opposite the reservoir electrode adjacent theinput electrode 42. Thenarrow path 48 of theelectrodes 7, 47 connects theelectrodes 7, 47 adjacent thereservoir electrodes 7, 43 to thewide balls 49 of the seven hexagonallytessellated electrodes 7, 47.First droplet 4 may be separated usingreservoir electrodes 7, 43 andseparation electrodes 7, 47₁Is actuated from thesample reservoir 44 into the droplet separation section 46 (step S22), so that thefirst droplet 4₁May be held partially over thebulb 49 of theseparation electrode 7, 47 and a portion of the droplet may be held over thereservoir portion 44. The result of this arrangement may be a squeezing of thefirst droplet 4 in a narrow path₁。

With particular reference to fig. 8, 9, 13 and 14, the first stage ofdroplet 4 separation will be described. Albeit thefirst droplet 4₁May remain over the wide sphere ofelectrode 49, the narrow path throughelectrode 48, and over thedroplet reservoir electrodes 7, 43, but theseparate electrodes 7, 47 that make up the narrow path and thereservoir electrode 7, 47 closest to thenarrow path 48 are inactive (step S23). As a result, thedroplet 4 is separated into asecond droplet 4₂And a third droplet 4₃(step S12), and thesecond droplet 4₂Remains in thereservoir portion 44 away from the separation portion 46 (step S24), and thethird droplet 4₃Is retained overbulb 49 of separatingportion 46.

With reference to fig. 8, 9, 14 and 15, the second phase ofdroplet 4 break-up will be described. By activating a row ofadjacent separation electrodes 7 from adjacent droplet reservoirs 4447, making thethird droplet 4₃Elongated byelectrode 7 at the center ofbulb 49, andelectrode 47 distal to bulb 49 (step S25). This action results in athird droplet 4₃Is returned through anarrow path 48. Twoseparate electrodes 47₁、47₃Arranged vertically and with aseparate electrode 47 arranged between thebulb 49 and thesample reservoir 44₂Adjacent to each other. Then activates the centrally located splitelectrode 47₂Twoexternal separation electrodes 47 on both sides₁、47₃And all theseparate electrodes 7, 47 except theseparate electrode 7, 47 located distal to the bulb ofelectrode 49 are switched to an inactive state (step S26). The process further drops thethird droplet 4₃Is divided and held at the first and thirddivided electrodes 47₁And 47₃At least onefourth droplet 4 above₄And afifth droplet 4, also called "sample droplet" 3₅(step S12, step S27). Optionally, in a second droplet splitting stage, theelectrode 7 or a set of consecutive electrodes adjacent to the ends of the activated separation electrode may be activated, so that thefifth droplet 4₅Or thesample droplet 3 is moved towards the incubation andamplification section 6.

When thesample droplet 3 is in the fractionatingportion 46 or near the fractionatingportion 46, the number ofparticles 21 in thesample 3 can be determined (step S13). This step may be performed using thesample inspection system 20 described previously. Alternatively, if the particles are biological cells, the number of biological cells in thesample 3 may be determined using other methods, such as electromechanical, electrical (e.g. measuring resistance using a coulter counter or CASY cell counter) or using flow cytometry. The number of biological cells within eachsample droplet 3 may be recorded inmemory 27. Other data about thesample 3, for example the type of cells and the reagents used or any other data required by the user, may also be stored in thememory 27 together with the number of biological cells.

With particular reference to fig. 10, after the number ofparticles 21 in thesample 3 has been determined (step S13 in fig. 8), thesample 3 may then be moved to an area or portion for further processing. For example, thesample droplet 3 mayMove to the incubation and amplification section 6 (step S31). Theelectrode 7 on which thesample droplet 3 is held may be recorded in thememory 27 at all times together with associated data required by the user, such as time, temperature andsample 3 identification data. Thedroplet 4 and/or thesample 3 are moved back to theseparation part 46 together or separately, and the process of isolating thesample 3 may be repeated until the desired number ofsamples 3 is obtained (steps S32 and S33). For example, if thesecond droplet 4₂Without depletion, the method returns to step S25 in fig. 9. If thesecond droplet 4₂Depletion, thefirst droplet 4 may be evaluated in step S33₁. If thefirst droplet 4₁Upon exhaustion, the method returns to step S14 in fig. 8. If the first droplet has not been depleted, the method returns to step S21 in FIG. 9.

The number ofisolated sample droplets 3 will depend on the size of thedevice 2, the method used in thefurther processing section 6 and the number required by the user. For example, the method used in the further processing section may be continuous, with thesample 3 being removed from theamplification row 50 once processing is complete, thereby allowingmore sample 3 to be moved to thefurther processing section 6.

Droplet recovery and reagent addition

With particular reference to fig. 16 and 17, thedevice 2 may have additional features that assist in the nucleic acid amplification process. For example,sample 3 may be returned tosample reservoir 44 and mixed withdroplets 4 viarecovery path 85. This may occur if no particles 21 (e.g. biological cells) or nucleic acids are detected in thesample 3. Therecovery path 85 may be formed by a series ofadjacent recovery electrodes 7, 86. Typically, the at least onerecovery electrode 86 is arranged adjacent to the at least oneseparation electrode 7, 47 and the at least onerecovery electrode 86 is arranged adjacent to the at least onereservoir electrode 7, 43, bypassing thedroplet separation section 46. There may be more than onerecovery lane 85, e.g.different recovery lanes 85 may be arranged at different locations on the device, e.g. the recovery lanes may be arranged to allowdroplets 4 and/orsample 3 to move from thefurther processing portion 6 of theextraction portion 56 to thereservoir electrode 43. Thedifferent recycling paths 85 may converge towards each other. The ability to recoverdroplets 4 and/orsample droplets 3 greatly reduces waste in preparingsamples 3 for further processing and increases efficiency sinceunsuitable samples 3 do not need further processing. Thesample 3, which has been evaluated and is considered unsuitable for further processing or recovery, can be discarded via a discard electrode (not shown) path and a waste portion (not shown). The waste fraction may also be an electrode.

Still referring to fig. 16 and 17, as well as fig. 8, thedevice 2 may also include ahybrid electrode 87. The mixingelectrode 87 is typically anelectrode 7 having a larger surface area than the separatingelectrode 47. Mixingelectrode 87 is placed adjacent to other types ofelectrodes 7, for example, mixingelectrode 87 may be arranged adjacent to one ormore separation electrodes 47, allowing actuation ofdroplet 4 and/orsample 3 into the same electrode, and allowingdroplet 4 and/orsample 3 to bind. This arrangement may allow for the optional addition of other reagents (step S14 in fig. 8), such as amplification buffers, cell lysis reagents, etc., todroplet 4 orsample droplet 3 at different stages of the process. For example, with particular reference to FIG. 16, thesixth droplet 4₆Can be held above theseparation electrode 47 and theseventh droplet 4₇May remain over thefurther electrode 7 on thedevice 2. Then the sixth andseventh droplets 4₆、4₇Can be moved to an electrode adjacent to thesame mixing electrode 87 using the methods described previously. When the mixingelectrode 87 is activated by thecontroller 8, the sixth andseventh droplets 4₆、4₇Merge into aneighth droplet 4₈。

Mixingelectrodes 87 may be arranged in a row to allow actuation ofdroplets 4 and/orsample 3 to other areas, portions or portions ofdevice 2, for example toreservoir 44 or incubation andamplification portion 6. The mixingelectrode 87 may be arranged adjacent toother mixing electrodes 87, whichother mixing electrodes 87 are configured to receivedroplets 4 and/orsamples 3 from different areas, portions or locations of thedevice 2. Thesample droplet 3 may optionally undergo a purification stage (step S15). The mixingelectrodes 87 may be of a suitable shape, for example, the mixingelectrodes 5 may be square (as shown in fig. 16), or they may be octagonal (as shown in fig. 17). The mixingelectrode 87 may be irregularly shaped to allow the path of theother electrodes 7 to be able to feed thedroplet 4 and/or thesample droplet 3 into the mixingelectrode 87.

Nucleic acid amplification

With reference to fig. 8 and fig. 18 to 22, the preparation and nucleic acid amplification ofsample 3 indevice 2 will now be described. With particular reference to fig. 19, after eachsample 3 has been isolated from thedroplet 4, thesample 3 is actuated individually to anelectrode 7 in one of theparallel rows 50 ofelectrodes 7 in thecell lysis portion 58. Thesample 3 may be held at theseelectrodes 7 until a desired number ofsamples 3 have been moved to thecell lysis section 58. One of theconnected rows 54 ofelectrodes 7 can be used to move thesample 3 between therows 50 if desired.

If necessary, a cell lysis process as described above may then be performed in thecell lysis section 58 to rupture the cell walls of the biological cells in the sample 3 (step S41). Cell lysis may be performed outside thedevice 2 and the product from the process is input into thedevice 2 for further processing, such as purification and amplification.Sample 3 may already contain nucleic acid ready for amplification, e.g. the nucleic acid may have been purified. After cell lysis, the contents ofsample 3 may then be subjected to a purification step (step S15) to prepare the nucleic acid for amplification.

With particular reference to FIG. 20, when the contents ofsample 3 are ready for amplification, the sample is actuated to a firstthermal control region 36 within the incubation and amplification section 6₁(step S42). Here, thesample 3 starts the amplification process (steps S16, S43). If the amplification process is isothermal, such as MDA, LAMP, 3SR, NASBA, SDA, or RCA, thesample 3 may remain in the firstthermal control zone 36₁Until the amplification process is complete.

If the amplification process is thermal cycle based, such as PCR, qPCR, and LCR,sample 3 may be moved betweenthermal control regions 36 to facilitate nucleic acid amplification. For example, for the denaturation step, the firstthermal control zone 36₁Adjusted to between 94 ℃ and 98 ℃ to denature the nucleic acids. Thesample 3 may then be actuated to the secondthermal control region 36₂The secondthermal control region 36₂Is adjusted to between 50 ℃ and 65 ℃ to perform annealing. The sample can then be dried3 to a thirdthermal control zone 36₃Between 75 ℃ and 80 ℃ for extension and elongation. Typically,sample 3 is actuated in approximately thirty cycles between these zones; however, the number of cycles may be more or less than thirty. As previously mentioned, the number oftemperature control zones 36 will depend on the process performed in the incubation andamplification section 6. The temperature at which the thermally controlledregion 36 is maintained may be a suitable temperature for facilitating the desired portion of the amplification reaction. Thesample 3 may be moved between the temperature controlledzones 36 as many times as the amplification reaction may take place and held at each temperature for as long as the reaction may take place. There may be a plurality ofsamples 3 at different stages of amplification on thedevice 2. Different forms of amplification may occur simultaneously on thedevice 2.

Referring specifically to fig. 21,sample 3 may be monitored at intervals or continuously to assess the status of the amplification cycle and quantify the amount of deoxyribonucleic acid (DNA) (steps S17 and S44). For example, nucleic acid amplification may be quantitative PCR (qPCR, also referred to as "real-time PCR"). For example, the fluorescence of thesample 3 can be measured to assess the amount of nucleic acid that has been amplified in the reaction. This process may be performed and controlled by thespecimen inspection system 20 described previously. For example, the fluorescence of a non-specific fluorescent dye inserted into double-stranded DNA can be detected and measured. Thesample detection system 20 may be used to record, process and analyze the level of fluorescence or another marker indicative of the amount of nucleic acid present in the reaction to determine the amount of nucleic acid in thesample 3. The amplification cycle will stop when the sample emits a level of fluorescence indicating that sufficient nucleic acid has been amplified for the intended use ofsample 3. Somesamples 3 may complete the cycle beforeother samples 3.

Once the amplification reaction is complete, thesample 3 can be moved to theelectrode 7, which is maintained at a temperature between 4 ℃ and 15 ℃. The amplified nucleic acids can be temporarily stored at these temperatures.

Referring specifically to fig. 21 and 22, oncesample 3 has undergone an amplification cycle or other process, the amount of nucleic acid product can be assessed, for example, by using the fluorescence measurements described above (step S17). First, second andthird samples 3₁、3₂、3₃Has a desired level of fluorescence to indicate that nucleic acid amplification has been successful and is therefore moved to the extraction electrode.Fourth sample 3₄Also has the required level of fluorescence to indicate that nucleic acid amplification has been successful, butsample 3₄Contains two biological cells, so it is moved to thewaste electrode 61.Other samples 3 were either not fully amplified or contained no cells or nucleic acids at the start of the process.

The measured fluorescence levels, as well as other information about thesample 3, may be fed back into theinstructions 16 or forwarded to the user to allow the program or user to decide to move the sample to the outlet electrode 57 (step S45) for extraction and further processing (steps S19, S46), such as deoxyribonucleic acid sequencing, or to move the droplets to the waste electrode 61 (step S18), where thesample 3 may be removed from thedevice 2. For example, in a high throughput process for single cell whole genome amplification,sample 3, single cell or multiple cells without cells may undergo lysis, amplification and incubation processes, but then be discarded to waste 61 without extraction, while the sample identified as having a single cell in step S13 is moved toextraction electrode 57. Thus, thedevice 2 can automatically move thesample 3 without single cells and anysample 3 whose nucleic acids have not been sufficiently amplified for further processing to thewaste electrode 61. With such adevice 2, the position of allsamples 3 and associated metadata, e.g. the number and type of cells orparticles 21 present in eachsample 3 before the cell lysis stage, can be easily stored. Such a system may eliminate the need for tracking markers, such as bar codes attached to the tube. Furthermore, thedevice 2 can be easily scaled, allowing a large number of sample droplets to be amplified simultaneously, and the quality of the product can be automatically assessed and selected for further processing.

Cell culture and incubation

Thedevice 2 may be used for culturing cells. For example, individual cells or groups of cells in thesample 3 may be transferred to the incubation andamplification section 6. Here, the cells may be immobilized by active (e.g., modifying thelid 77 to enable physical entrapment in the porous matrix) or passive cell immobilization (not shown). The incubation andamplification section 6 can be thermally regulated by athermal control system 34 to a temperature that promotes cell culture, allowing cells to grow and divide to form a larger cell population. For example, the temperature of the incubation andconditioning portion 6 may be controlled between 37 ℃ and 38 ℃, however, the temperature range will depend on the requirements of one or more cells in thesample 3. Depending on the evaluation of thespecimen inspection system 20, these cell populations may be moved to theextraction electrode 57 for further processing, or they may be moved to waste.

Additional reagents used in the culture, growth and handling of cells (such as cell release and cell nutrient solutions) may be added to thesample 3 before, during or after incubation to aid in the movement, culture, examination and evaluation of the cell population. Additional reagents may be added to thesample 3 containing one or more cells via connecting electrodes (not shown) arranged adjacent to the connectingrow 54 and/or the incubation andamplification row 50. Additional reagents may be added to thesample 3 before thesample 3 is moved to the incubation andamplification part 6 via the mixingelectrode 87. By splitting thesample 3 using a method similar to the second phase ofdroplet 4 splitting described above, cellular waste can be removed from the sample in the incubation andamplification section 6. Additional buffers, nutrients or other reagents may then be added to the cell-containingsample 3 to dilute the waste concentration in thesample 3.

Modifying

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

Although claims have been formulated in this application to particular combinations of features, it should be understood that the scope of the disclosure of the present invention also includes any novel feature or any novel combination of features disclosed herein either explicitly or implicitly or any generalisation thereof, whether or not it relates to the same invention as presently claimed in any claim and whether or not it mitigates any or all of the same technical problems as does the present invention. The applicants hereby give notice that new claims may be formulated to such features and/or combinations of such features during the prosecution of the present application or of any further application derived therefrom.

Claims (15)

Translated fromChinese

1.一种方法，包括：1. A method comprising:激活介质上电润湿器件(2)中的第一组连续电极、第二组连续电极和第三组连续电极(7)，第三组电极插在第一组电极和第二组电极之间，以在所述第一组和第二组电极之间形成路径(48)，从而使得第一液滴(4₁)保持在所述第一组电极、第二组电极和第三组电极上方；A first set of continuous electrodes, a second set of continuous electrodes and a third set of continuous electrodes (7) in the electrowetting device (2) are activated, the third set of electrodes being interposed between the first set of electrodes and the second set of electrodes , to form a path (48) between the first and second sets of electrodes such that a_first droplet (41) remains over the first, second and third sets of electrodes ;去激活所述第三组电极中的至少一个电极，以便断开所述第一组电极和所述第二组电极之间的路径，并使所述第一液滴分成分别保持在所述第一组电极上方的第二液滴和所述第二组电极上方的第三液滴(4₂，4₃)；Deactivating at least one electrode of the third set of electrodes to break the path between the first set of electrodes and the second set of electrodes and to separate the first droplets into remaining at the first set of electrodes, respectively. a second droplet over a set of electrodes and a third droplet over the second set of electrodes (4₂ , 4₃ );激活第四组连续电极，所述第四组连续电极包括所述第二组电极中的至少一个电极，并且形成具有第一和第二相对端的一行连续电极，以使所述第三液滴拉长；activating a fourth set of continuous electrodes comprising at least one electrode of the second set of electrodes and forming a row of continuous electrodes having first and second opposing ends to pull the third droplet long;激活与第四组电极的第一端相邻的第五组连续电极，并去激活所述第四组电极中的在第一端和第二端之间的的至少一个电极，以及激活所述第四组电极的第二端处或邻近的至少一个电极，以使所述第三液滴分成第四液滴(4₄)和第五液滴(4₅，3)。activating a fifth set of continuous electrodes adjacent to the first end of the fourth set of electrodes, deactivating at least one electrode of the fourth set of electrodes between the first end and the second end, and activating the At least one electrode at or adjacent the second end of the fourth set of electrodes to separate the third droplet into a_fourth droplet (44) and a_fifth droplet (45,3).2.根据权利要求1所述的方法，所述方法还包括：2. The method of claim 1, further comprising:使用传感器确定所述第五液滴内的粒子(21)的数量；using a sensor to determine the number of particles (21) within the fifth droplet;根据所述第五液滴中的粒子的数量：According to the number of particles in the fifth droplet:将所述第五液滴移动到第一处理部分；或者moving the fifth droplet to the first processing section; or经由包括回收电极(86)的回收路径(85)将所述第五液滴移动到第二处理部分。The fifth droplet is moved to the second processing section via a recovery path (85) comprising a recovery electrode (86).3.根据权利要求1或2所述的方法，所述方法还包括：3. The method of claim 1 or 2, further comprising:激活第六组连续电极和第七组连续电极，以便使第六液滴和第七液滴(4)分别保持在每组电极上方，其中每组电极被布置成为在它们之间设置有间隔，并且还被布置成与去激活的一组连续混合电极(87)相邻；以及activating the sixth set of continuous electrodes and the seventh set of continuous electrodes such that the sixth and seventh droplets (4) are held over each set of electrodes, respectively, wherein each set of electrodes is arranged with a space therebetween, and also arranged adjacent to a deactivated set of continuous mixing electrodes (87); and此后，激活该组混合电极并去激活所述第六组连续电极或第七组连续电极中的至少一组，以使保持在所述第六组连续电极和第七组连续电极上方的所述液滴结合成保持在至少一组连续电极和/或该组混合电极上方的单个液滴；Thereafter, the set of hybrid electrodes is activated and at least one of the sixth set of continuous electrodes or the seventh set of continuous electrodes is deactivated, so that the the droplets coalesce into a single droplet held over at least one set of continuous electrodes and/or the set of mixed electrodes;其中所述第六组连续电极和第七组连续电极能够包括来自所述第一至第五组连续电极中的任一组的电极。Wherein the sixth set of continuous electrodes and the seventh set of continuous electrodes can comprise electrodes from any of the first to fifth sets of continuous electrodes.4.根据权利要求2或3所述的方法，其中，通过使用相机(22)捕获保持在电极上方的液滴的图像，并在处理器(12)中使用机器视觉分析所述图像，来确定所述液滴中粒子的数量。4. A method according to claim 2 or 3, wherein the determination is made by capturing an image of the droplet held above the electrode using a camera (22) and analyzing the image using machine vision in the processor (12) The number of particles in the droplet.5.根据权利要求2至4中任一项所述的方法，其中所述粒子是生物细胞。5. The method of any one of claims 2 to 4, wherein the particles are biological cells.6.根据权利要求2至5中任一项所述的方法，所述方法还包括：6. The method of any one of claims 2 to 5, further comprising:激活电极以便对移至第一处理部分的样本液滴进行第一过程，其中所述第一过程是热调节过程。The electrodes are activated to perform a first process on the sample droplet moved to the first processing section, wherein the first process is a thermal conditioning process.7.一种计算机程序(16)，其在由至少一个处理器(12)执行时，使所述至少一个处理器执行根据权利要求1至6中任一项所述的方法。7. A computer program (16) which, when executed by at least one processor (12), causes the at least one processor to perform the method according to any one of claims 1 to 6.8.一种计算机程序产品，包括存储根据权利要求7所述的计算机程序的计算机可读介质(13)。8. A computer program product comprising a computer readable medium (13) storing the computer program according to claim 7.9.一种装置，包括：9. An apparatus comprising:介质上电润湿器件(2)；以及an electrowetting device on dielectric (2); and控制器(8)；controller(8);所述控制器被配置为激活所述介质上电润湿器件中的第一组连续电极、第二组连续电极和第三组连续电极(7)，第三组电极插入第一组电极和第二组电极之间，以在所述第一组电极和第二组电极之间形成路径(48)，从而使得第一液滴(4₁)保持在第一组电极、第二组电极和第三组电极上方；The controller is configured to activate a first set of continuous electrodes, a second set of continuous electrodes and a third set of continuous electrodes (7) in the electrowetting device on a dielectric, the third set of electrodes being inserted into the first set of electrodes and the third set of electrodes. between the two sets of electrodes to form a path (48) between the first set of electrodes and the second set of electrodes so that the_first droplet (41) is held between the first set of electrodes, the second set of electrodes and the second set of electrodes Above the three sets of electrodes;去激活所述第三组电极中的至少一个电极，从而断开所述第一组电极和第二组电极之间的路径，并使所述第一液滴分成分别保持在所述第一组电极和第二组电极上方的第二液滴和第三液滴(4₂,4₃)；Deactivating at least one electrode of the third set of electrodes, thereby breaking the path between the first set of electrodes and the second set of electrodes, and causing the first droplets to separate into each remaining in the first set the second and third droplets (4₂ , 4₃ ) above the electrodes and the second set of electrodes;激活第四组连续电极，所述第四组连续电极包括所述第二组电极中的至少一个电极，并且形成具有第一和第二相对端的一行连续电极，以使所述第三液滴拉长；activating a fourth set of continuous electrodes comprising at least one electrode of the second set of electrodes and forming a row of continuous electrodes having first and second opposing ends to pull the third droplet long;激活与第四组电极的第一端相邻的第五组连续电极，并去激活所述第四组电极中的在第一端和第二端之间的至少一个电极，以及激活所述第四组电极的第二端处或邻近的至少一个电极，以使所述第三液滴分成第四液滴(4₄)和第五液滴(4₅，3)。activating a fifth set of continuous electrodes adjacent to the first end of the fourth set of electrodes, deactivating at least one electrode of the fourth set of electrodes between the first end and the second end, and activating the fourth set of electrodes At least one electrode at or adjacent the second end of the four sets of electrodes to separate the third droplet into a_fourth droplet (44) and a_fifth droplet (45,3).10.根据权利要求9所述的装置，还包括处理器和传感器，以用于确定液滴(4，3)中的粒子的数量；以及10. The apparatus of claim 9, further comprising a processor and a sensor for determining the number of particles in the droplet (4, 3); and控制器，所述控制器被配置成，根据所述第五液滴中的粒子的数量：a controller configured to, based on the number of particles in the fifth droplet:激活和去激活电极以将所述第五液滴移至第一处理部分；或者activating and deactivating electrodes to move the fifth droplet to the first treatment section; or激活和去激活连续回收电极(86)的路径(85)，以将所述第五液滴移至第二处理部分。The path (85) of the continuous recovery electrode (86) is activated and deactivated to move the fifth droplet to the second processing section.11.根据权利要求9或10所述的装置，还包括相机(22)、镜头(23)和处理器(12)，其中通过使用相机和镜头捕获保持在电极上方的液滴的图像并在处理器(12)中使用机器视觉分析所述图像来确定液滴中的粒子的数量。11. Apparatus according to claim 9 or 10, further comprising a camera (22), a lens (23) and a processor (12), wherein images of the droplets held above the electrodes are captured and processed by using the camera and the lens Machine vision is used in the device (12) to analyze the image to determine the number of particles in the droplet.12.根据权利要求9至11中任一项所述的装置，还包括：12. The apparatus of any one of claims 9 to 11, further comprising:与所述第四组电极相邻布置的第一处理部分(6)，其包括一组连续的热控区(36)，以促进核酸扩增；以及a first processing portion (6) disposed adjacent to said fourth set of electrodes, comprising a set of contiguous thermal control zones (36) to facilitate nucleic acid amplification; and所述控制器被配置为激活和去激活电极，以便对移至所述第一处理部分的样本液滴进行第一过程，其中所述第一过程是单细胞核酸扩增。The controller is configured to activate and deactivate electrodes to perform a first process on the sample droplet moved to the first processing portion, wherein the first process is single-cell nucleic acid amplification.13.根据权利要求9至12中任一项所述的装置，其中所述电极或连续电极的组是六边形或八边形的。13. The device of any one of claims 9 to 12, wherein the electrode or group of continuous electrodes is hexagonal or octagonal.14.根据权利要求9至13中任一项所述的装置，其中，所述控制器还被配置成：14. The apparatus of any one of claims 9 to 13, wherein the controller is further configured to:激活第六组连续电极和第七组连续电极，以便使第六液滴和第七液滴(3)分别保持在每组电极上方，其中每组电极被布置成在它们之间设置有间隔，并且还被布置成与去激活的一组连续混合电极(57)相邻，以及；activating the sixth set of continuous electrodes and the seventh set of continuous electrodes such that the sixth and seventh droplets (3) are held over each set of electrodes, respectively, wherein each set of electrodes is arranged with a space therebetween, and also arranged adjacent to the deactivated set of continuous mixing electrodes (57), and;激活该组混合电极并去激活所述第六组连续电极或所述第七组连续电极中的至少一组，以使保持在所述第六组连续电极和所述第七组连续电极上方的液滴结合成保持在至少一组连续电极和/或该组混合电极上方的单个液滴；Activating the set of hybrid electrodes and deactivating at least one of the sixth set of continuous electrodes or the seventh set of continuous electrodes so that the electrodes held above the sixth set of continuous electrodes and the seventh set of continuous electrodes the droplets coalesce into a single droplet held over at least one set of continuous electrodes and/or the set of mixed electrodes;其中所述第六组连续电极和所述第七组连续电极能够包括来自所述第一组连续电极至所述第五组连续电极中任一组的电极。Wherein the sixth set of continuous electrodes and the seventh set of continuous electrodes can include electrodes from any of the first set of continuous electrodes to the fifth set of continuous electrodes.15.一种系统(1)，包括：15. A system (1) comprising:根据权利要求9至14中任一项所述的装置(2)；以及The device (2) according to any one of claims 9 to 14; and包括可操作地连接到所述控制器(8)的存储器(13)的计算机系统(11)，所述计算机系统被配置为执行根据权利要求1至8中任一项所述的方法，并在所述存储器中记录至少一个液滴的位置。A computer system (11) comprising a memory (13) operatively connected to the controller (8), the computer system being configured to perform the method according to any one of claims 1 to 8, and in The position of at least one droplet is recorded in the memory.

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