Digital microfluidic chip, and method for performing droplet generation and library preparation using same

Abstract

A digital microfluidic chip, and a method for performing droplet generation and library preparation using same. The digital microfluidic chip comprises: a droplet generation structure, which is configured to generate droplets having various volumes, wherein the droplet generation structure comprises one or more droplet generation modules, each droplet generation module is configured to generate at least one of a first droplet, a second droplet and a third droplet, and the first droplet, the second droplet and the third droplet have a first volume, a second volume and a third volume, respectively, which volumes are different from each other; a droplet receiving module, which is configured to implement at least one of mixing and reaction of a plurality of droplets; and an interconnection module, which is configured to drive the first droplet, the second droplet and the third droplet, and to connect the droplet generation structure to the droplet receiving module.

Description

Digital microfluidic chip and method for droplet generation and library preparation using the same Technical Field

[0001] Embodiments of the present disclosure relate to a digital microfluidic chip and methods for performing droplet generation and library preparation using the same. Background Art

[0002] Microfluidics is a technology that can precisely control and manipulate microscale fluids. It can integrate basic operations such as sample preparation, reaction, separation, and detection in biochemical processes onto a centimeter-scale chip. This technology is generally used in the analysis of trace drugs in the fields of biology, chemistry, and pharmaceuticals. Digital microfluidic chips use droplets as the manipulation objects, controlling the movement of droplets through electric field forces, thermal forces, electrostatic forces, and the like, and can independently control each droplet. Microfluidics technology requires small amounts of samples and reagents, has a simple structure, and is easy to integrate on a large scale. Droplet manipulation is more convenient, and droplet size and shape are controllable. It has the advantages of miniaturization, integration, low cost, high sensitivity, and high flexibility. It greatly speeds up the movement, extraction, separation, mixing, and detection of samples, and shows great prospects in many fields such as physics, chemistry, biology, and medicine.

[0003] Digital microfluidic chips are particularly well-suited for complex biochemical reactions. Passive digital microfluidic chips have become the mainstream chip solution for commercial digital microfluidic products due to their significant cost advantages. However, passive digital microfluidic chips suffer from issues such as a limited number of controllable electrodes and limited flexibility in liquid manipulation volume. Biochemical processes may require droplets of multiple volumes and types, with a wide range of droplet volumes. Therefore, improving the droplet manipulation volume range and flexibility of digital microfluidic chips remains a current technical challenge.

[0004] Summary of the Invention

[0005] According to at least one embodiment of the present disclosure, a digital microfluidic chip is provided, comprising: a droplet generating structure configured to generate droplets having various volumes, and the droplet generating structure comprises: one or more droplet generating modules, wherein each droplet generating module is configured to generate at least one of a first droplet, a second droplet, and a third droplet, and the first droplet, the second droplet, and the third droplet respectively have a first volume, a second volume, and a third volume that are different from each other; a droplet receiving module configured to achieve at least one of mixing and reaction of multiple droplets; and an interconnection module configured to drive the first droplet, the second droplet, and the third droplet, and connect the droplet generating structure to the droplet receiving module.

[0006] In the digital microfluidic chip provided by at least one embodiment of the present disclosure, the interconnection module includes: one or more droplet driving modules, wherein each droplet driving module is configured to drive at least one of the first droplet, the second droplet, and the third droplet, and the droplet volume that can be driven by a single driving electrode in the droplet driving module is the first volume, the second volume, and the third volume, respectively.

[0007] In the digital microfluidic chip provided in at least one embodiment of the present disclosure, the one or more droplet driving modules include: at least one first droplet driving module, wherein the single driving electrode in each first droplet driving module is configured to drive the first droplet; at least one second droplet driving module, wherein the single driving electrode in each second droplet driving module is configured to drive the second droplet; and at least one third droplet driving module, wherein the single driving electrode in each third droplet driving module is configured to drive the third droplet.

[0008] In the digital microfluidic chip provided in at least one embodiment of the present disclosure, the second volume is larger than the minimum volume of droplets that can be driven by the first droplet driving module, and smaller than the maximum volume of droplets that can be driven by the first droplet driving module; the third volume is larger than the minimum volume of droplets that can be driven by the second droplet driving module, and smaller than the maximum volume of droplets that can be driven by the second droplet driving module.

[0009] In the digital microfluidic chip provided in at least one embodiment of the present disclosure, the first volume is smaller than the second volume and the third volume, the second volume and the third volume are both integer multiples of the first volume, and the second volume and the third volume are different.

[0010] In the digital microfluidic chip provided in at least one embodiment of the present disclosure, the second volume is twice the first volume, and the third volume is five times the first volume; the first volume is the minimum unit volume of droplets that can be manipulated by the digital microfluidics, and the digital microfluidic chip is configured to manipulate liquids in a volume range from the first volume to twenty times the first volume, with a step size of the minimum unit volume.

[0011] In the digital microfluidic chip provided in at least one embodiment of the present disclosure, the one or more droplet generation modules include: at least one first droplet generation module, wherein each first droplet generation module is configured to generate the first droplet; at least one second droplet generation module, wherein each second droplet generation module is configured to generate the second droplet; and at least one third droplet generation module, wherein each third droplet generation module is configured to generate the third droplet.

[0012] In the digital microfluidic chip provided by at least one embodiment of the present disclosure, each of the first droplet generation module, the second droplet generation module, and the third droplet generation module includes a first drive electrode, a second drive electrode, a third drive electrode, a fourth drive electrode, a fifth drive electrode, and a sixth drive electrode; and the drive electrode with the smallest area in each droplet generation module can accommodate and drive a droplet volume that is approximately equal to the droplet volume of a single droplet generated by the corresponding droplet generation module.

[0013] In the digital microfluidic chip provided by at least one embodiment of the present disclosure, in each of the first droplet generation module to the third droplet generation module, the second drive electrode, the third drive electrode, the fourth drive electrode and the sixth drive electrode have the same first area as each other, and are arranged in sequence along the first sub-direction, and the sixth drive electrode is connected to the drive electrode in the interconnection module; the fifth drive electrode includes two fifth drive sub-electrodes electrically connected to each other, and the two fifth drive sub-electrodes are arranged on opposite sides of the second drive electrode and the third drive electrode in a second sub-direction perpendicular to the first sub-direction, and each fifth drive sub-electrode has a second area; and the first drive electrode is arranged on a side of the second drive electrode away from the third drive electrode in the first sub-direction, and is connected to the second drive electrode and the fifth drive electrode, and the first drive electrode has a third area; wherein the first area is smaller than the second area, and the second area is smaller than the third area.

[0014] In the digital microfluidic chip provided by at least one embodiment of the present disclosure, each of the first droplet generation module and the second droplet generation module further includes an additional drive electrode, which is located on a side of the first drive electrode away from the second drive electrode and the fifth drive electrode, and the additional drive electrode has the same third area as the corresponding first drive electrode.

[0015] In the digital microfluidic chip provided by at least one embodiment of the present disclosure, each of the first droplet generation module and the second droplet generation module further includes an additional dummy electrode, which is arranged on a side of the additional driving electrode away from the first driving electrode.

[0016] In the digital microfluidic chip provided by at least one embodiment of the present disclosure, the first area, second area, and third area of ​​the corresponding driving electrodes in the first droplet generation module are respectively smaller than the first area, second area, and third area of ​​the corresponding driving electrodes in the second droplet generation module, and the first area, second area, and third area of ​​the corresponding driving electrodes in the second droplet generation module are respectively smaller than the first area, second area, and third area of ​​the corresponding driving electrodes in the third droplet generation module.

[0017] In the digital microfluidic chip provided in at least one embodiment of the present disclosure, the multiple droplet generation modules include: at least one fourth droplet generation module, wherein each fourth droplet generation module is configured to generate at least two of the first droplet, the second droplet and the third droplet.

[0018] In the digital microfluidic chip provided by at least one embodiment of the present disclosure, each fourth droplet generation module includes a first drive electrode, a second drive electrode, a third drive electrode, a fourth drive electrode, a fifth drive electrode, a sixth drive electrode, a first additional drive electrode, a second additional drive electrode, and two third additional drive electrodes; the second drive electrode, the third drive electrode, the fourth drive electrode, the sixth drive electrode, the first additional drive electrode, and the second additional drive electrode are arranged in sequence along a first sub-direction, and the two third additional drive electrodes are arranged on opposite sides of the sixth drive electrode along a second sub-direction perpendicular to the first sub-direction; the second drive electrode to the fourth drive electrode have the same first main body area, and the sum of the areas of the sixth drive electrode and the two third additional drive electrodes is approximately equal to the first main body area; the first additional drive electrode and the second additional drive electrode have the same second main body area; the fifth drive electrode includes two fifth drive sub-electrodes, which are arranged on opposite sides of the second drive electrode and the third drive electrode in the second sub-direction; and the first drive electrode is arranged on a side of the second drive electrode and the fifth drive electrode away from the fourth drive electrode.

[0019] In the digital microfluidic chip provided by at least one embodiment of the present disclosure, the fourth droplet generation module is configured to generate the second droplet, the third droplet or an additional droplet, the volume of the additional droplet is approximately equal to the third volume minus the second volume, and the volume of the droplet that can be accommodated and driven by each drive electrode with the second main body area is approximately equal to the second volume of the second droplet, and the volume of the droplet that can be accommodated and driven by a single drive electrode or a combination of drive electrodes with the first main body area is approximately equal to the third volume of the third droplet; or the fourth droplet generation module is configured to generate the first droplet or the second droplet, and the volume of the droplet that can be accommodated and driven by each drive electrode with the second main body area is approximately equal to the first volume of the first droplet, and the volume of the droplet that can be accommodated and driven by a single drive electrode or a combination of drive electrodes with the first main body area is approximately equal to the second volume of the second droplet.

[0020] In the digital microfluidic chip provided by at least one embodiment of the present disclosure, the second additional driving electrode is connected to the driving electrode in the interconnection module.

[0021] The digital microfluidic chip provided in at least one embodiment of the present disclosure further includes a fourth additional drive electrode and a fifth additional drive electrode, which are arranged along the first sub-direction on a side of the second additional drive electrode away from the first additional drive electrode, and the fourth additional drive electrode and the fifth additional drive electrode have the same third main body area, and the third main body area is smaller than the second main body area.

[0022] In the digital microfluidic chip provided in at least one embodiment of the present disclosure, the droplet receiving module is configured to at least achieve mixing and a first reaction of liquids within a first mixing volume range and to achieve mixing and a second reaction of liquids within a second mixing volume range.

[0023] In the digital microfluidic chip provided in at least one embodiment of the present disclosure, the droplet receiving module includes: a first reaction zone, configured to at least achieve mixing of liquids within the first mixing volume and the first reaction, wherein the first reaction zone is connected to a third droplet driving module in the interconnection module.

[0024] In the digital microfluidic chip provided in at least one embodiment of the present disclosure, the first reaction zone includes a plurality of first reaction electrodes arranged in an array, and the volume of droplets that can be accommodated by each first reaction electrode is greater than the first volume and the second volume and smaller than the third volume, and the maximum value of the first mixed volume range of mixed droplets that can be accommodated by the plurality of first reaction electrodes is an integer multiple of the third volume.

[0025] The digital microfluidic chip provided in at least one embodiment of the present disclosure further includes: a second reaction zone, configured to achieve mixing of liquids within the second mixing volume and the second reaction, wherein the second reaction zone includes a plurality of second reaction electrodes, and the volume of droplets that can be accommodated and driven by each second reaction electrode is an integer multiple of the third volume.

[0026] In the digital microfluidic chip provided by at least one embodiment of the present disclosure, the second reaction area and the third droplet driving module share the same electrode array.

[0027] In the digital microfluidic chip provided in at least one embodiment of the present disclosure, the second reaction zone includes a temperature control zone, and is configured to control the liquid in the temperature control zone to react at a temperature within a predetermined temperature range.

[0028] In the digital microfluidic chip provided in at least one embodiment of the present disclosure, the temperature control zone includes a first temperature control zone, a second temperature control zone and a third temperature control zone, which are respectively configured to control the liquid located in the corresponding area to react at temperatures within a first temperature range, a second temperature range and a third temperature range that are different from each other.

[0029] In the digital microfluidic chip provided by at least one embodiment of the present disclosure, the droplet generating structure and the interconnection module are located between the first reaction area and the second reaction area.

[0030] In the digital microfluidic chip provided in at least one embodiment of the present disclosure, the first reaction zone serves as a common reaction zone and is configured to achieve mixing of liquids within the first mixing volume range and the first reaction and to achieve mixing of liquids within the second mixing volume range and the second reaction in different operation steps.

[0031] In the digital microfluidic chip provided in at least one embodiment of the present disclosure, the droplet generating structure further includes a droplet trimming module, which is connected to the one or more droplet generating modules and is configured to trim the droplets from the one or more droplet generating modules into droplets having a target volume.

[0032] In the digital microfluidic chip provided in at least one embodiment of the present disclosure, the droplet cutting module and the first droplet driving module in the interconnection module share the same electrode array, and the electrode array is directly connected to the first droplet generating module.

[0033] In the digital microfluidic chip provided in at least one embodiment of the present disclosure, the droplet cutting module includes a plurality of main cutting electrodes, and the volume of droplets that can be accommodated and driven by each main cutting electrode is substantially equal to the first volume.

[0034] In the digital microfluidic chip provided in at least one embodiment of the present disclosure, the multiple main cutting electrodes are arranged along a first direction, and the end main cutting electrodes among the multiple main cutting electrodes located at the end of the droplet cutting module in the first direction are connected to the interconnection module.

[0035] In the digital microfluidic chip provided by at least one embodiment of the present disclosure, one or more first droplet generation modules among the multiple droplet generation modules for generating the first droplets are located on one side or opposite sides of the droplet cutting module in a second direction perpendicular to the first direction.

[0036] In the digital microfluidic chip provided in at least one embodiment of the present disclosure, the droplet cutting module further includes an auxiliary cutting electrode, which is connected to the main cutting electrode close to the interconnection module and is connected to the interconnection module.

[0037] In the digital microfluidic chip provided in at least one embodiment of the present disclosure, the multiple main cutting electrodes have the same first width as each other, the auxiliary cutting electrodes have a second width, and the second width is greater than the first width, and the first width and the second width are widths along the first direction.

[0038] In the digital microfluidic chip provided by at least one embodiment of the present disclosure, the auxiliary cutting electrodes are located on opposite sides of the main cutting electrode close to the interconnection module in a second direction perpendicular to the first direction.

[0039] In the digital microfluidic chip provided by at least one embodiment of the present disclosure, the number of the second droplet driving modules is greater than the number of the first droplet driving modules and the number of the third droplet driving modules.

[0040] In the digital microfluidic chip provided in at least one embodiment of the present disclosure, the droplet generation module for generating the second droplet and the third droplet in the one or more droplet generation modules is directly connected to one or more second droplet driving modules.

[0041] In the digital microfluidic chip provided in at least one embodiment of the present disclosure, it includes a first substrate, wherein the first substrate includes: a first base substrate; a first conductive layer, located on one side of the first base substrate; a second conductive layer, located on a side of the first conductive layer away from the base substrate, and corresponding parts of the first conductive layer and the second conductive layer are electrically connected to each other; a dummy layer, located between the first conductive layer and the second conductive layer, and is electrically floating or grounded; and a first hydrophobic layer, located on a side of the second conductive layer away from the first base substrate.

[0042] In the digital microfluidic chip provided by at least one embodiment of the present disclosure, the second conductive layer includes the droplet generating structure, the droplet receiving module and multiple driving electrodes of the interconnection module, and the multiple driving electrodes are spaced apart from each other; the dummy layer includes multiple dummy electrodes spaced apart from each other, and the outer contour of the positive projection of each dummy electrode on the first substrate coincides with the outer contour of the positive projection of the corresponding driving electrode among the multiple driving electrodes on the substrate.

[0043] In the digital microfluidic chip provided by at least one embodiment of the present disclosure, the multiple dummy electrodes each have an opening, and the multiple driving electrodes are respectively connected to the first conductive layer through a plurality of conductive through holes, and the multiple conductive through holes are respectively located in the openings corresponding to the multiple dummy electrodes and separated from the multiple dummy electrodes.

[0044] In the digital microfluidic chip provided by at least one embodiment of the present disclosure, the first conductive layer includes a plurality of conductive traces, and the plurality of driving electrodes are connected to the binding electrodes through the plurality of conductive traces.

[0045] In the digital microfluidic chip provided in at least one embodiment of the present disclosure, the first conductive layer and the second conductive layer include transparent conductive materials, and the dummy layer includes opaque conductive materials.

[0046] In the digital microfluidic chip provided by at least one embodiment of the present disclosure, the dummy layer includes a metal material.

[0047] In the digital microfluidic chip provided in at least one embodiment of the present disclosure, the dummy layer further includes one or more additional dummy electrodes of the droplet generation module, and the orthographic projection of the additional dummy electrodes on the first substrate does not overlap with the second conductive layer.

[0048] The digital microfluidic chip provided in at least one embodiment of the present disclosure also includes: a second substrate, arranged opposite to the first substrate; wherein the second substrate includes: a second base substrate; a third conductive layer, located on one side of the second base substrate; and a second hydrophobic layer, located on the side of the third conductive layer away from the second base substrate; and a droplet channel, located between the first hydrophobic layer of the first substrate and the second hydrophobic layer of the second substrate; wherein the second substrate is provided with a plurality of sample inlets passing through the second substrate, and the plurality of sample inlets are respectively arranged corresponding to the plurality of droplet generation modules; for the droplet generation module including the additional dummy electrode, the orthographic projection of the additional dummy electrode on the main surface of the first substrate facing the second substrate overlaps with the orthographic projection of the corresponding sample inlet on the main surface of the first substrate.

[0049] In the digital microfluidic chip provided in at least one embodiment of the present disclosure, the one or more droplet generation modules include a droplet generation module without an additional dummy electrode, and the orthographic projection of the sample inlet corresponding to the droplet generation module on the main surface of the first substrate is close to the orthographic projection of the driving electrode of the droplet generation module on the main surface of the first substrate, and does not overlap with the orthographic projection of the driving electrode.

[0050] At least one embodiment of the present disclosure provides a method for generating droplets of various volumes using the droplet generating structure in the digital microfluidic chip described in any one of the above items, including at least one of the following: using a droplet generating module to generate one or more first droplets each having a first volume; using a droplet generating module to generate one or more second droplets each having a second volume; using a droplet generating module to generate one or more third droplets each having a third volume, the first volume being smaller than the second volume, and the second volume being smaller than the third volume; using a droplet generating module and a droplet cutting module in combination to generate at least one of one or more fourth droplets each having a fourth volume and one or more fifth droplets each having a fifth volume, the fourth volume and the fifth volume being larger than the second volume and smaller than the third volume; and mixing at least two of the one or more first droplets, the one or more second droplets, the one or more third droplets, the one or more fourth droplets, and the one or more fifth droplets to obtain droplets with other target volumes.

[0051] In the above method provided by at least one embodiment of the present disclosure, the combination of the droplet generation module and the droplet trimming module to generate the fourth droplet includes: using the third droplet generation module to generate an initial droplet having the third volume; and trimming two trimmed droplets each having the first volume from the initial droplet by the droplet trimming module, and moving the two trimmed droplets to a waste liquid area, wherein the remaining droplets after the trimming of the initial droplet form the fourth droplet.

[0052] In the above method provided by at least one embodiment of the present disclosure, the generation of the fifth droplet by combining the droplet generation module and the droplet trimming module includes: using the third droplet generation module to generate an initial droplet having the third volume; and trimming a trimmed droplet having the first volume from the initial droplet by the droplet trimming module, and moving the trimmed droplet to a waste liquid area, wherein the remaining droplets after the trimming of the initial droplet form the fifth droplet.

[0053] At least one embodiment of the present disclosure provides a method for preparing a gene library using the digital microfluidic chip described in any one of the above items, comprising: adding a deoxyribonucleotide sample solution, a ligase buffer, a linker solution, and a ligase solution to the corresponding droplet generation modules in the multiple droplet generation modules, and generating sample droplets, ligase buffer droplets, linker droplets, and ligase droplets through the droplet generation structure; moving the sample droplets, the ligase buffer droplets, the linker droplets, and the ligase droplets to the droplet receiving module through the interconnection module, and the sample droplets, the ligase buffer droplets, the linker droplets, and the ligase droplets are mixed and reacted in the first reaction zone of the droplet receiving module to form a sample droplet. The invention relates to a method for preparing a mixed droplet; performing deoxyribonucleotide adsorption and elution on the sample mixed droplet to obtain a first deoxyribonucleotide chain; adding an amplification solution and a primer solution to corresponding droplet generation modules in the multiple droplet generation modules, and generating amplification droplets and primer droplets through the droplet generation structure; moving the amplification droplet and the primer droplet to the droplet receiving module through the interconnection module, and the amplification droplet and the primer droplet undergo polymerase chain reaction with the first deoxyribonucleotide chain in the droplet receiving module to form amplified deoxyribonucleotide mixed droplets; and performing deoxyribonucleotide adsorption and elution on the amplified deoxyribonucleotide mixed droplet to obtain an amplified deoxyribonucleotide chain.

[0054] In the above method provided by at least one embodiment of the present disclosure, the sample droplet has a sixth volume, and generating the sample droplet includes: adding a deoxyribonucleotide sample solution at a third droplet generation module, and generating a first sample droplet and a second sample droplet each having a third volume through the third droplet generation module, moving the second sample droplet to the droplet trimming module through the interconnection module, and trimming the second sample droplet into a trimmed sample droplet having the second volume and a third sample droplet having the remaining volume through the droplet trimming module, the first sample droplet and the third sample droplet constituting the sample droplet having the sixth volume.

[0055] In the above method provided by at least one embodiment of the present disclosure, generating the ligase buffer droplet, the linker droplet, and the ligase droplet includes: adding a ligase buffer at a second droplet generation module, and generating a ligation buffer droplet with a second volume through the second droplet generation module; adding a ligase solution at the second droplet generation module, and generating two ligase sub-droplets each with a second volume through the second droplet generation module, the two ligase sub-droplets constituting the ligase droplet; adding a linker solution at the first droplet generation module, and generating a linker droplet with a first volume through the first droplet generation module. BRIEF DESCRIPTION OF THE DRAWINGS

[0056] In order to more clearly illustrate the technical solutions of the embodiments of the present disclosure, the drawings of the embodiments will be briefly introduced below. Obviously, the drawings in the following description only relate to some embodiments of the present disclosure, rather than limiting the present disclosure.

[0057] Figure 1A is a schematic top view of a digital microfluidic chip according to some embodiments of the present disclosure; Figure 1B is a schematic top view of an upper substrate in a digital microfluidic chip according to some embodiments of the present disclosure; and Figure 1C is a schematic cross-sectional view of the digital microfluidic chip taken along line II' in Figure 1A according to some embodiments of the present disclosure.

[0058] 2A to 2C illustrate top views of driving electrode structures in a digital microfluidic chip according to some embodiments of the present disclosure.

[0059] 3A to 3C are schematic top views of various droplet generation modules in a digital microfluidic chip according to some embodiments of the present disclosure.

[0060] 4A to 4C are schematic top views of various droplet generation modules in a digital microfluidic chip according to some embodiments of the present disclosure.

[0061] 5A to 5K are schematic top views of a driving method for generating droplets of various volumes using a droplet generation module according to some embodiments of the present disclosure.

[0062] FIG6 is a schematic top view of a portion of a droplet receiving module according to some embodiments of the present disclosure.

[0063] 7A to 7D are schematic top views of various electrode arrays in a digital microfluidic chip according to some embodiments of the present disclosure.

[0064] 8A to 8C are schematic diagrams showing the use of a digital microfluidic chip to generate droplets of target volume through a droplet generation module and a droplet cutting module.

[0065] FIG9A shows a top view of each module in a digital microfluidic chip according to some other embodiments of the present disclosure; FIG9B shows an enlarged plan view of a droplet cutting module in the digital microfluidic chip of FIG9A .

[0066] 10A and 10B are schematic diagrams of droplet tailoring using the droplet tailoring module shown in FIG. 9A and FIG. 9B according to some embodiments of the present disclosure.

[0067] 11A and 11B are schematic top views of modules in a digital microfluidic chip according to other embodiments of the present disclosure.

[0068] 12A to 12I are schematic top views showing methods for preparing a gene library using a digital microfluidic chip according to some embodiments of the present disclosure. DETAILED DESCRIPTION

[0069] To make the purpose, technical solutions, and advantages of the embodiments of the present disclosure more clear, the technical solutions of the embodiments of the present disclosure will be clearly and completely described below in conjunction with the accompanying drawings of the embodiments of the present disclosure. Obviously, the described embodiments are part of the embodiments of the present disclosure, not all of the embodiments. Based on the described embodiments of the present disclosure, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present disclosure.

[0070] Unless otherwise defined, the technical or scientific terms used in this disclosure should have the usual meanings understood by persons of ordinary skill in the field to which this disclosure belongs. The words "first", "second" and similar terms used in this disclosure do not indicate any order, quantity or importance, but are only used to distinguish different components. Words such as "include" or "comprise" mean that the elements or objects preceding the word include the elements or objects listed after the word and their equivalents, without excluding other elements or objects. Words such as "connect" or "connected" are not limited to physical or mechanical connections, but may include electrical connections, whether direct or indirect.

[0071] Various embodiments of the present disclosure provide a digital microfluidic chip that can solve technical problems of existing digital microfluidic chips such as low flexibility in liquid manipulation volume, a small number of controllable droplet volume types, and a small span; the digital microfluidic chip of the embodiments of the present disclosure can generate droplets of multiple volume types and can realize the manipulation of droplets of multiple volume types and large spans, with high droplet manipulation flexibility, thus being applicable to more biochemical reaction processes.

[0072] Figure 1A is a schematic top view of a digital microfluidic chip according to some embodiments of the present disclosure; Figure 1B is a schematic top view of an upper substrate in a digital microfluidic chip according to some embodiments of the present disclosure; and Figure 1C is a schematic cross-sectional view of the digital microfluidic chip taken along line II' in Figure 1A according to some embodiments of the present disclosure.

[0073] Referring to FIG. 1 , in some embodiments, a digital microfluidic chip 500 includes a droplet generating structure GS, a droplet receiving module RM, and an interconnect module IM. The droplet generating structure GS is configured to generate droplets of various volumes and may include one or more droplet generating modules. For example, each droplet generating module is configured to generate at least one of a first droplet, a second droplet, and a third droplet, wherein the first droplet, the second droplet, and the third droplet have first, second, and third volumes that are different from each other, respectively. The droplet receiving module RM is configured to achieve at least one of mixing and reaction of the multiple droplets. The interconnect module IM is configured to drive the first droplet, the second droplet, the third droplet, and other droplets of various volumes and connect various modules and / or regions, for example, connecting the modules in the droplet generating structure GS to each other and connecting the droplet generating structure GS to the droplet receiving module RM and / or connecting different regions in the droplet receiving module RM, thereby driving operations such as the movement of droplets between the modules and / or regions.

[0074] In a microfluidic chip, the more types of volumes of individual droplets that can be generated by a droplet generation module, the simpler and more efficient the droplet manipulation is. In addition, in order to improve the utilization efficiency of the chip area, it is necessary to set as few droplet generation modules as possible. In the embodiment of the present disclosure, taking into account the above two factors, the types of initial single droplet volumes that can be generated by multiple droplet generation modules are set to three types, namely the first volume, the second volume and the third volume. By coordinating the use of various modules, the flexibility of the droplet manipulation volume of the microfluidic chip can be improved, and the effect of a wide range of controllable droplet volumes can be achieved. It can also make droplet manipulation simple, the operation efficiency is high, and the chip area utilization rate is high. Here, the initial single droplet volume refers to the initial volume of a single droplet generated by the droplet generation module, that is, the volume of the single droplet that has not been cut and / or mixed after being generated by the droplet generation module.

[0075] In some embodiments, the interconnect module 124 may include one or more droplet drive modules, wherein each droplet drive module is configured to drive at least one of the first droplet, the second droplet, and the third droplet, and the droplet volumes that can be driven by a single drive electrode in the droplet drive module are the first volume, the second volume, and the third volume, respectively. For example, the first volume is smaller than the second volume and the third volume, and the droplet drive module for driving the first droplet can be directly connected to the droplet generation module for generating the first droplet.

[0076] In some embodiments, the one or more droplet drive modules may include: at least one droplet drive module IM1, at least one droplet drive module IM2, and at least one droplet drive module IM3. The droplet drive modules IM1, IM2, and IM3 may be referred to as the first droplet drive module, the second droplet drive module, and the third drive module, respectively. For example, a single drive electrode in each first droplet drive module IM1 is configured to drive a first droplet, a single drive electrode in each second droplet drive module IM2 is configured to drive a second droplet, and a single drive electrode in each third droplet drive module IM3 is configured to drive a third droplet. For example, the volume of droplets that can be driven by each drive electrode in the first droplet drive module IM1 is a first volume, the volume of droplets that can be driven by each drive electrode in the second droplet drive module IM2 is a second volume, and the volume of droplets that can be driven by each drive electrode in the third droplet drive module IM2 is a third volume.

[0077] In some embodiments, the volume of droplets that can be driven by a single drive electrode in each drive module is the minimum volume of droplets that can be driven by the drive module; that is, the minimum volume of droplets that can be driven by the droplet drive modules IM1, IM2, and IM3 are the first volume, the second volume, and the third volume, respectively; it should be understood that each droplet drive module can include multiple drive electrodes, and can drive droplets by the coordinated operation of one or more drive electrodes. In other words, in addition to being configured to drive the first droplet, the second droplet, and the third droplet, the droplet drive modules IM1, IM2, and IM3 can also drive droplets of other volumes through the corresponding multiple drive electrodes. For example, considering the accuracy of droplet manipulation, the volume of droplets that can be accommodated and driven by the three drive electrodes in each droplet drive module is defined as the maximum volume of droplets that can be driven by the droplet drive module; that is, the maximum volume of droplets that can be driven by the droplet drive modules IM1, IM2, and IM3 is three times the first volume, three times the second volume, and three times the third volume, respectively. Therefore, the volume range of droplets that can be driven by the droplet driving module IM1 can be from the first volume to three times the first volume; the volume range of droplets that can be driven by the droplet driving module IM2 can be from the second volume to three times the second volume; and the volume range of droplets that can be driven by the droplet driving module IM3 can be from the third volume to three times the third volume.

[0078] In some embodiments, the second volume is greater than the minimum volume of droplets that can be driven by the first droplet drive module, and less than the maximum volume of droplets that can be driven by the first droplet drive module; the third volume is greater than the minimum volume of droplets that can be driven by the second droplet drive module, and less than the maximum volume of droplets that can be driven by the second droplet drive module.

[0079] In some embodiments, the first volume is smaller than the second and third volumes; for example, the first, second, and third volumes increase in order. The first volume may be the smallest operational unit of the microfluidic chip, i.e., the minimum volume at which droplets can be manipulated. In some embodiments, region D directly connected to the droplet generation module (e.g., droplet generation module B-1) that generates droplets of the first volume may be or include a first droplet drive module IM1.

[0080] In some embodiments, the droplet generation structure GS further includes one or more droplet tailoring modules CM, connected to the one or more droplet generation modules and configured to tailor droplets from the one or more droplet generation modules into droplets having a target volume. For example, the droplet tailoring module CM can be connected to the one or more droplet generation modules via an interconnect module IM1, or the droplet tailoring module CM can be directly connected to the one or more droplet generation modules.

[0081] In some embodiments, the droplet tailoring module CM and the first droplet drive module IM1 in the interconnection module may share the same electrode array; for example, the area D directly connected to the generation module that generates the first volume droplets (for example, the droplet generation module B-1) may be the first droplet drive module IM1, and may be reused as the droplet tailoring module CM, that is, the droplet generation module for generating the first droplets may be directly connected to the droplet tailoring module CM. The droplet tailoring module CM includes a plurality of tailoring drive electrodes, and each tailoring drive electrode may drive a droplet with a volume that is substantially equal to the first volume, but the present disclosure is not limited thereto. In other embodiments, the droplet tailoring module CM may also further include a single tailoring drive electrode that can drive a droplet with a volume greater than the first volume.

[0082] In some embodiments, the second volume and the third volume are both integer multiples of the first volume, and the second volume and the third volume are different from each other; for example, the second volume is twice the first volume, and the third volume is five times the first volume. In some embodiments, the first volume is the minimum unit volume of the droplets that can be manipulated by the digital microfluidic chip, and the digital microfluidic chip can manipulate a volume range from the first volume to several tens of times (for example, twenty times) the first volume, with a step size of the minimum unit volume of liquid. In this article, the "step size" of the liquid refers to the volume difference between adjacent controllable droplets.

[0083] For example, the droplet generation module may include a droplet generation module configured to generate droplets with a minimum operating unit volume of 1 μL, 2 μL, and 5 μL, and through the coordinated operation of the droplet generation module and the droplet trimming module, at least a plurality of droplets of different volumes with a volume of 1 μL to 20 μL and a step size of 1 μL (i.e., a volume of 1 μL, 2 μL, 3 μL, 4 μL, 5 μL, 6 μL, 7 μL, 8 μL, 9 μL, 10 μL, 11 μL, 12 μL, 13 μL, 14 μL, 15 μL, 16 μL, 17 μL, 18 μL, 19 μL, and 20 μL) can be generated.

[0084] In some embodiments, the digital microfluidic chip 500 may include at least a sample inlet area, a liquid reaction area, a liquid trimming area, a waste liquid area, and a sampling area. The digital microfluidic chip 500 is, for example, a passive digital microfluidic chip. The sample inlet area is the area where the sample solution is introduced into the digital microfluidic chip. The sample inlet area corresponds to the area where the droplet generation module is located, and the droplet generation module can also be referred to as the sample inlet module. The liquid reaction area may include at least part of the droplet receiving module, for example, it may include area C and area A shown in Figure 1A, and areas C and A can also be referred to as reaction areas C and A. The liquid trimming area may include at least a droplet trimming module CM; in some embodiments, droplet trimming can also be implemented in each droplet generation module. The waste liquid area is used to receive waste droplets, such as waste droplets trimmed by the droplet trimming module. The sampling area is the area where target substances (e.g., target liquid, product, etc.) are drawn out from the digital microfluidic chip. The target substance is, for example, waste liquid or a product formed after the sample solution undergoes a series of biochemical reaction processes in the digital microfluidic chip. In some embodiments, the waste liquid area and the sampling area can be selected from the sample injection module according to needs, but other modules besides the above modules can also be separately set up to serve as the waste liquid area and the sampling area.

[0085] In some embodiments, the digital microfluidic chip 500 includes a main region R1, a peripheral region R2, and a binding region R3. The peripheral region R2 is located on one side of the main region R1 in a direction D2, for example, and the binding region R3 is located on a side of the peripheral region R2 away from the main region R1. Modules such as a droplet generating structure GS, a droplet receiving module RM, and an interconnect module IM are disposed in the main region R1. A plurality of conductive traces (e.g., conductive traces t1 and t2 shown in the figure) extend from corresponding modules in the main region R1 through the peripheral region R2 to the binding region R3 to electrically connect the drive electrodes in each module to corresponding binding electrodes in a binding electrode region F located in the binding region R3. The binding electrodes are electrically connected to a driver integrated circuit (IC), which applies corresponding drive signals to the drive electrodes in each module via the binding electrodes and the plurality of conductive traces.

[0086] Referring to Figures 1A to 1C, in some embodiments, the droplet generation structure GS, droplet receiving module RM, interconnect module IM, and other modules in the digital microfluidic chip 500 all include similar laminated structures. For example, the digital microfluidic chip 500 includes a first substrate S1, a second substrate S2, and a droplet channel DC. The first substrate S1 and the second substrate S2 are arranged opposite each other and spaced apart from each other, with the droplet channel DC disposed between the first substrate S1 and the second substrate S2. The first substrate and the second substrate may also be referred to as the lower substrate and the upper substrate, respectively. Each of these modules in the microfluidic chip 500 may be composed of the laminated structure at corresponding positions in the first substrate S1 and the second substrate, as well as corresponding portions of the droplet channel DC. During use of the digital microfluidic chip, a drive signal is applied to the drive electrodes in the corresponding modules to cause a droplet LD to move in the droplet channel DC. For example, the digital microfluidic chip drives the movement of the droplet LD using the principle of dielectric wetting, driving the droplet LD in a specific direction by changing the potential between the drive electrode and the droplet LD.

[0087] Referring to FIG. 1C , in some embodiments, the first substrate S1 may include a base substrate 100, a conductive layer 101, an insulating layer 102, a dummy layer M1, an insulating layer 105, a conductive layer 106, a dielectric layer 107, and a hydrophobic layer 108. Conductive layer 101 is disposed on one side of base substrate 100, and conductive layer 106 is disposed on a side of conductive layer 101 away from base substrate 100, with conductive layer 106 and corresponding portions of conductive layer 101 being electrically connected to each other. Dummy layer M1 is disposed between conductive layer 101 and conductive layer 106 and is electrically isolated from conductive layers 101 and 106. In some embodiments, dummy layer M1 is electrically floating, i.e., not connected to any other conductive components and / or voltage sources; alternatively, dummy layer M1 may be grounded.

[0088] For example, the conductive layer 106 may include a plurality of drive electrodes DE, portions of a plurality of conductive traces (e.g., the horizontally extending conductive trace t2 in FIG. 1A ), and / or portions of the binding electrodes; the conductive layer 101 may include a conductive connection pad CP, a plurality of conductive traces (e.g., the vertically extending conductive trace t1 in FIG. 1A ), and at least a portion of the binding electrodes in the binding electrode region F; the conductive traces t1 and t2 may be electrically connected to each other via vias (not shown) in the insulating layers 105 and 102; the conductive connection pad CP may be located at an end of the conductive trace t1, and the drive electrode DE may be electrically connected to the conductive connection pad CP via a conductive via V, thereby connecting to the conductive traces t1 and t2, and further connected to the binding electrodes via the conductive traces t1 and t2. The dummy layer M1 may include a plurality of dummy electrodes 103a.

[0089] Adjacent conductive patterns in the conductive layer 102 , the conductive layer 106 , and the dummy layer M1 are isolated by an insulating layer and / or a dielectric layer. For example, the insulating layer 102 is arranged on the base substrate 100 and covers the side walls of the conductive layer 101 and the surface thereof away from the base substrate 100; the dummy layer M1 is located on the side of the insulating layer 102 away from the base substrate 100 and is electrically isolated from the conductive layer 101 by the insulating layer 102; the insulating layer 105 is located on the side of the insulating layer 102 away from the base substrate 100 and covers the side walls of the dummy layer M1 and the surface thereof away from the base substrate 100; the conductive layer 106 is arranged on the side of the insulating layer 105 away from the base substrate 100 and is electrically isolated from the dummy layer M1 by the insulating layer 105; the dielectric layer 107 is located on the side of the insulating layer 105 away from the base substrate 100 and covers the side walls of the conductive layer 106 and the surface thereof away from the base substrate 100; the hydrophobic layer 108 is located on the side of the dielectric layer 107 away from the base substrate 100 and is isolated from the conductive layer 106 by the dielectric layer 107.

[0090] In some embodiments, the base substrate 100 may be or include a glass substrate. The conductive layers 101 and 106 may include the same or different conductive materials. For example, both conductive layers 101 and 106 may include transparent conductive materials such as indium tin oxide (ITO) or indium zinc oxide (IZO). The thickness of the conductive layers 101 and 106 may be the same or different, and may be, for example, greater than or equal to 52 nanometers (nm).

[0091] The material of dummy layer M1 is different from that of conductive layers 101 and 106. For example, dummy layer M1 is opaque and may include an opaque conductive material, such as a metal. For example, dummy layer M1 may include a metal material such as molybdenum (Mo) or a molybdenum-aluminum (Mo / Al) alloy. The thickness of dummy layer M1 may be the same as or different from that of conductive layers 101 and 106. For example, the thickness of dummy layer M1 may be greater than or equal to 52 nm.

[0092] The insulating layers 102 and 105 can each be selected from insulating materials such as silicon oxide, silicon nitride, silicon oxynitride, etc., and can have a thickness greater than or equal to 300 nm; for example, in some examples, the insulating layers 102 and 105 are both made of silicon nitride material and each has a thickness of 600 nm, but the present disclosure is not limited to this.

[0093] The dielectric layer 107 may include an inorganic dielectric material, an organic dielectric material, or a combination thereof; for example, the dielectric layer 107 may be selected from one or more of silicon oxide, silicon nitride, SU8, and polyimide (PI); in some examples, the dielectric layer 107 uses PI material and may have a thickness of approximately 28 microns (μm), but the present disclosure is not limited thereto.

[0094] The hydrophobic layer 108 may comprise a fluorine-containing material, such as Teflon, CYTOP, etc. The thickness of the hydrophobic layer 108 may be greater than or equal to 100 nm, and the coverage of the hydrophobic layer 108 may be substantially the same as the coverage of the dielectric layer 107. That is, the orthographic projection of the hydrophobic layer 108 on the base substrate 100 may substantially overlap with the orthographic projection of the dielectric layer 107 on the base substrate 100. For example, the dielectric layer 107 and the hydrophobic layer 108 may entirely cover the underlying material layer on the base substrate 100.

[0095] Continuing with reference to Figures 1A to 1C, in some embodiments, the second substrate S2 includes a base substrate 200, a conductive layer 206, and a hydrophobic layer 208. The conductive layer 206 is located on one side of the base substrate 200, and the hydrophobic layer 208 is located on a side of the conductive layer 206 away from the base substrate 200. A droplet channel DC is defined, for example, between the hydrophobic layer 108 of the first substrate S1 and the hydrophobic layer 208 of the second substrate S2.

[0096] The base substrate 200 may be or include a glass substrate. The material and thickness of the conductive layer 206 may be the same as or different from those of the conductive layers 101 and 106. For example, the conductive layer 206 may include a conductive material such as ITO or IZO, and may have a thickness greater than or equal to 52 nm and a sheet resistance less than approximately 150 Ω / □. The candidate materials and thickness ranges for the hydrophobic layer 208 are substantially the same as those for the hydrophobic layer 108 and are not further described here.

[0097] In some embodiments, the conductive layer 206 extends continuously along one side of the base substrate 200, for example, covering the entire surface of the base substrate 200 adjacent to the first substrate S1. The first substrate S1 may include a ground electrode G, which may be included in the conductive layer 106 and electrically isolated from other conductive structures in the conductive layer 106, such as the drive electrodes. A conductive structure (not shown) is provided between the first substrate S1 and the second substrate S2 to electrically connect the ground electrode G of the first substrate S1 to the conductive layer 206 of the second substrate S2, thereby grounding the conductive layer 206. The conductive structure may be or include conductive foam and may have a thickness of, for example, approximately 500 μm. The ground electrode G and the conductive structure are, for example, provided in the peripheral region R2.

[0098] In some embodiments, a sealing glue 300 is provided between the first substrate S1 and the second substrate S2. The sealing glue 300 is used to align the first and second substrates and support the thickness of the box. At least a portion of the sealing glue 300 is provided along the periphery of the first substrate S1 and the second substrate S2 and surrounds the main region R1 of the microfluidic chip. The droplet channel DC is defined, for example, by the facing surfaces of the first substrate S1 and the second substrate S2 and the sidewalls of the sealing glue 300. For example, the sealing glue 300 may have a width of approximately 2 mm and a thickness of approximately 500 μm, and the spacing between the sealing glue 300 and the ground electrode G (for example, in the direction D2) may be greater than 200 μm. The edge of the portion of the sealing glue 300 away from the peripheral region R2 and the binding region R3 may be roughly aligned with the edge of the first substrate and the second substrate in a direction perpendicular to the main surface of the substrate.

[0099] In some embodiments, the second substrate S2 includes one or more liquid adding holes 201, which pass through the second substrate S2 and are connected to the droplet channel DC; the liquid adding holes 201 are used to add environmental solutions (for example, oil) to the droplet channel DC, and the liquid adding holes 201 can also be called oil adding holes; in some embodiments, four oil adding holes 201 are arranged at the four corners of the main region R1, but the present disclosure is not limited to this.

[0100] In some embodiments, the second substrate S2 further includes sample inlets 202a, 202b, and 202c; the sample inlets 202a, 202b, and 202c extend through the second substrate S2 and communicate with the droplet channel DC for adding sample solutions, reaction solutions, and the like to the droplet channel DC. It should be understood that the solution applied by the sample inlets 202a, 202b, and 202c is insoluble in the environmental solution added by the liquid addition port 201. The sample inlets 202a, 202b, and 202c are respectively disposed at positions corresponding to the liquid generation modules B-1, B-2, and B-3. It should be understood that, in different steps of the biochemical process, each sample inlet can also be reused as a sample outlet for removing the target substance from the opening.

[0101] 1A , in some embodiments, an alignment mark H, such as a cross mark, may be provided on the first substrate S1 , but the present disclosure is not limited thereto. The alignment mark H may be an alignment mark for chip cutting and dielectric layer attachment.

[0102] Continuing with reference to Figures 1A to 1C, in some embodiments, each of the droplet generating structure GS, droplet receiving module RM, and interconnect module IM in the digital microfluidic chip 500 may include one or more electrode array regions and a laminated structure (i.e., a portion of the laminated structure of the first substrate, the second substrate, and the droplet channel) that overlaps the electrode array region in a direction perpendicular to the chip's main surface. Each electrode array region may include a plurality of drive electrode structures DS spaced apart from each other. The plurality of drive electrode structures DS are located in the first substrate S1, and each drive electrode structure DS includes a drive electrode DE, a dummy electrode 103a, and a conductive connection pad CP, respectively located in the conductive layer 106, the dummy layer M1, and the conductive layer 101. As shown in Figure 1A, each drive electrode structure DS has a generally rectangular or square planar shape in a top view, and the area of ​​the drive electrode structures DS in different modules may vary. The area of ​​a drive electrode structure DS is approximately equal to the area of ​​its drive electrode DE.

[0103] 2A to 2C illustrate top views of the driving electrode structure DS according to some embodiments of the present disclosure.

[0104] Referring to Figures 1C and 2A , in some embodiments, the outer contours of the drive electrode DE and the dummy electrode 103a may substantially overlap. That is, the outer contour of the orthographic projection of the drive electrode DE onto the main surface of the base substrate 100 may substantially overlap the outer contour of the orthographic projection of the dummy electrode 103a onto the main surface of the base substrate 100. In other words, the outer sidewalls of the drive electrode DE and the outer sidewalls of the dummy electrode 103a are substantially aligned with each other in a direction perpendicular to the main surface of the base substrate 100. The dummy electrode 103a has an opening 103h through which the drive electrode DE passes to connect to the conductive connection pad CP. For example, the drive electrode DE is electrically connected to the conductive connection pad CP via a conductive via V, which is connected to the conductive trace T that connects to the bonding electrode. The conductive via V fills the vias of the insulating layers 102 and 105 and is located in the opening 103h of the dummy electrode 103a. A portion of the insulating layer 105 is located between the dummy electrode 103 a and the conductive via V, such that the dummy electrode 103 a and the conductive via V are spaced apart from each other and electrically isolated.

[0105] In some embodiments, the conductive layers 101 and 106 are made of transparent conductive materials to minimize the absorption of visible light by the microfluidic chip, thereby improving the optical performance of the chip, so that the microfluidic chip of this embodiment can meet the needs of fluorescence detection. In some embodiments, the insulating layer is also transparent. However, if both the driving electrode and the insulating layer are made of transparent materials, the position of the driving electrode cannot be determined by optical methods. In this way, if the position of the driving electrode cannot be accurately determined, it will be impossible to determine whether an error occurs in the sample addition process during the sample addition operation (for example, manual sample addition using a pipette), which will be detrimental to the use of the chip. On the other hand, when the first substrate and the second substrate are aligned or cut, other opaque graphics other than the alignment mark structure are required to complete image capture, so as to achieve automated production of the chip while ensuring its accuracy. In addition, when the multiple conductive traces of the conductive layer 101 are wired, multiple conductive traces may appear under the driving electrode (as shown in Figure 1A), and the multiple conductive traces may include conductive traces corresponding to other driving electrodes, which will cause signal crosstalk between the driving electrodes and affect the manipulation of the droplets; for example, if there is a conductive trace connected to other driving electrodes (or called a crosstalk trace) under a certain driving electrode, the conductive trace forms a capacitor with the driving electrode. When the conductive trace is energized, the coupling effect of the capacitor will cause the driving electrode to have a coupled voltage, causing signal crosstalk, thereby affecting the manipulation of the droplets.

[0106] The disclosed embodiment solves the aforementioned problem by providing a dummy electrode 103a. Because the dummy electrode 103a is opaque and coincides with the outer contour of the drive electrode DE, the position of the dummy electrode 103a can be determined optically, thereby accurately determining the location of the corresponding drive electrode DE. Furthermore, an opening 103h is provided in the middle of the dummy electrode 103a, thereby ensuring optical performance at the location of the droplet. Furthermore, the dummy electrode 103a is made of metal and is electrically floating or grounded, effectively shielding the signal from the conductive layer 101 and avoiding or reducing signal crosstalk between different drive electrodes. Specifically, the dummy electrode 103a can shield the crosstalk from other conductive traces in the conductive layer 101 that are not connected to the corresponding drive electrode DE. This improves the accuracy and reliability of droplet manipulation by the drive electrodes in the microfluidic chip.

[0107] In some embodiments, the size and dimensions of the opening of the dummy electrode 103a can be adjusted according to product design and needs. For example, as shown in FIG2A , in some examples, the size of the opening 103h of the dummy electrode 103a corresponds to the minimum field of view diameter of a fluorescence microscope. For example, the opening 103h can be a square with a side length of approximately 50 μm. In this example, the opening range of the dummy electrode 103a is minimized, thereby increasing the area of ​​the dummy electrode. This can maximize the signal shielding of the dummy electrode from the crosstalk traces in the conductive layer 101 while meeting the optical performance of the drive structure.

[0108] Referring to FIG. 2B , in other examples, the opening 103h of the dummy electrode 103a is relatively large. This maximizes the transparency of the drive electrode structure DS while ensuring that the drive electrode position can be visually determined, thereby meeting the requirements of fluorescence detection over a wide field of view. For example, the boundary of the opening 103h of the dummy electrode 103a is indented approximately 100 microns from the boundary of the drive electrode DS. In other words, the distance L1 between the outer wall of the dummy electrode 103a and the inner wall defining the opening 103a is approximately 100 microns.

[0109] Referring to FIG. 2C , in yet other embodiments, the opening 103h of the dummy electrode 103a may be circular in shape, and the minimum distance between the boundary of the opening 103h and the boundary of the corresponding drive electrode DE in a direction parallel to the main surface of the substrate must be greater than or equal to 100 microns to ensure human visual recognition. In other words, the minimum distance L1 between the outer sidewall of the dummy electrode 103a and the inner sidewall defining the opening 103h is greater than or equal to 100 microns.

[0110] It should be understood that the shape and size of the opening of the dummy electrode 103a described above are merely illustrative and are not intended to limit the present disclosure. The shape and size of the dummy electrode can be designed based on actual application requirements. For example, when a wide field of view of fluorescence detection is required, the embodiment of FIG2B is preferred; when a wide field of view of fluorescence detection is not required, the embodiment of FIG2A is preferred.

[0111] This embodiment uses the cross-sectional structure at the interconnection module IM as an example to illustrate the stacked structure of the microfluidic chip. It should be understood that other modules in the microfluidic chip, such as the droplet generation module, have similar stacked structures and have the same features as described above with reference to Figures 1C and 2A to 2C.

[0112] In some embodiments, as shown in FIG1A , the plurality of droplet generation modules may include at least one droplet generation module B-1, at least one droplet generation module B-2, and at least one droplet generation module B-3, each configured to generate a first droplet, a second droplet, and a third droplet, respectively. Droplet generation modules B-1, B-2, and B-3 may also be referred to as a first droplet generation module, a second droplet generation module, and a third droplet generation module, respectively. It should be understood that the number of droplet generation modules can be designed and adjusted based on actual application needs and can be the same or different. Each droplet generation module can implement sample addition, removal, storage, and droplet generation.

[0113] 3A to 3C respectively illustrate top views of droplet generation modules B-1, B-2, and B-3 according to some embodiments of the present disclosure.

[0114] Referring to Figures 3A to 3C, in some embodiments, each of the droplet generation modules B-1, B-2, and B-3 may include drive electrodes 1, 2, 3, 4, 5, and 6. Drive electrodes 1, 2, 3, 4, 5, and 6 may be referred to as the first drive electrode, the second drive electrode, the third drive electrode, the fourth drive electrode, the fifth drive electrode, and the sixth drive electrode, respectively. It should be understood that each drive electrode in each droplet generation module corresponds to a drive electrode structure DS as shown in Figure 1C, and for the sake of simplicity, Figures 3A to 3C only show the drive electrodes located on the upper layer, while structures such as dummy electrodes, conductive through-holes, and conductive connection pads located between the corresponding drive electrodes and the base substrate are not shown in Figures 3A to 3C. For the corresponding structures, reference can be made to the descriptions of Figures 1C and 2A to 2C above. The relative positional relationships between the drive electrodes of each droplet generation module described herein can also be interpreted as the relative positional relationships between the corresponding drive electrode structures.

[0115] In some embodiments, in each of droplet generation modules B-1, B-2, and B-3, drive electrodes 2, 3, 4, and 6 are sequentially arranged along a first sub-direction d1 and have the same first area. Drive electrode 6 is located at an end of the corresponding droplet generation module and is configured to connect (e.g., directly connect) to drive electrodes in other modules or regions. Drive electrode 5 includes two electrically connected drive sub-electrodes (or fifth drive sub-electrodes) 5a and 5b. Drive sub-electrodes 5a and 5b are arranged on opposite sides of drive electrodes 2 and 3 in a second sub-direction d2 perpendicular to the first sub-direction d1, and each drive sub-electrode 5a / 5b has a second area. Drive electrode 1 is arranged on a side of drive electrode 2 away from drive electrode 3 in the first sub-direction d1 and is connected to drive electrodes 2 and 5. Drive electrode 1 may have a third area. For example, the first area is smaller than the second area, and the second area is smaller than the third area. Among multiple drive electrodes, adjacent drive electrodes are spaced apart from each other. It should be noted that, in this document, direct connection of two or more drive electrodes means that the two or more drive electrodes are in close proximity to each other, and connection of two or more drive electrodes includes situations where the two or more drive electrodes are in close proximity to each other or are separated by other electrodes. The connection does not limit the two or more drive electrodes to being physically or electrically connected to each other, unless it is explicitly defined as a physical connection or electrical connection. The drive electrodes are connected to each other, for example, to enable storage of droplets on the drive electrodes and / or to drive droplet movement between the drive electrodes.

[0116] In some embodiments, each of the droplet generation modules B-1 and B-2 may further include an additional drive electrode 0. The additional drive electrode 0 is located on the side of the drive electrode 1 away from the drive electrode 2 in the first sub-direction d1, and may have a third area substantially the same as that of the drive electrode 1. In some embodiments, one or more droplet generation modules may further include an additional dummy electrode. For example, each of the droplet generation modules B-1 and B-2 may further include an additional dummy electrode 103b, which is located on the side of the additional drive electrode 0 away from the drive electrode 1 in the first sub-direction d1. In some embodiments, the additional dummy electrode 103b is located in the dummy layer M1 shown in Figure 1C, that is, it is arranged in the same layer as the dummy electrode 103a in each drive electrode structure DS. In this article, "the same layer arrangement" of two patterns means that the two patterns are formed from the same material layer through the same patterning process. In some embodiments, no corresponding driving electrode is provided on the side of the additional dummy electrode 103b away from the base substrate 100 , that is, the orthographic projection of the additional dummy electrode 103b on the base substrate 100 does not overlap with the driving electrode and / or the conductive layer 106 where it is located.

[0117] In some embodiments, the additional dummy electrode 103b serves as an alignment mark for the sample inlet of the second substrate, used to identify the location of the corresponding droplet generation module to facilitate sample insertion. For example, as shown in FIG1A , for droplet generation modules B-1 and B-2 having additional dummy electrodes 103b, the orthographic projections of the additional dummy electrodes 103b of the droplet generation modules B-1 and B-2 on the main surface of the first substrate S1 overlap (partially or completely overlap) with the orthographic projections of the corresponding sample inlets 202a and 202b on the main surface of the first substrate S1 facing the second substrate S2.

[0118] In some embodiments, the microfluidic chip may include a droplet generation module without an additional dummy electrode, such as the droplet generation module B-3. For such a droplet generation module without an additional dummy electrode, the corresponding sample inlet may be set at a position adjacent to but not overlapping with its drive electrode. For example, for the droplet generation module B-3 that does not include an additional dummy electrode, the orthographic projection of the corresponding sample inlet 202c on the main surface of the first substrate S1 is located near the orthographic projection of the drive electrode 1 of the droplet generation module B-3 on the main surface of the first substrate S1, but does not overlap with the orthographic projection of the drive electrode 1 on the main surface of the first substrate S1.

[0119] In some embodiments, the drive electrode with the smallest area (or may be referred to as the main electrode) in each droplet generation module can accommodate and drive a droplet volume that is approximately equal to the droplet volume of a single droplet generated by the corresponding droplet generation module. For example, if droplet generation modules B-1, B-2, and B-3 are respectively configured to generate droplets having a first volume (e.g., 1 μL), a second volume (e.g., 2 μL), and a third volume (e.g., 5 μL), then the drive electrode with the smallest area (e.g., drive electrodes 2, 3, 4, and 6) in droplet generation module B-1 can accommodate and drive a droplet volume that is approximately equal to the first volume; the drive electrode with the smallest area (e.g., drive electrodes 2, 3, 4, and 6) in droplet generation module B-2 can accommodate and drive a droplet volume that is approximately equal to the second volume; and the drive electrode with the smallest area (e.g., drive electrodes 2, 3, 4, and 6) in droplet generation module B-3 can accommodate and drive a droplet volume that is approximately equal to the third volume.

[0120] In some examples, as shown in Figures 3A to 3C , in each droplet generation module, drive electrodes 2, 3, 4, and 6 can all be square electrodes, each having a side length SL. Each drive sub-electrode in drive electrode 5 can be a rectangular electrode, having a long side along the first sub-direction d1 and a short side along the second sub-direction d2, wherein the long side has a length approximately equal to twice the side length SL of the square electrode in the same generation module, and the short side has a length approximately equal to the side length SL of the corresponding square electrode. Drive electrode 1 or 0 can be a rectangular electrode, having a short side along the first sub-direction d1 and a long side along the second sub-direction d2, wherein the short side has a length approximately equal to the side length SL of the corresponding square electrode, and the long side has a length approximately equal to three times the side length SL of the corresponding square electrode. In some embodiments, the additional dummy electrode 103b can have a shape different from that of the drive electrode, for example, a trapezoidal shape. In some examples, the additional dummy electrode 103b may be an isosceles trapezoid having a parallel short side (or upper base) and a long side (or lower base) extending along the second sub-direction d2. The short side has a length that is approximately 1.5 times the side length SL of the square electrode in the same generation module, and the long side has a length that is approximately 3 times the side length SL of the corresponding square electrode. In this context, "two dimensional features (side length, area, width, length, volume, etc.) are approximately equal" may include the two dimensional features being completely equal, and may also include the case where the difference between each dimensional feature and the average of the two dimensional features is less than 5% to 20% of the average, such as less than 5%, 10%, 15%, or 20% of the average. For example, "the side length of a rectangular electrode is approximately equal to the side length of two square electrodes" may include the case where the side length of the rectangular electrode is equal to the sum of the side lengths of the two square electrodes and the spacing between the two square electrodes. It should be understood that the shapes and sizes of the aforementioned drive electrodes, additional drive electrodes, and / or additional dummy electrodes are merely illustrative and are not intended to be limiting. For example, in some embodiments, the additional dummy electrodes may also be in a circular, square, or other shape.

[0121] In some embodiments, adjacent drive electrodes are spaced apart from each other by a spacing p1, and the spacing between different adjacent drive electrodes in the multiple drive electrodes of each module in the chip may be approximately equal. The additional dummy electrode 103b is spaced apart from the adjacent drive electrode 0 by a spacing p2, and the spacing p2 between different adjacent electrodes in each module may be approximately equal. The spacings p1 and p2 may be different from each other, for example, the spacing p2 is greater than the spacing p1. In some examples, the spacing p1 between adjacent drive electrodes is approximately 25 μm, and the spacing p2 between the additional dummy electrode 103b and the adjacent drive electrode 0 is approximately 200 μm, but the present disclosure is not limited to this.

[0122] In some embodiments, the droplet generation modules B-1, B-2, and B-3 are respectively configured to generate droplets of different volumes, and the areas of the corresponding drive electrodes in the droplet generation modules B-1, B-2, and B-3 are different. For example, the first area, second area, and third area of ​​the corresponding drive electrode in the droplet generation module B-1 are respectively smaller than the first area, second area, and third area of ​​the corresponding drive electrode in the droplet generation module B-2, and the first area, second area, and third area of ​​the corresponding drive electrode in the droplet generation module B-2 are respectively smaller than the first area, second area, and third area of ​​the corresponding drive electrode in the droplet generation module B-3. The area of ​​each drive electrode in each droplet generation module can be set according to the volume of a single droplet generated by the droplet generation module configuration. For example, in a digital microfluidic chip, the droplet volume V can be estimated using the following formula:

[0123] Where θ is the contact angle of the droplet, δ is the cell thickness of the digital microfluidic chip, i.e., the distance between the first substrate S1 and the second substrate S2, and a is the radius of the droplet-solid-liquid interface. For example, the droplet-solid-liquid interface radius a can be calculated based on the volume of a single droplet generated by the droplet generation module configuration. The side length SL and area of ​​the smallest square electrode in the droplet generation module can be calculated based on the value of a, and the corresponding dimensions of other drive electrodes and / or additional dummy electrodes in the droplet generation module can be inferred.

[0124] For example, droplet generation modules B-1, B-2, and B-3 are configured to generate droplets with volumes of 1 μL, 2 μL, and 5 μL, respectively. Using the above formula, it is deduced that the side lengths SL of the square drive electrodes 2, 3, 4, and 6 included in droplet generation modules B-1, B-2, and B-3 are 1084 μm, 1550 μm, and 2474 μm, respectively. The sizes of other electrodes in each droplet generation module can be determined based on the multiples of the side length SL described above. In some embodiments, due to the relatively large size of droplet generation module B-3, the additional drive electrode 0 and the additional dummy electrode 103b are omitted in droplet generation module B-3, thereby saving chip area, but the present disclosure is not limited to this. In other embodiments, the droplet generation module B-3 may also include an additional drive electrode 0 and an additional dummy electrode 103b; or one or more of the droplet generation modules B-1 and B-2 may also omit the additional drive electrode 0 and the additional dummy electrode 103b, as long as each droplet generation module can generate droplets of corresponding volume.

[0125] When using a digital microfluidic chip for sample loading, a pipette is used to inject the required solution from the corresponding sample loading hole, and then a drive signal is applied to the corresponding drive electrode in the droplet generation module to complete the sample loading operation and the generation of droplets. For example, for droplet generation modules B-1 and B-2, after injecting the sample solution from the corresponding sample loading hole, the drive electrodes 0 and 1 are opened, and then the drive electrodes 0 and 1 are closed, and the drive electrodes 2, 3, and 5 are opened to complete the sample loading operation. After the sample loading operation is completed, the droplet generation operation includes: first opening the drive electrodes 2, 3, 4, and 6 so that the droplets move to the drive electrodes 2, 3, 4, and 6, and then closing the drive electrode 4 and opening the drive electrode 5 so that the droplets split into target volume droplets located on the drive electrode 6 and the remaining volume droplets located on the drive electrodes 2, 3, and 5, thereby achieving the generation of target volume droplets. The target volume droplets can subsequently be driven and moved to other modules through the interconnected modules. For the droplet generation module B-3, after the sample solution is injected from the sample inlet, the drive electrodes 1, 2, and 3 are opened, and then the drive electrode 1 is closed and the drive electrode 5 is opened, thereby completing the sample injection operation. The droplet generation operation of the droplet generation module B-3 is roughly the same as that of the above-mentioned droplet generation modules B-1 and B-2, and will not be repeated here. It should be understood that in this article, "opening the drive electrode" means applying a drive signal to the drive electrode, that is, energizing the drive electrode; "closing the drive electrode" means not applying a drive signal to the drive electrode, that is, not energizing or turning off the power.

[0126] In the above embodiments, each droplet generation module is configured to generate droplets of a single volume type, but the present disclosure is not limited thereto. In other embodiments, one or more droplet generation modules in a digital microfluidic chip are configured to generate droplets of different volume types. For example, the digital microfluidic chip may include one or more fourth droplet generation modules, each of which is configured to generate at least two of a first droplet having a first volume, a second droplet having a second volume, and a third droplet having a third volume.

[0127] 4A to 4C illustrate schematic plan views of droplet generation modules (eg, a fourth droplet generation module) according to further embodiments of the present disclosure.

[0128] Referring to FIG. 4A , in some embodiments, droplet generation module B-4 may include drive electrodes 1, 2, 3, 4, 5, 6b, an additional drive electrode 1', an additional drive electrode 2', and two additional drive electrodes 3'. Additional drive electrodes 1', 2', and 3' may also be referred to as a first additional drive electrode, a second additional drive electrode, and a third additional drive electrode, respectively. In droplet generation module B-4, the relative positional relationship of drive electrodes 1-5 is substantially the same as the relative positional relationship of drive electrodes 1-5 in the aforementioned droplet generation module.

[0129] For example, drive electrodes 2, 3, 4, 6b, 1', and 2' are sequentially arranged along a first sub-direction d1. Two additional drive electrodes 3' are arranged on opposite sides of drive electrode 6b in a second sub-direction d2 perpendicular to the first sub-direction d1. The two drive sub-electrodes 5a and 5b of drive electrode 5 are arranged on opposite sides of drive electrodes 2 and 3 in the second sub-direction d2. Drive electrode 1 is arranged on a side of drive electrodes 2 and 5 away from drive electrode 4 in the first sub-direction d1. In droplet generation module B-4, additional drive electrode 2' is located at an end and can be connected to drive electrodes in other modules or regions such as the interconnect module. For example, it can be connected to droplet drive module IM2 in the interconnect module.

[0130] In some embodiments, the drive electrodes 2, 3, and 4 are square electrodes and have the same first main body area; the additional drive electrodes 1' and 2' may have the same second main body area; in this embodiment, the drive electrode 6b is a rectangular electrode, and the sum of the areas of the drive electrode 6b and the two additional drive electrodes 3' is approximately equal to the first main body area; the second main body area is smaller than the first main body area. In some embodiments, the first main body area and the second main body area respectively correspond to the volumes of the two droplets generated by the droplet generation module B-4 configuration. Here, the sum of the areas of the drive electrode 6b and the two additional drive electrodes 3' being approximately equal to the first main body area may include the case where the sum of the areas of the drive electrode 6b and the two additional drive electrodes 3' is completely equal to the first main body area, and may also include the case where the sum of the areas of the drive electrode 6b and the two additional drive electrodes 3' and the area of ​​the gap area between the drive electrode 6b and the two additional drive electrodes 3' is equal to the first main body area.

[0131] In the fourth droplet generation module B-4, drive electrode 1 and additional drive electrode 1' are electrically connected to each other, drive electrode 2 and additional drive electrode 2' are electrically connected to each other, and drive electrode 3 and additional drive electrode 3' are electrically connected to each other. The electrically connected drive electrodes receive the same drive signal, that is, they are powered on or off at the same time.

[0132] For example, in some embodiments, droplet generation module B-4 is configured to generate a second droplet having a second volume and a third droplet having a third volume. In this embodiment, each drive electrode having the second body area (i.e., additional drive electrodes 1', 2') can accommodate and drive a droplet having a volume substantially equal to the second volume of the second droplet, and a single drive electrode having the first body area (i.e., drive electrodes 2, 3, 4) or a combination of drive electrodes (i.e., a combination of drive electrode 6 and two additional drive electrodes 3') can accommodate and drive a droplet having a volume substantially equal to the third volume of the third droplet. For example, droplet generation module B-4 can also be configured to generate an additional droplet having a volume equal to, for example, the third volume minus the second volume.

[0133] For example, the dimensions of drive electrodes 1-5 can be the same as those of the corresponding drive electrodes in droplet generation module B-3 shown in FIG3C . Drive electrode 6b has a long side along the first sub-direction d1 and a short side along the second sub-direction d2, wherein the long side has a length approximately equal to the side length of square electrodes 2-4, and the short side has a length approximately equal to the side length of electrodes 1' and 2'. For example, when the second and third volumes are 2 μL and 5 μL, respectively, drive electrode 6' has a long side of approximately 2474 μm and a short side of approximately 1550 μm. Drive electrode 3' has a long side along the first sub-direction d1 and a short side along the second sub-direction d2, wherein the long side has a length of approximately 2474 μm and the short side has a length of approximately 437 μm. Additional drive electrodes 1' and 2' can be square electrodes having the same dimensions as the square electrodes in droplet generation module B-2, for example, additional drive electrodes 1' and 2' have a side length of approximately 1550 μm.

[0134] In this embodiment, droplets having a second volume (eg, 2 μL) or a third volume (eg, 5 μL) are generated by droplet generation module B- 4 as shown in FIG. 5A to FIG. 5F .

[0135] Referring to Figures 5A to 5C , in some embodiments, after the sample loading operation is completed, drive electrodes 2, 3, and 5 are energized, thereby driving the droplet to move to drive electrodes 2, 3, and 5. The shape of droplet LD0 is shown in Figure 5A . Next, drive electrode 5 is de-energized, and drive electrodes 4 and 6b are energized; that is, drive electrodes 2, 3, 4, 6b and additional drive electrode 3' are in an open state, thereby driving droplet LD0 to move to the open drive electrode. The state of droplet LD0 driven in this step is shown in Figure 5B . Thereafter, drive electrode 4 is de-energized, and drive electrode 5 is energized, causing droplet LD0 to split in opposite directions, driven by drive electrodes 3' and 6b and drive electrodes 2, 3, and 5, into droplet LD1 and droplet LD2. Droplet LD1 is the target droplet having a third volume (e.g., 5 μL).

[0136] Referring to Figures 5A, 5B, and 5D-5F, in some embodiments, drive electrodes 2, 3, and 5 are energized, thereby driving a droplet to move to drive electrodes 2, 3, and 5. The shape of droplet LD0 is shown in Figure 5A. Next, drive electrode 5 is de-energized, and drive electrodes 4 and 6b are energized; that is, drive electrodes 2, 3, 4, 6b and additional drive electrode 3' are in an open state, thereby driving droplet LD0 to move to the open drive electrode. The state of droplet LD0 driven in this step is shown in Figure 5B. Referring to Figures 5B and 5D, drive electrodes 2 and 3 are de-energized, and drive electrode 1 is energized; that is, drive electrodes 1, 4, and 6b are in an open state. This causes droplet LD0 to split in opposite directions, into droplet LD1' and droplet LD2, driven by drive electrodes 4 and 6b and drive electrode 1, respectively. It should be understood that Figure 5D illustrates the intermediate stage of droplet splitting, so that a portion of droplet LD2 remains on drive electrode 2. After splitting, droplet LD2 moves entirely to drive electrode 1.

[0137] Referring to Figures 5D to 5E, thereafter, the drive electrode 4 is de-energized, and the additional drive electrode 2' and the drive electrode 2 are energized; that is, the drive electrodes 1, 2, 6b and the additional drive electrodes 1' and 2' are in an open state; therefore, the droplet LD1' moves onto the drive electrodes 1', 2', 6b, and the droplet LD2 moves partially from the drive electrode 1 to the drive electrode 2, that is, is located above both the drive electrodes 1 and 2. 5E and 5F , the driving electrodes 1 and 1' are powered off, and the driving electrodes 3, 3', and 4 are powered, that is, the driving electrodes 2, 2', 3, 3', 4, and 6b are in an open state, thereby driving part of the droplet LD1' to move in a direction away from the additional driving electrode 2' and split into droplets LD1 and LD3. The droplet LD3 is located on the additional driving electrode 2' and has a second volume (for example, 2 μL), and the droplet LD1 is located on the driving electrode 6b and the additional driving electrode 3' and has approximately a third volume (for example, 5 μL). Since the driving electrode 6b and the additional driving electrode 3' are large enough to accommodate the droplet LD1, although the driving electrode 4 is open, the droplet LD1 will not move onto the driving electrode 4. In addition, the droplet LD2 is driven by the driving electrodes 2 / 3 / 4 and moves onto the driving electrodes 2 and 3. Similarly, since the driving electrodes 2 and 3 are large enough to accommodate the droplet LD2, the droplet LD2 will not move onto the driving electrode 4, but the present disclosure is not limited to this. In this step, the driving electrode 4 is powered to increase the driving force on the droplet LD1 ′, thereby facilitating the splitting of the droplet LD1 ′.

[0138] In some embodiments, a driving method for generating additional droplets by the droplet generation module B-4' may include, for example, generating a droplet LD1 having a third volume (e.g., 5 μL) through the steps of Figures 5A to 5C , splitting the droplet LD1 into a droplet having a second volume (e.g., 2 μL) and an additional droplet, where the volume of the additional droplet is the third volume minus the second volume (e.g., 3 μL). For example, after the droplet LD1 is generated in the step of Figure 5C , the electrodes 3' and 6b are de-energized, and the electrodes 1' and 2' are energized, thereby driving the droplet LD1 to move onto the electrodes 1' and 2'. Since the volume of the droplet LD1 is greater than the total volume that can be accommodated by the electrodes 1' and 2', part of the droplet LD1 remains on the electrode 6b. Thereafter, the electrode 1' is de-energized, and the electrode 6b is energized, thereby splitting the droplet LD1 into a droplet having the second volume located on the electrode 2' and an additional droplet located on the electrode 6b.

[0139] Figure 4B shows a plan view of a droplet generation module B-4' according to some other embodiments of the present disclosure. The relative positional relationship of the multiple drive electrodes in the droplet generation module B-4' is substantially the same as the relative positional relationship of the multiple drive electrodes of the droplet generation module B-4 shown in Figure 4A, with the difference being that the size of each drive electrode in the droplet generation module B-4' is different from that of the droplet generation module B-4. For example, in some embodiments, the droplet generation module B-4' is configured to generate a first droplet having a first volume and a second droplet having a second volume; in this embodiment, each drive electrode having a second main body area (i.e., additional drive electrodes 1', 2') can accommodate and drive a droplet volume that is substantially equal to the first volume of the first droplet (e.g., 1 μL), and a single drive electrode having a first main body area (i.e., drive electrode 2, 3, or 4) or a combination of drive electrodes (i.e., a combination of drive electrode 6b and two additional drive electrodes 3') can accommodate and drive a droplet volume that is substantially equal to the second volume of the second droplet (e.g., 2 μL).

[0140] In this embodiment, the dimensions of additional drive electrodes 1' and 2' are substantially the same as those of the square electrodes in droplet generation module B-1 shown in FIG3A . The dimensions of drive electrodes 2, 3, and 4 are substantially the same as those of the square electrodes in droplet generation module B-2 shown in FIG3B . The dimensions of the other drive electrodes are set accordingly. The driving method for generating droplets of the first volume or the second volume using droplet generation module B-4' is substantially the same as that described in FIG5A through FIG5F and will not be further described here.

[0141] FIG4C shows a schematic plan view of a droplet generation module B-4″ according to some other embodiments of the present disclosure.

[0142] Referring to FIG. 4C , in some embodiments, droplet generation module B-4″ is configured to generate droplets of three volume types (e.g., a first volume, a second volume, and a third volume). For example, droplet generation module B-4″ adds a fourth additional drive electrode (e.g., electrode 4') and a fifth additional drive electrode (e.g., electrode 5') to droplet generation module B-4. The fourth and fifth additional drive electrodes are arranged on a side of the second additional drive electrode away from the first additional drive electrode in sub-direction d1, and the fourth and fifth additional drive electrodes have the same third main body area, which is smaller than the second main body area. The third main body area may correspond to the minimum droplet volume that can be generated by droplet generation module B-4″. Furthermore, in this example, to improve droplet generation accuracy, the second additional drive electrode is split into electrode 2' and two additional electrodes 6', and the sum of the areas of electrodes 2' and 6' is approximately equal to the second main body area. The other structures of droplet generation module B-4″ are similar to those of droplet generation module B-4 and are not further described here. In some embodiments, in the droplet generating module B-4", the drive electrode (or a combination of drive electrodes) having a first main body area, a second main body area, and a third main body area can accommodate and drive droplet volumes equal to the third volume, the second volume, and the first volume, respectively. For example, the additional drive electrodes 4' and 5' are both positive direction electrodes and have the same size as the square electrodes in the droplet generating module B-1. The additional drive electrode 2' has a long side extending along the first sub-direction d1 and a short side extending along the second sub-direction d2, and the long side is approximately equal to the side length of the electrode 1', and the short side is approximately equal to the side length of the electrode 4'. Electrodes with similar labels are electrically connected to each other, for example, electrodes 2 and 2' are electrically connected, electrodes 4 and 4' are electrically connected, electrodes 5 and 5' are electrically connected, and electrodes 6b and 6' are electrically connected.

[0143] In some embodiments, the operation process of using the droplet generation module B-4" to generate droplets with the second volume and the third volume is substantially similar to the operation process of using the droplet generation module B-4 in Figures 5A to 5F, and is not repeated here.

[0144] In some embodiments, the generation of droplets having a first volume (eg, 1 μL) by the droplet generation module B- 4 ″ may use the driving method shown in FIG. 5G to 5K .

[0145] For example, referring to FIG5G , after the sample loading operation is completed, the droplet LD0 is driven onto electrodes 2, 3, 4, 6b, and 3' using the same method as shown in FIG5A and FIG5B ; referring to FIG5G to FIG5H , then, electrodes 3, 3', and 4 are powered off, and electrode 5 is powered on, that is, electrodes 2, 5, and 6b are in an open state to split the droplet LD0 into droplets LD1 and LD2; referring to FIG5H to FIG5I , electrodes 5 and 6b are powered off, and electrodes 1, 1' is powered, thereby driving the droplet LD1 to move to electrodes 1' and 2', and the droplet LD2 moves to electrodes 1 and 2; referring to Figures 5I and 5J, electrodes 1 and 1' are powered off, and electrodes 4, 4', 5, and 5' are powered, thereby driving the droplet LD1 to move to electrodes 2', 4', and 5'; referring to Figures 5J and 5K, electrode 4' is powered off, and electrode 6' is powered, thereby splitting the droplet LD1 into droplets LD3 and LD4, and droplet LD3 has a first volume.

[0146] It should be noted that in each embodiment of the present disclosure, the first sub-direction d1 described with respect to each droplet generation module refers to the arrangement direction of the corresponding drive electrodes (e.g., main electrodes 2, 3, 4, 6) in the droplet generation module, and correspondingly, the second sub-direction d2 is a direction perpendicular to the arrangement direction. In a microfluidic chip, the first sub-directions d1 of multiple droplet generation modules may be the same or different. For example, as shown in FIG1A , the first sub-directions d1 (i.e., the arrangement direction of the corresponding electrodes) of droplet generation modules B-2 and B-3 are the same, for example, they are both the same as direction D1, while the first sub-direction d1 (i.e., the arrangement direction of the corresponding electrodes) of droplet generation module B-1 is different from that of droplet generation modules B-2 and B-3, for example, it is approximately perpendicular. The sub-directions of each droplet generation module can be set and adjusted according to product design and requirements, and the present disclosure is not limited to this.

[0147] Referring to Figure 1A, in some embodiments, the droplet generation module B-1 is directly connected to the area D (including the first droplet drive module IM1); in an embodiment where the digital microfluidic chip includes a droplet generation module B-4' or B-4", the droplet generation module B-4' or B-4" (for example, an additional drive electrode 2' or 5' located at its end) may also be directly connected to the first droplet drive module IM1; for example, at least part of the first droplet drive module IM1 may be reused as a droplet cropping module CM. In an alternative embodiment, the first droplet drive module IM1 may not be reused as the droplet cropping module CM, and the droplet cropping module CM may be separately set in other areas.

[0148] In some embodiments, the first droplet drive module IM1 includes a plurality of drive electrodes, each of which has approximately the same size and is arranged along a direction D1. One or more droplet generation modules B-1 are directly connected to the first droplet drive module IM1. When there are multiple droplet generation modules B-1, the multiple droplet generation modules B-1 can be arranged on opposite sides of the first droplet drive module IM1 in a direction D2 perpendicular to the direction D1, so that a relatively large number of droplet generation modules B-1 can be provided. Directions D1 and D2 can be referred to as the first direction and the second direction, respectively; the drive electrodes 6 at the ends of the droplet generation module B-1 are directly connected to the corresponding electrodes of the first droplet drive module IM1, and the arrangement direction of the main electrodes in the droplet generation module B-1 (i.e., the first sub-direction d1) is approximately perpendicular to the arrangement direction of the electrodes in the first droplet drive module IM1 (i.e., the first direction D1).

[0149] In some embodiments, the droplet receiving module RM is configured to receive multiple droplets generated by multiple droplet generation modules and to perform operations such as mixing and reacting the multiple droplets. For example, the droplet receiving module RM can be configured to at least perform mixing and a first reaction of liquids within a first mixing volume range, and to perform mixing and a second reaction of liquids within a second mixing volume range, where the first mixing volume range is different from the second mixing volume range. Furthermore, droplet splitting operations can be performed on droplets in the droplet receiving module RM. Droplet splitting performed in this module is, for example, used to separate large droplets into smaller droplets for liquid transfer.

[0150] In some embodiments, the droplet receiving module RM includes a region C and a region A, which can be respectively referred to as a first reaction zone C and a second reaction zone A. In some embodiments, the first reaction zone C is configured to at least achieve mixing and a first reaction of liquids within a first mixing volume.

[0151] FIG6 shows an enlarged schematic diagram of the first reaction zone C according to some embodiments of the present disclosure.

[0152] Referring to FIG. 1A and FIG. 6 , in some embodiments, the first reaction zone C includes a plurality of electrodes (or reaction electrodes) RE arranged in an array. For example, the plurality of reaction electrodes RE are arranged in a 3×3 array along directions D1 and D2 , but the present disclosure is not limited thereto. The reaction electrodes RE may also be referred to as first reaction electrodes.

[0153] In some embodiments, the volume of droplets that each reaction electrode RE can accommodate and / or drive is greater than the first volume and the second volume and less than the third volume. The maximum value of the first mixed volume range of mixed droplets that can be accommodated in the first reaction zone C composed of multiple reaction electrodes RE is, for example, an integer multiple of the third volume. Region E located on the side of the reaction zone C and directly connected to the reaction zone C includes multiple drive electrodes arranged along the direction D1. The volume of droplets that each drive electrode can accommodate and drive can be the same as or different from the volume of droplets that can be accommodated and / or driven by each reaction electrode RE; in some embodiments, the volume of droplets that each drive electrode can accommodate and drive can be greater than the volume of droplets that can be accommodated and / or driven by each reaction electrode RE. For example, the volume of droplets that each of the connecting electrodes can accommodate and drive can be approximately equal to the third volume. For example, region E can be or include a droplet drive module IM3.

[0154] In some examples, the reaction zone C is composed of 3×3 reaction electrodes with a side length of 1645 μm, and is configured to achieve mixing, reaction and other operations of droplets with a maximum mixing volume of 20 uL. The volume of the controllable droplets of a single reaction electrode RE is approximately 2.3 uL. However, the present disclosure is not limited to this. It should be understood that the size and number of electrodes in the reaction zone can be set and adjusted according to the actual product design and needs; for example, the reaction zone may include more or fewer reaction electrodes, and the reaction electrodes may have other types of shapes and / or adopt other types of arrangements, so as to achieve mixing, reaction and other operations of liquids with larger or smaller mixing volumes.

[0155] In some embodiments, the second reaction zone A is configured to achieve mixing and a second reaction of liquids within the second mixing volume. The second reaction zone may include multiple second reaction electrodes. The multiple second reaction electrodes may be arranged, for example, along direction D1, and the volume of droplets that each second reaction electrode can accommodate and drive is, for example, an integer multiple of the third volume, and may be larger than the volume of droplets that a single electrode in region C or E can accommodate and drive, but the present disclosure is not limited to this.

[0156] In some embodiments, when performing a droplet reaction operation, it may include the use of magnetic beads for purification operations, or part of the reaction needs to be carried out within a suitable temperature range. In some embodiments, the droplet receiving module (e.g., the reaction zone) may include a magnetic control area to facilitate the manipulation of the magnetic bead droplets. In some embodiments, the reaction zone may include a temperature control area so that the liquid located in the temperature zone can react at a temperature within a predetermined temperature range. For example, the first reaction zone C may include a magnetic control area, and the second reaction zone A may include a temperature control area. In some examples, the second reaction zone A may include a first temperature control area, a second temperature control area, and a third temperature control area, which are respectively configured to control the liquid located in the corresponding area to react at a temperature within a first temperature range, a second temperature range, and a third temperature range; the first temperature range, the second temperature range, and the third temperature range are different from each other. For example, a second reaction zone A including three temperature control areas can be used to perform a three-temperature polymerase chain reaction (PCR amplification reaction), but the present disclosure is not limited thereto. In alternative embodiments, the second reaction zone may include two temperature-controlled zones (e.g., for two-step PCR amplification reactions), a single temperature-controlled zone (e.g., for constant-temperature PCR amplification reactions), or more temperature-controlled zones according to actual application requirements.

[0157] In some embodiments, the interconnection module IM includes multiple droplet drive modules, thereby realizing the interconnection between multiple droplet generation modules, droplet receiving modules, droplet cutting modules and / or other areas. For example, the interconnection module IM may include droplet drive modules IM1, IM2 and IM3; the droplet drive modules in the interconnection module IM may be arranged along direction D1 and / or direction D2, and may be connected to each other. For example, the droplet drive modules IM1 and IM3 may be connected through the droplet drive module IM2. The droplet generation module for generating the second droplet in the droplet generation module may be connected to the droplet drive module IM2, and the droplet generation module for generating the third droplet may be connected to the droplet drive module IM2 or IM3. Reaction areas A and C may be connected to at least one of the droplet drive modules IM1, IM2, and IM3, respectively.

[0158] For example, droplet generation modules B-2 and B-3 are both directly connected to droplet drive module IM2, and reaction zone C is directly connected to droplet drive modules IM2 and IM3. For example, droplet drive module IM3 can be arranged on opposite sides of reaction zone C in direction D1. In some embodiments, direct connection between reaction zone C and multiple different types of droplet drive modules in interconnect module IM can increase the flexibility of droplet movement paths and / or shorten the droplet movement paths.

[0159] It should be understood that, with the exception of the additional dummy electrodes in the droplet generation module, the electrode arrays in each module of the digital microfluidic chip 500 all include drive electrode structures DS, as shown in FIG1C , with adjacent drive electrode structures spaced a certain distance apart. For simplicity, FIG1A does not explicitly illustrate the gaps between adjacent electrode structures DS. However, it should be understood that the presence of gaps between adjacent electrode structures ensures that adjacent electrode structures are spaced apart from each other and do not contact each other, thereby enabling reliable and effective droplet manipulation.

[0160] In some embodiments, as shown in FIG1A , among the multiple modules in the digital microfluidic chip 500, the reaction areas A and C may be arranged at both ends, and the droplet generation structure and the interconnection module may be arranged between the reaction areas A and C. This allows the droplet generation module to be kept away from the temperature control or magnetic control area, so that after the droplets are generated from the droplet generation module, they will not pass through the temperature control or magnetic control area before entering the reaction area for reaction, thereby avoiding the temperature control or magnetic control area from adversely affecting the manipulation and / or properties of the droplets before the droplets react.

[0161] In some embodiments, the droplet trimming module CM is located in a relatively central position, so that the distance between each droplet generation module and the droplet trimming module CM is relatively short, thereby making the movement path of the droplets to be trimmed relatively short during trimming.

[0162] In some embodiments, the electrode array areas other than the droplet generation module and the reaction area C of the digital microfluidic chip 500 can select electrode arrays with appropriate sizes according to needs, and the sizes of the driving electrodes in different modules can be different, and the sizes of multiple driving electrodes in the same module can also be the same or different.

[0163] 7A to 7D are schematic enlarged plan views of various electrode arrays included in a digital microfluidic chip according to some embodiments of the present disclosure.

[0164] Referring to Figures 7A to 7D , in some embodiments, the digital microfluidic chip 500 includes electrode arrays AE1, AE2, AE3, and AE4 in different regions. The electrode arrays AE1, AE2, AE3, and AE4 each include multiple electrodes E1, E2, E3, and E4 arranged in rows or columns. Within each electrode array, the multiple electrodes are spaced a certain distance apart and are generally aligned with one another in the electrode arrangement direction. It should be understood that for ease of description, Figures 7A to 7D illustrate the multiple electrodes of the electrode arrays AE1-AE4 as being arranged in the same direction. However, within the chip, each electrode array can be arranged in the direction D1 or D2 shown in Figure 1A , or in any other suitable direction, as desired.

[0165] In each of the electrode arrays AE1-AE4, the plurality of driving electrodes may have substantially the same shape and size and be spaced apart by a predetermined distance. In some embodiments, the electrodes E1-E4 may all be square electrodes, and the electrodes E1-E4 may have different sizes. For example, the areas of the electrodes E1-E4 may increase in sequence and may be designed to have appropriate sizes based on actual application requirements. In other words, the volume of controllable droplets in the electrode arrays AE1-AE4 increases in sequence. In some embodiments, in each of the electrode arrays AE1-AE4, the minimum volume of droplets that can be driven by the electrode array is the volume of droplets that can be accommodated and driven by a single electrode in the electrode array, and the maximum volume of droplets that can be driven by the electrode array is the volume of droplets that can be accommodated and driven by three electrodes in the electrode array.

[0166] For example, in electrode array AE1, a single electrode E1 can drive a droplet with a volume substantially equal to the first volume. The maximum droplet volume that electrode array AE1 can drive is three times the first volume (i.e., corresponding to a droplet occupying three electrodes E1). Therefore, electrode array AE1 can drive droplets with a volume ranging from the first volume (corresponding to a droplet occupying one electrode E1) to three times the first volume (corresponding to a droplet occupying one to three electrodes E1).

[0167] In the electrode array AE2, a single electrode E2 can drive droplets with a volume substantially equal to the second volume. The maximum droplet volume that the electrode array AE2 can drive is three times the second volume (i.e., the corresponding droplet occupies three electrodes E2). Therefore, the electrode array AE2 can drive droplets with a volume ranging from the second volume to three times the second volume (corresponding to the droplet occupying 1-3 electrodes E2).

[0168] In the electrode array AE3, a single electrode E3 can drive droplets with a volume substantially equal to the third volume. The maximum droplet volume that the electrode array AE3 can drive is three times the third volume (i.e., the corresponding droplet occupies three electrodes E3). Therefore, the electrode array AE3 can drive droplets with a volume ranging from the third volume to three times the third volume (corresponding to the droplet occupying 1-3 electrodes E3).

[0169] In the electrode array AE4, a single electrode E4 can drive droplets with a volume that is an integer multiple of the third volume, for example, twice the third volume. The maximum droplet volume that the electrode array AE4 can drive is six times the third volume (i.e., corresponding to a droplet occupying three electrodes E4). Therefore, the droplet volume that the electrode array AE4 can drive ranges from twice the third volume to six times the third volume (corresponding to a droplet occupying 1-3 electrodes E4).

[0170] In some examples, the first volume, the second volume, and the third volume are 1 μL, 2 μL, and 5 μL, respectively, and electrodes E1-E4 are all square electrodes; accordingly, the side length of electrode E1 is approximately 1084 μm, the interval between adjacent electrodes E1 is approximately 25 μm, and the electrode array AE1 can achieve the movement of 1-3 μL droplets; the side length of electrode E2 is approximately 1550 μm, the interval between adjacent electrodes E2 is approximately 25 μm, and the electrode array AE2 can achieve the movement of 2-6 μL droplets; the side length of electrode E3 is approximately 2474 μm, the interval between adjacent electrodes E3 is approximately 25 μm, and the electrode array AE3 can achieve the movement of 5-15 μL droplets; the side length of electrode E4 is approximately 3500 μm, the interval between adjacent electrodes E4 is approximately 25 μm, and the electrode array AE4 can achieve the movement of 10-30 μL droplets. In some embodiments, for the larger electrode arrays AE3 and AE4, in order to improve the accuracy of droplet manipulation, the droplets are allowed to occupy a maximum of 2 electrodes in the electrode arrays AE3 and AE4 during droplet manipulation, that is, the electrode array AE3 corresponds to the movement of 5-10 μL droplets, and the electrode array AE4 corresponds to the movement of 10-20 μL droplets.

[0171] Referring to Figure 1A and Figures 7A to 7D, in some embodiments, to ensure that droplets generated by multiple droplet generation modules can be driven by the electrode array, the connection and arrangement between the droplet generation modules and the electrode array must comply with the following rules: In the droplet generation modules and electrode arrays connected to each other, the minimum volume of droplets generated by the droplet generation modules must be less than or equal to the maximum volume of droplets that can be driven by the electrode array, and greater than or equal to the minimum volume of droplets that can be driven by the electrode array. Therefore, droplet generation module B-1 can be connected to electrode array AE1, droplet generation module B-2 can be connected to electrode array AE1 or AE2, and droplet generation module B-3 can be connected to electrode array AE2 or AE3. After the above conditions are met, the positions of the droplet generation modules can be further set according to the characteristics of the biochemical reaction and chip layout.

[0172] In some embodiments, to ensure that large-span droplet manipulation can be achieved in a digital microfluidic chip, the connection, arrangement, and movement of droplets between electrode arrays follow the following rules: (1) When droplets on a small-size electrode array need to be directly driven by a large-size electrode array, the maximum droplet volume that can be driven by the small-size electrode array must be greater than the minimum droplet volume that can be driven by the large-size electrode array; (2) When droplets on a small-size electrode array do not need to be directly driven by a large-size electrode array, the droplets on the small-size electrode array are driven by the electrodes in the large-size electrode array and can move unidirectionally toward the large-size electrode array, and only when the cumulative volume of the moving droplets is greater than the minimum droplet volume that can be driven by the large-size electrode array can the accumulated droplets move between the electrodes in the large-size electrode array; (3) When droplets on a large-size electrode array need to be directly driven by a small-size electrode array, the droplets on the large-size electrode array must be split into droplets with a volume within the droplet volume range that can be driven by the small-size electrode array before they can move between the electrodes of the small-size electrode array. After the above conditions are met, the position of the electrode array can be further set according to actual application requirements (for example, characteristics of biochemical reactions and chip layout, etc.).

[0173] It should be noted that here, the small-size electrode array and the large-size electrode array are relative to two electrode arrays connected to each other; for example, the first electrode array and the second electrode array are connected to each other, and the size of a single electrode in the first electrode array is smaller than the size of a single electrode in the second electrode array, then the first electrode array is called the small-size electrode array, and the second electrode array is called the large-size electrode array.

[0174] Referring to Figures 1A and 7A to 7D, in some embodiments, based on the above-described rules, in the digital microfluidic chip 500, region D (i.e., droplet drive module IM1) directly connected to droplet generation module B-1 utilizes electrode array AE1. The volume of a single droplet generated in droplet generation module B-1 corresponds to (e.g., is the same as) the minimum volume of a droplet that can be driven by a single electrode in electrode array AE1. In some embodiments, droplet trimming module CM1 also includes electrode array AE1, and droplet trimming module CM1 and droplet trimming module CM1 may share the same electrode array AE1, but the present disclosure is not limited thereto. Droplet drive module IM2 in interconnect module IM utilizes electrode array AE2. Droplet drive module IM2 can connect various modules and / or regions, as well as droplet drive modules IM1 and IM3. Reaction zone C utilizes the electrode array shown in Figure 6. Region E (i.e., droplet drive module IM3) located to the side of reaction zone C utilizes electrode array AE3. Reaction zone A utilizes electrode array AE4. In some embodiments, the reaction zone C is directly connected to electrode arrays AE2 and AE3 of different sizes (i.e., different drive capabilities) to increase the flexibility of the droplet movement path and / or shorten the droplet movement path. In other embodiments, the interconnection module IM may also include electrode arrays of other sizes based on actual application requirements.

[0175] In some embodiments, among the multiple droplet actuation modules of the interconnect module IM, the droplet actuation module IM1 using the electrode array AE1 is primarily configured to connect to the droplet generation module B-1; the droplet actuation module IM3 using the electrode array AE3 is primarily configured to connect to the reaction area C; and the droplet actuation module IM2 using the electrode array AE2 is primarily configured to connect to the other modules and / or areas, as well as to connect the droplet actuation modules IM1 and IM3. In some embodiments, in the interconnect module IM, the number of droplet actuation modules IM2 is greater than the number of droplet actuation modules IM1 and the number of droplet actuation modules IM3.

[0176] In some embodiments, to accommodate as many biochemical processes as possible, as many droplet generation modules as possible can be provided in the digital microfluidic chip, and the droplet generation modules need to be as far away as possible from the temperature-controlled magnetic control region (e.g., located in the reaction zone) to avoid the temperature-controlled magnetic control region from adversely affecting the droplets before the reaction. In some embodiments, because the volume of a single droplet generated by the droplet generation module B-1 (i.e., the minimum droplet volume that the module can generate) is consistent with (e.g., approximately the same as) the minimum volume of a droplet that can be driven in region D, the droplet generation module B-1 needs to be directly connected to region D to enable the movement of the droplets generated by the droplet generation module B-1. In order to maximize the number of droplet generation modules B-1 and improve the flexibility of droplet generation, that is, to provide as many droplet generation modules B-1 as possible, the droplet generation modules B-1 can be provided on opposite sides of region D in direction D2 and can be evenly distributed. In some embodiments, because the interconnect modules 1M connecting the various regions primarily utilize electrode arrays AE2, and the droplet generation modules B-2 are configured to generate a single droplet volume (i.e., the minimum droplet volume that the module can generate) that is consistent with the minimum droplet volume that can be driven by electrode array AE2, a larger number of droplet generation modules B-2 may be provided. For example, in some embodiments, the number of droplet generation modules B-2 is greater than the number of droplet generation modules B-1 and / or greater than the number of droplet generation modules B-3.

[0177] On the other hand, the multiple droplet generation modules need to include at least droplet generation modules that can generate the first volume, the second volume, and the third volume; the multiple droplet generation modules may include generation modules that can only generate droplets of a single volume type (for example, droplet generation modules B-1, B-2, B-3), generation modules that can generate droplets of multiple volume types (for example, droplet generation modules (or can be called fourth droplet generation modules) B-4, B-4', B-4"), or a combination thereof. For example, when the droplet generation modules are all generation modules for droplets of a single volume type, the droplet generation modules include at least one droplet generation module B-1, at least one droplet generation module B-2, and at least one droplet generation module B-3; in other examples, the droplet generation modules may include at least one first droplet generation module B-1 and at least one droplet generation module B-4; or, the droplet generation modules may include at least one third droplet generation module B-3 and at least one droplet generation module B-4'; or, the droplet generation modules may include at least one droplet generation module B-4".

[0178] In the example shown in FIG1A , the digital microfluidic chip 500 includes two first droplet generation modules B-1, three second droplet generation modules B-2, and one third droplet generation module B-3. In some examples, the droplet cutting module CM is located between reaction zones A and C. A portion of the interconnect module IM (e.g., the portion located on the left in FIG1A ) connects reaction zones A and C. The droplet cutting module CM can be connected to a drive electrode located in the middle of the portion of the interconnect module IM. One of the multiple second droplet generation modules B-2 and the droplet generation module B-3 are located on the same side of the droplet cutting module CM in direction D1, thereby ensuring that the path required for droplets to move in various operation steps is the shortest. In some embodiments, multiple droplet generation modules connected to the portion of the interconnect module extending in the same direction are located on the same side of the portion of the interconnect module; for example, as shown in FIG1A . Droplet generation modules B-2 and B-3 connected to the portion of the interconnect module IM extending in direction D1 are both located to the left of the portion of the interconnect module IM, thereby saving chip area.

[0179] In some embodiments, the settings of the first volume, the second volume, and the third volume can be set according to the droplet manipulation volume required to be achieved in the actual application of the chip; for example, in some examples, the digital microfluidic chip is configured to achieve the manipulation of droplets with a volume of 1 μL-20 μL and a step size of 1 μL, which can be applied to gene library preparation, for example. Based on this application requirement, in library preparation, the required volume of various reagents is generally less than 5μL, and volumes of 1-5μL are involved. In order to obtain droplets with a volume of 1μL-20μL and a step size of 1μL, a 1μL droplet operation unit is necessary, so the first volume is set to 1μL; considering the driving ability of the driving electrode on the droplets, the volume of the next-level droplet operation unit (i.e., the second volume) needs to be less than 3μL. If a 3μL operation unit is used, both 2ul droplets and 1uL droplets cannot be driven by an electrode array with a single electrode capable of controlling a droplet volume of 3uL. If a 2uL operation unit is used, 1uL droplets cannot be driven by an electrode array with a single electrode capable of controlling a droplet volume of 2uL. Taking into account the connection between each area, if a single electrode can control a droplet volume of 1uL as the connecting electrode array between each area, then the droplet volume that can be driven by the connecting electrode array is only 1-3uL, and the number of electrodes required is relatively large, which will lead to a decrease in the efficiency of droplet driving and an increase in the number of binding electrodes. Therefore, in order to improve the driving ability and driving efficiency of the connecting electrode array for droplets, a 2uL droplet volume is selected as the volume of the next-level droplet operation unit, that is, the second volume is set to 2uL. In addition, in order to reduce the number of droplet operation steps, the volume of the next-level operation unit (that is, the third volume) is set to 5uL.

[0180] When the first volume, the second volume, and the third volume are set to 1 μL, 2uL, and 5uL, respectively, the above-mentioned electrode arrangement is adopted, and the acquisition and operation of droplets of 1-20uL with a step size of 1uL can be achieved in the digital microfluidic chip 500. For example, in the example shown in Figure 1A, 1 μL droplets can be generated by the droplet generation module B-1, 2uL droplets can be generated by the droplet generation module B-2, and 5uL droplets can be generated by the droplet generation module B-3. In addition, 4uL droplets can be obtained by splitting 1uL droplets from 5uL droplets, and 3uL droplets can be obtained by splitting 2uL droplets from 5ul droplets (i.e., two 1uL droplets). The above-mentioned droplet splitting operations (or called clipping operations) can all be achieved by the droplet clipping module CM. Alternatively, 4uL droplets can also be obtained by generating two 2uL droplets in the droplet generation module B-2 and mixing the two droplets. A 6uL droplet can be obtained by mixing a 5uL droplet with a 1uL droplet, a 7uL droplet can be obtained by mixing a 5uL droplet with a 2uL droplet, an 8uL droplet can be obtained by mixing a 5uL droplet with a 3uL droplet, a 9uL droplet can be obtained by mixing a 5uL droplet with a 4uL droplet, a 10uL droplet can be obtained by generating two 5uL droplets in the droplet generation module B-3 and mixing the two droplets, and droplets of 10uL or more can be obtained by arranging and combining droplets of different volumes.

[0181] In some embodiments, a method for generating droplets of multiple volume types using a digital microfluidic chip may include at least one of the following: using a droplet generation module (e.g., droplet generation module B-1, B-4', or B-4") to generate one or more first droplets each having a first volume; using a droplet generation module (e.g., droplet generation module B-2, B-4, B-4", or B-4") to generate one or more second droplets each having a second volume; using a droplet generation module (e.g., droplet generation module B-3, B-4, B-4', B-4") to generate one or more third droplets each having a third volume; using a droplet generation module (e.g., droplet generation module B-4, B-4', B-4") to generate one or more third droplets each having a third volume. At least one of one or more second droplets, each having a second volume, and one or more third droplets, each having a third volume; using a droplet generation module (e.g., droplet generation modules B-3, B-4, B-4', B-4") and a droplet trimming module in combination to generate at least one of one or more fourth droplets, each having a fourth volume, and one or more fifth droplets, each having a fifth volume, wherein the fourth volume and the fifth volume are greater than the second volume and smaller than the third volume; and mixing at least two of the one or more first droplets, the one or more second droplets, the one or more third droplets, the one or more fourth droplets, and the one or more fifth droplets to obtain droplets with other target volumes. In some examples, the first volume, second volume, third volume, fourth volume, and fifth volume are 1 μL, 2 μL, 5 μL, 3 μL, and 4 μL, respectively.

[0182] In some embodiments, generating the fourth droplet using the third droplet generation module and the droplet trimming module in combination includes: using the third droplet generation module to generate an initial droplet having the third volume; and trimming two trimmed droplets each having the first volume from the initial droplet by the droplet trimming module, and moving the two trimmed droplets to a waste area, wherein the remaining droplets after trimming of the initial droplet form the fourth droplet.

[0183] In some embodiments, generating the fifth droplet using the third droplet generation module and the droplet trimming module in combination includes: using the third droplet generation module to generate an initial droplet having the third volume; and trimming a trimmed droplet having the first volume from the initial droplet by the droplet trimming module, and moving the trimmed droplet to a waste area, wherein the remaining droplet after trimming of the initial droplet forms the fifth droplet.

[0184] 8A to 8C illustrate an example of using the digital microfluidic chip shown in FIG. 1A to generate droplets of a target volume through a droplet generation module B-3 and a droplet trimming module CM.

[0185] Referring to Figure 8A , in some embodiments, a droplet LD0 having a third volume is generated by droplet generation module B-3. By sequentially applying drive signals (i.e., energizing) to corresponding drive electrodes in interconnect module IM connected to droplet generation module B-3, droplet LD0 is moved through interconnect module IM to region D (i.e., droplet cutting module CM). For example, droplet cutting module CM includes drive electrodes 1, 2, 3, 4, 5, ... arranged along direction D1; electrode 0 in interconnect module IM is directly connected to droplet cutting module CM, for example, directly connected to electrode 1. The movement path of droplet LD0 is shown in Figure 8A , and Figure 8B shows an enlarged schematic diagram of droplet LD0 after it has moved to region D. In some embodiments, when the droplet LD0 moves close to the droplet cutting module CM, the electrodes 1-4 are opened, so that the droplet LD0 moves onto the electrodes 1-4; in some examples, because the volume of the droplet LD0 (i.e., the third volume) is greater than the droplet volume that the electrodes 1-4 can accommodate (i.e., four times the first volume), part of the droplet LD0 is still located on the electrode 0. The state of the droplet LD0 at this step is shown in Figure 8B; referring to Figures 8B and 8C, then, the electrode 3 is closed and the electrode 0 is opened, so that the droplet LD0 splits into droplets LD1 and LD2; thereafter, the droplet LD2 is driven to move to the waste area, thereby obtaining a droplet LD1 with a target volume. In this example, the droplet generation module B-1 can be reused as a waste area, and the discarded droplet LD2 can be removed to the module. That is, through the operations of Figures 8A to 8C, the droplet LD2 with the first volume is cut from the droplet LD0 with the third volume, thereby obtaining a droplet LD1 with a target volume (i.e., the third volume minus the first volume). In some examples, the first volume is 1 μL and the third volume is 5 μL, that is, the above steps can obtain a droplet with a target volume of 4 μL. In some examples, the above steps can be repeated to cut out a waste droplet of the first volume from the droplet LD1, thereby obtaining a droplet with a target volume of, for example, 3 μL.

[0186] In the above embodiment, the multiple electrodes in the droplet trimming module CM have the same size and are arranged along a single direction, but the present disclosure is not limited thereto.

[0187] Figure 9A shows a plan view of various modules in a digital microfluidic chip 500' according to other embodiments of the present disclosure. Digital microfluidic chip 500' includes a droplet trimming module CM'. Figure 9B shows an enlarged plan view of the droplet trimming module CM' in Figure 9A. The droplet trimming module CM' is similar in structure to the droplet trimming module CM of the aforementioned embodiment, differing in that it also includes auxiliary trimming electrodes. The remaining structures of digital microfluidic chip 500' are similar to those of digital microfluidic chip 500 and are not further described here.

[0188] 9A and 9B , in some embodiments, the droplet trimming module CM′ includes a plurality of electrodes ce1 and one or more auxiliary electrodes ce2; the electrodes ce1 may also be referred to as main electrodes or main trimming electrodes. The auxiliary electrodes ce2 may also be referred to as auxiliary trimming electrodes. Similar to the aforementioned embodiments, the plurality of main electrodes ce1 have the same dimensions (e.g., width, area, etc.) as one another and are arranged, for example, along direction D1. One or more auxiliary trimming electrodes are arranged on one or opposite sides of the main electrode array in a direction (e.g., direction D2) perpendicular to the arrangement direction of the main electrodes ce1 (e.g., direction D1). For example, the auxiliary trimming electrodes may be connected to the trimming drive electrodes ce1 near the interconnect module and may be connected to the interconnect module IM. The dimensions of the auxiliary trimming electrodes ce2 may be the same as or different from those of the main electrodes ce1. In this embodiment, at least a portion of the droplet trimming module CM′ (e.g., the main electrodes) may share the same electrode array with the droplet drive module IM1.

[0189] In some embodiments, the main electrode ce1 has a width w1, and the auxiliary cutting electrode ce2 has a width w2, and the width w2 is greater than the width w1, for example, twice the width w1. The widths w1 and w2 are the widths along the main electrode arrangement direction (for example, direction D1). For example, the main electrode ce1 is a square electrode with a side length w1, and the auxiliary cutting electrode ce2 is a rectangular electrode with a long side extending along the main electrode arrangement direction (for example, direction D1) and a short side extending in a direction perpendicular to the main electrode arrangement direction (for example, direction D2). For example, the short side and long side of the auxiliary cutting electrode ce2 may be approximately equal to one and two times the length of the side of the main electrode ce1, respectively.

[0190] For example, among the multiple main electrodes ce1, the electrodes 1 located at both ends of the droplet cutting module CM' in the direction D1 are directly connected to the driving electrodes of the interconnection module (for example, the driving electrodes of the droplet driving module IM2), respectively, and the electrode 2 is located on the side of the electrode 1 away from the interconnection module IM and is directly connected to the electrode 1. The one or more main electrodes located at the end of the droplet cutting module CM' can be referred to as end cutting electrodes or end main cutting electrodes. In some embodiments, the auxiliary cutting electrodes are located on opposite sides of the end main cutting electrodes (for example, electrodes 1 and 2) in the direction D2, and auxiliary cutting electrodes ce2 can be provided at the ends of the droplet cutting module CM' connected to the interconnection module (for example, both ends), that is, the droplet cutting module CM' may include four auxiliary cutting electrodes ce2. The auxiliary cutting electrodes ce2 are adjacent to and directly connected to the end main cutting electrodes and the interconnection module. In some embodiments, the accuracy of droplet cutting can be improved by providing auxiliary cutting electrodes. It should be understood that the shape, size and number of the auxiliary cutting electrodes ce2 shown in Figures 9A and 9B are only for illustration, and the present disclosure is not limited thereto.

[0191] 10A and 10B illustrate plan views of droplet trimming using the droplet trimming module CM' shown in FIG. 9A and FIG. 9B according to some embodiments of the present disclosure.

[0192] Referring to Figure 10A , a droplet LD0 is moved to a droplet shearing module CM'. For example, electrodes 1-4 are turned on, allowing the droplet LD0 to move onto electrodes 1-4. The droplet LD0 can be a droplet from droplet generation module B-3, a sheared droplet, or a mixed droplet. If the droplet LD0 is from droplet generation module B-3, a portion of the droplet LD0 also resides on an electrode connected to electrode 1 in the interconnect module, though this is not shown for simplicity. After the droplet LD0 is moved onto electrodes 1-4, electrode 3 is turned off and electrode 6 is turned on. That is, electrodes 1, 2, 4, and 6 are turned on, causing the droplet LD0 to split into droplet LD1 and droplet LD2, driven by electrodes 1, 2, and 6, and electrode 4, respectively. Droplet LD2 then moves to the waste area, while droplet LD1 remains at the target volume.

[0193] In this embodiment, during droplet splitting, compared to the aforementioned embodiments of Figures 8B and 8C , which used electrodes 0, 1, and 2 to apply driving force, this embodiment uses electrodes 1, 2, and 6 to apply driving force to the droplet. Electrode 6 is closer to the droplet, thereby better driving the droplet splitting and improving the accuracy of droplet clipping, that is, improving the accuracy of the obtained droplet volume. In this context, a higher accuracy of the obtained droplet volume means a smaller difference between the droplet volume and the target volume. In this embodiment, the structure of the droplet clipping module is similar to that of the droplet generation module, and the volume accuracy of the droplets clipped by the droplet clipping module is similar to the volume accuracy of the droplets generated by the droplet generation module.

[0194] Figures 11A and 11B illustrate schematic plan views of digital microfluidic chips according to other embodiments of the present disclosure. For the sake of simplicity, Figures 11A and 11B only illustrate the droplet manipulation modules within the digital microfluidic chip, without specifically illustrating other components. It should be understood that these other components are similar to those in the previous embodiments and can be referenced to the structure shown in Figure 1A.

[0195] Referring to Figure 11A, the various driving modules in the digital microfluidic chip 600a are similar to those in the previous embodiment. The following describes only the differences between this embodiment and the previous embodiment. In the digital microfluidic chip 600a, the droplet generation structure may include a droplet generation module B-1, a droplet generation module B-2, a droplet generation module B-3, and a droplet cutting module CM. The interconnection module IM includes droplet driving modules IM1, IM2, and IM3. For example, the area D directly connected to the droplet generation module B-1 is the droplet driving module IM1, and can be reused as the droplet cutting module CM. The droplet receiving module includes a reaction area C. The area E located on one side of the reaction area C may be or include a droplet driving module IM3, and the droplet driving module IM3 is directly connected to the reaction area C. The modules are connected to each other through the interconnection module IM, and the arrangement rules between the modules are the same as those described above. In this embodiment, reaction zone C can serve as the first reaction zone and has substantially the same function as reaction zone C in the previous embodiment; while zone E (i.e., droplet drive module IM3) can be reused as the second reaction zone to achieve the same function as reaction zone A in the previous embodiment. That is, droplet drive module IM3 and the second reaction zone can share the same electrode array. For example, zone E can include a temperature-controlled area for performing reactions that require temperature control, such as PCR amplification. In this example, reaction zone C can be configured to achieve mixing and a first reaction of liquids within a first mixing volume, while zone E is configured to achieve mixing and a second reaction of liquids within a second mixing volume. In other embodiments, the droplet receiving module includes a single reaction zone C, and reaction zone C can be used to perform multiple types of reactions. For example, reaction zone C can include a magnetic control area, a temperature-controlled area, etc. In this example, reaction zone C can serve as a common reaction zone and be configured to achieve mixing and a first reaction of liquids within the first mixing volume, and to achieve mixing and a second reaction of liquids within the second mixing volume, in different operation steps.

[0196] In some embodiments, the module unit MU1 shown in FIG11A can serve as the smallest module unit (e.g., the smallest reaction unit) of a microfluidic chip, and can be combined and / or expanded according to actual biochemical process and other application requirements to achieve compatibility with more biochemical processes and other application requirements. For example, the module unit in FIG11A can include multiple expansion connection points AP, BP, CP, DP, and EP; here, "expansion connection points" refer to locations that can be further connected to other electrode arrays and / or other modules to further expand the module unit.

[0197] For example, referring to Figures 7A to 7D and Figure 11A, the extended connection points AP and BP can be further connected to electrode arrays AE1, AE2, or AE3; the extended connection points CP and DP can be further connected to electrode arrays AE1, AE2, or AE3; and the extended connection point EP can be further connected to electrode array AE2. The modular units can be expanded according to actual application needs.

[0198] FIG11B shows a schematic plan view of a droplet manipulation module in a digital microfluidic chip 600 b according to other embodiments of the present disclosure. The module is an expanded module based on the module unit shown in FIG11A .

[0199] 11B , the droplet manipulation module of the digital microfluidic chip 600 b includes a module unit MU1 and a module unit MU2 connected to each other; the module unit MU2 has a similar structure to the module unit MU1 , and the two module units can share an area D as a droplet trimming module CM.

[0200] It should be understood that FIG11B is only an example of module unit expansion, and the present disclosure is not limited thereto. Various combinations and expansions of modules can be performed according to actual application needs.

[0201] In some embodiments of the present disclosure, a method for droplet mixing and / or reaction using a digital microfluidic chip is provided. The method may include: generating sample droplets and / or various required droplets through a droplet generating structure (e.g., one or more droplet generating modules and / or droplet cutting modules), driving these droplets to move to a droplet receiving module (e.g., a reaction zone) through a driving electrode of an interconnected module, and causing these droplets to mix and / or react in the droplet receiving module. In some embodiments, multiple steps of mixing and / or reaction are required, and different mixing and reaction steps may be performed, for example, in different areas of the droplet receiving module, and the method may include driving mixed droplets and / or reacted droplets from one reaction zone to another reaction zone to perform the next step of the reaction. The present disclosure does not limit the application fields and reaction types of digital microfluidic chips.

[0202] In some embodiments of the present disclosure, a method for preparing a gene library using a digital microfluidic chip is provided, the method may include: adding a deoxyribonucleotide sample solution, a ligase buffer, a linker solution, and a ligase solution to corresponding droplet generation modules in a plurality of droplet generation modules, and generating sample droplets, ligase buffer droplets, linker droplets, and ligase droplets through a droplet generation structure; moving the sample droplets, ligase buffer droplets, linker droplets, and ligase droplets to a droplet receiving module through an interconnection module, and mixing and reacting the sample droplets, ligase buffer droplets, linker droplets, and ligase droplets in a first reaction zone of the droplet receiving module to form a sample mixture. The method comprises the steps of: combining droplets; adsorbing and eluting deoxyribonucleotides on the sample mixed droplets to obtain a first deoxyribonucleotide chain; adding an amplification solution and a primer solution to corresponding droplet generation modules in the plurality of droplet generation modules, and generating amplification droplets and primer droplets through a droplet generation structure; moving the amplification droplets and primer droplets to a droplet receiving module through an interconnection module, wherein the amplification droplets and primer droplets undergo a polymerase chain reaction with the first deoxyribonucleotide chain in the droplet receiving module to form amplified deoxyribonucleotide mixed droplets; and adsorbing and eluting deoxyribonucleotides on the amplified deoxyribonucleotide mixed droplets to obtain an amplified deoxyribonucleotide chain.

[0203] In some embodiments, the sample droplet has a sixth volume, and generating the sample droplet includes: adding a deoxyribonucleotide sample solution at a third droplet generation module, and generating a first sample droplet and a second sample droplet, each having a third volume, through the third droplet generation module, moving the second sample droplet to a droplet trimming module through an interconnection module, and trimming the second sample droplet into a trimmed sample droplet having the second volume and a third sample droplet having a remaining volume through the droplet trimming module, the first sample droplet and the third sample droplet constituting a sample droplet having the sixth volume.

[0204] In some embodiments, generating ligase buffer droplets, linker droplets, and ligase droplets includes: adding ligase buffer at a second droplet generation module, and generating a ligase buffer droplet having a second volume through the second droplet generation module; adding a ligase solution at the second droplet generation module, and generating two ligase sub-droplets, each having a second volume, through the second droplet generation module, the two ligase sub-droplets constituting a ligase droplet; adding a linker solution at the first droplet generation module, and generating a linker droplet having a first volume through the first droplet generation module.

[0205] Figures 12A through 12I illustrate methods for preparing a gene library using a digital microfluidic chip 500, according to some embodiments of the present disclosure. The following examples illustrate droplet generation modules B-1, B-2, and B-3 configured to generate 1 μL, 2 μL, and 5 μL droplets, respectively. The droplet movement paths in each step are indicated by the arrows in the corresponding figures.

[0206] Referring to Figure 12A, deoxyribonucleic acid (DNA) sample droplets are obtained through a droplet generation structure (for example, one or more droplet generation modules and / or droplet shearing modules), for example, 8uL DNA sample droplets are obtained; in order to minimize the number of operation steps and reduce the driving distance of the droplets, this step uses the droplet generation module B-3, the droplet generation module B-1 module located on the side of area D close to the droplet generation module B-3 (that is, located on the lower side of the middle area in the figure), area D (droplet shearing module CM), the reaction area C in the droplet receiving module and the droplet driving module IM3 on one side thereof, wherein the droplet generation module B-1 module is used as a waste liquid pool in this step. For example, a DNA sample solution is injected from the sample inlet corresponding to the droplet generation module B-3, and a 5uL DNA sample droplet is generated by the droplet generation module B-3; the 5uL sample droplet is moved to the droplet cutting module CM through the driving electrode of the interconnection module IM (for example, the droplet driving module IM2), and 2uL of waste droplets are cut out from the 5uL sample droplet by the droplet cutting module CM and moved to the waste liquid pool, thereby obtaining a 3uL DNA sample droplet SD1; the 3uL DNA sample droplet SD1 is moved to the electrode array on the left side of the chip; thereafter, a 5uL DNA sample droplet SD2 is generated again by the droplet generation module B-3, and the sample droplet SD2 is driven by the electrodes of the droplet driving modules IM2 and IM3 in the interconnection module IM to move to the reaction area C.

[0207] Referring to FIG12B , a ligase buffer droplet is generated by a droplet generation structure, and the ligase buffer droplet is driven to move to a droplet receiving module by a driving electrode in an interconnecting module, and the ligase buffer droplet and the DNA sample droplet are mixed in the droplet receiving module (e.g., a reaction zone). In some embodiments, in order to minimize the number of operation steps and reduce the driving distance of the droplet, this step uses the droplet generation module B-2 and area D (i.e., a droplet cutting module) on the left to generate the ligase buffer droplet. For example, the ligase buffer is added from the sample inlet corresponding to the droplet generation module B-2, and a 2uL ligase buffer droplet SD3 is generated by the droplet generation module B-2; the ligase buffer droplet SD3 is driven by an electrode in the interconnecting module IM to move to the position of the DNA sample droplet SD1 (the 3uL droplet generated in the previous step), so that the 2uL ligase buffer droplet SD3 and the 3uL DNA sample droplet SD1 are mixed and then the mixed droplet is driven to move to the reaction zone C of the droplet receiving module. The droplet movement path is shown by the arrow in FIG12B . In this example, the 3uL DNA sample droplet SD1 was not moved to the reaction zone in the previous step. Instead, it was mixed with the ligase buffer droplet SD3 in this step and then moved to reaction zone C. This allows the mixed droplet to be moved to reaction zone C via the droplet drive module IM3. This shortens the droplet's movement path, reduces the number of electrodes the droplet crosses, and improves efficiency. In other embodiments, the 3uL DNA sample droplet SD1 can also be moved to reaction zone C in the previous step. In this case, the 2uL ligase buffer droplet SD3 must be moved to reaction zone C in this step via area D and the interconnection module between area D and reaction zone C. In actual applications, the droplet movement path can be flexibly selected according to needs.

[0208] Referring to Figure 12C, the droplet generation module generates linker droplets and ligase mixed droplets, and these droplets are mixed and moved to the droplet receiving module (e.g., reaction zone C). In some examples, in order to reduce reagent waste, this step uses the droplet generation module B-2 on the left and the droplet generation module B-1 on the side of the reaction zone A in area D (i.e., the upper side of the middle area in the figure) to generate droplets. For example, after taking out the liquid (i.e., the liquid remaining in the previous step) from the droplet generation module B-2 on the left, the ligase mixture is added, and two 2uL (i.e., 4uL) ligase mixed droplets SD4 are generated by the droplet generation module B-2; the linker solution is added at the droplet generation module B-1, and a 1uL linker droplet SD5 is generated by the droplet generation module B-1; then the three droplets (i.e., two 2uL ligase mixed droplets SD4 and one 1uL linker droplet SD5) are mixed and moved to area C, and kept at room temperature for 20 minutes. The movement path of the droplets is shown by the arrow in Figure 12C. In other embodiments, a 4uL ligase droplet can also be obtained by generating a 5uL droplet using the droplet generation module B-3 and cutting out a 1uL droplet from the 5uL droplet. However, using the droplet generation module B-2 to generate two 2uL droplets to obtain a 4uL ligase droplet can avoid reagent waste.

[0209] Referring to Figure 12D, magnetic bead droplets are obtained by the droplet generation structure and driven to move to the droplet receiving module. A purification operation is then performed, for example, in the reaction zone C of the droplet receiving module, so that the DNA is adsorbed onto the magnetic beads. The liquid is then discarded, leaving behind the magnetic beads adsorbed with DNA. For example, the residual liquid in the droplet generation module B-3 is removed; then, magnetic beads (or purification magnetic beads) are added to the droplet generation module B-3 to generate 5uL droplets; the magnetic bead droplets are driven to move to area C to mix with the droplets moved to the reaction zone C in the previous step and a purification operation is performed, so that the DNA is adsorbed onto the magnetic beads. Next, the liquid in the reaction zone C is driven to move to the waste liquid area, leaving behind the magnetic beads with DNA chains.

[0210] Referring to Figure 12E , elution buffer droplets are obtained by the droplet generation structure and driven by the interconnecting module to the droplet receiving module (e.g., reaction zone C), where they mix with the magnetic beads carrying the DNA strands and complete the elution of the DNA, i.e., detaching the DNA strands from the beads. For example, this step requires obtaining a 3uL elution buffer droplet. To reduce the number of sample injection modules used, the droplet generation module B-2 and the droplet cutting module CM in the lower right corner of the figure can be used to obtain the elution buffer droplets. Elution buffer is added to the droplet generation module B-2, and two 2uL (i.e., 4uL) elution buffer droplets are generated by the droplet generation module B-2. These elution buffer droplets are driven to the droplet cutting module CM, and a 1uL waste droplet SD7' is cut from the droplet to obtain a 3uL elution buffer droplet SD7. The elution buffer droplet SD7 is then moved to zone C to complete the DNA elution and obtain a DNA droplet SD8, as shown in Figure 12F .

[0211] Referring to Figure 12F, amplification droplets and primer droplets are generated by the droplet generation structure, and the amplification droplets, primer droplets, and the purified DNA droplets obtained in the previous step are driven to the reaction area (e.g., reaction area A) in the droplet receiving module to perform a PCR amplification reaction. For example, the residual liquid in the droplet generation module B-3 is removed, the amplification mixed solution is added, and a 5uL amplification droplet SD9 is generated by the droplet generation module B-3; the residual liquid in the left droplet generation module B-2 is removed, the primer solution is added, and a 2uL primer droplet SD10 is generated by the droplet generation module B-2; the DNA droplet SD8, the amplification droplet SD9, and the primer droplet SD10 are driven to the reaction area A. Afterwards, a PCR amplification reaction is performed in the reaction area A. In some examples, a three-step PCR amplification reaction is used, and the reaction zone includes three temperature control zones. For example, the reaction is performed at 98°C for 45 seconds in the first temperature control zone, followed by 30 cycles of reaction. The cycle reaction conditions are 98°C for 15 seconds, then 60°C for 30 seconds, and then 72°C for 30 seconds.

[0212] 12G , the liquid after the amplification reaction in reaction zone A is moved to reaction zone C. For example, the liquid in reaction zone A is split into two droplets SD11a and SD11b, and the two droplets are driven to move to reaction zone C by the driving electrodes of the interconnection module.

[0213] Referring to Figure 12H , after the amplification reaction is complete, magnetic bead droplets are generated by the droplet generation structure and driven to reaction zone C of the droplet receiving module to purify the amplified DNA solution. The liquid is then discarded, leaving behind the magnetic beads with the amplified DNA. For example, the residual liquid in droplet generation module B-3 is removed, magnetic beads are added, and two 5uL purification droplets SD12 are generated. The magnetic beads and purification droplet SD12 are then moved to reaction zone C to mix with droplets SD11a and SD11b obtained after the amplification reaction in the previous step and undergo purification, allowing the amplified DNA strands to be adsorbed onto the magnetic beads. The liquid is then driven to the waste liquid area, leaving behind the magnetic beads with the amplified DNA strands.

[0214] Referring to Figure 12I, elution buffer droplets are generated by the droplet generation structure, and the elution buffer droplets are driven to move to the reaction area C of the droplet receiving module to elute the amplified DNA and obtain the amplified DNA chain. For example, the two droplet generation modules B-2 on the right can be used in this step. The residual liquid in the droplet generation module B-2 in the lower right corner is removed, the eluent is added, and two 2uL elution droplets SD13 are generated by the droplet generation module, and the elution droplets SD13 are driven to move to the reaction area C. After that, a magnetic control operation is performed, the magnetic beads are discarded and the liquid product (including the amplified DNA chain) is left. The liquid product is moved to the droplet generation module B-2 in the upper right corner, and the product is taken out, thereby completing the gene library preparation.

[0215] It should be understood that Figures 12A to 12I are only examples of microfluidic chip applications, and the present disclosure is not limited thereto. The microfluidic chip of the present disclosure can be applied to any suitable field and can realize the manipulation of droplets of various volumes and large spans.

[0216] There are a few points to note:

[0217] (1) The drawings of the embodiments of the present disclosure only relate to the structures related to the embodiments of the present disclosure, and other structures may refer to conventional designs.

[0218] (2) Unless there is any conflict, the features of the same embodiment and different embodiments of the present disclosure may be combined with each other.

[0219] The above are only specific embodiments of the present disclosure, but the scope of protection of the present disclosure is not limited thereto. Any changes or substitutions that can be easily conceived by a person skilled in the art within the technical scope disclosed in this disclosure should be included in the scope of protection of the present disclosure. Therefore, the scope of protection of the present disclosure should be based on the scope of protection of the claims.

Claims

1. A digital microfluidic chip, comprising: A droplet generation structure is configured to generate droplets having various volumes, and the droplet generation structure comprises: One or more droplet generation modules, wherein each droplet generation module is configured to generate at least one of a first droplet, a second droplet, and a third droplet, wherein the first droplet, the second droplet, and the third droplet have first volumes, second volumes, and third volumes that are different from each other, respectively; a droplet receiving module configured to achieve at least one of mixing and reacting the plurality of droplets; and The interconnection module is configured to drive the first droplet, the second droplet, and the third droplet, and connect the droplet generating structure to the droplet receiving module.

2. The digital microfluidic chip according to claim 1, wherein the interconnection module comprises: One or more droplet driving modules, wherein each droplet driving module is configured to drive at least one of the first droplet, the second droplet, and the third droplet, and the droplet volume that can be driven by a single driving electrode in the droplet driving module is the first volume, the second volume, and the third volume, respectively.

3. The digital microfluidic chip according to claim 2, wherein the one or more droplet driving modules include: at least one first droplet driving module, wherein a single driving electrode in each first droplet driving module is configured to drive the first droplet; at least one second droplet driving module, wherein a single driving electrode in each second droplet driving module is configured to drive the second droplet; as well as At least one third droplet driving module, wherein a single driving electrode in each third droplet driving module is configured to drive the third droplet.

4. The digital microfluidic chip according to claim 3, wherein the second volume is larger than the minimum volume of the droplets that can be driven by the first droplet driving module, and smaller than the maximum volume of the droplets that can be driven by the first droplet driving module; The third volume is larger than a minimum volume of a droplet that can be driven by the second droplet driving module, and smaller than a maximum volume of a droplet that can be driven by the second droplet driving module. 5 . The digital microfluidic chip according to claim 1 , wherein the first volume is smaller than the second volume and the third volume, the second volume and the third volume are both integer multiples of the first volume, and the second volume and the third volume are different.

6. The digital microfluidic chip according to claim 5, wherein the second volume is twice the first volume, and the third volume is five times the first volume; the first volume is the minimum unit volume of droplets that can be manipulated by the digital microfluidics, and the digital microfluidic chip is configured to manipulate a volume range of from the first volume to twenty times the first volume, with a step size of the minimum unit volume of liquid.

7. The digital microfluidic chip according to claim 1, wherein the one or more droplet generation modules include: at least one first droplet generation module, wherein each first droplet generation module is configured to generate the first droplet; at least one second droplet generation module, wherein each second droplet generation module is configured to generate the second droplet; as well as At least one third droplet generation module, wherein each third droplet generation module is configured to generate the third droplet.

8. The digital microfluidic chip according to claim 7, wherein Each of the first droplet generation module, the second droplet generation module, and the third droplet generation module includes a first drive electrode, a second drive electrode, a third drive electrode, a fourth drive electrode, a fifth drive electrode, and a sixth drive electrode; and The driving electrode with the smallest area in each droplet generation module can accommodate and drive a droplet volume that is substantially equal to the droplet volume of a single droplet generated by the corresponding droplet generation module.

9. The digital microfluidic chip according to claim 8, wherein in each of the first droplet generation module to the third droplet generation module, the second drive electrode, the third drive electrode, the fourth drive electrode and the sixth drive electrode have the same first area as each other, and are arranged in sequence along the first sub-direction, and the sixth drive electrode is connected to the drive electrode in the interconnection module; The fifth driving electrode includes two fifth driving sub-electrodes electrically connected to each other, and the two fifth driving sub-electrodes are arranged on opposite sides of the second driving electrode and the third driving electrode in a second sub-direction perpendicular to the first sub-direction, and each fifth driving sub-electrode has a second area; and The first driving electrode is arranged on a side of the second driving electrode away from the third driving electrode in the first sub-direction and is connected to the second driving electrode and the fifth driving electrode, and the first driving electrode has a third area; The first area is smaller than the second area, and the second area is smaller than the third area.

10. The digital microfluidic chip according to claim 9, wherein each of the first droplet generation module and the second droplet generation module further includes an additional driving electrode located on a side of the first driving electrode away from the second driving electrode and the fifth driving electrode, and the additional driving electrode has the same third area as the corresponding first driving electrode. 11 . The digital microfluidic chip according to claim 10 , wherein each of the first droplet generation module and the second droplet generation module further comprises an additional dummy electrode disposed on a side of the additional driving electrode away from the first driving electrode.

12. The digital microfluidic chip according to any one of claims 9 to 11, wherein the first area, second area, and third area of ​​the corresponding driving electrodes in the first droplet generation module are respectively smaller than the first area, second area, and third area of ​​the corresponding driving electrodes in the second droplet generation module, and the first area, second area, and third area of ​​the corresponding driving electrodes in the second droplet generation module are respectively smaller than the first area, second area, and third area of ​​the corresponding driving electrodes in the third droplet generation module.

13. The digital microfluidic chip according to claim 1, wherein the plurality of droplet generation modules comprises: At least one fourth droplet generation module, wherein each fourth droplet generation module is configured to generate at least two of the first droplet, the second droplet, and the third droplet.

14. The digital microfluidic chip according to claim 13, wherein Each fourth droplet generation module includes a first drive electrode, a second drive electrode, a third drive electrode, a fourth drive electrode, a fifth drive electrode, a sixth drive electrode, a first additional drive electrode, a second additional drive electrode and two third additional drive electrodes; The second driving electrode, the third driving electrode, the fourth driving electrode, the sixth driving electrode, the first additional driving electrode, and the second additional driving electrode are sequentially arranged along a first sub-direction, the two third additional driving electrodes are arranged on opposite sides of the sixth driving electrode along a second sub-direction perpendicular to the first sub-direction, the second driving electrode to the fourth driving electrode have the same first main body area, and the sum of the areas of the sixth driving electrode and the two third additional driving electrodes is substantially equal to the first main body area; the first additional driving electrode and the second additional driving electrode have the same second main body area; The fifth driving electrode comprises two fifth driving sub-electrodes, which are arranged on opposite sides of the second driving electrode and the third driving electrode in the second sub-direction; The first driving electrode is disposed on a side of the second driving electrode and the fifth driving electrode that is away from the fourth driving electrode.

15. The digital microfluidic chip according to claim 14, wherein the fourth droplet generation module is configured to generate the second droplet, the third droplet or an additional droplet, the volume of the additional droplet is substantially equal to the third volume minus the second volume, and the volume of the droplet that can be accommodated and driven by each driving electrode having the second main body area is substantially equal to the second volume of the second droplet, and the volume of the droplet that can be accommodated and driven by a single driving electrode or a combination of driving electrodes having the first main body area is substantially equal to the third volume of the third droplet; or The fourth droplet generation module is configured to generate the first droplet or the second droplet, and each driving electrode having the second main body area can accommodate and drive a droplet volume that is approximately equal to the first volume of the first droplet, and a single driving electrode or a combination of driving electrodes having the first main body area can accommodate and drive a droplet volume that is approximately equal to the second volume of the second droplet. 16 . The digital microfluidic chip according to claim 14 , wherein the second additional driving electrode is connected to the driving electrode in the interconnection module.

17. The digital microfluidic chip according to claim 14, further comprising a fourth additional driving electrode and a fifth additional driving electrode, which are arranged along the first sub-direction on a side of the second additional driving electrode away from the first additional driving electrode, and the fourth additional driving electrode and the fifth additional driving electrode have the same third main body area, and the third main body area is smaller than the second main body area.

18. The digital microfluidic chip according to claim 1, wherein the droplet receiving module is configured to at least achieve mixing and a first reaction of liquids within a first mixing volume range and to achieve mixing and a second reaction of liquids within a second mixing volume range.

19. The digital microfluidic chip according to claim 18, wherein the droplet receiving module comprises: A first reaction zone is configured to at least achieve mixing of the liquid within the first mixing volume and the first reaction, wherein the first reaction zone is connected to a third droplet driving module in the interconnection module.

20. The digital microfluidic chip according to claim 19, wherein the first reaction zone comprises a plurality of first reaction electrodes arranged in an array, and each first reaction electrode can accommodate a droplet volume that is greater than the first volume and the second volume and less than the third volume, and a maximum value of a first mixed volume range of mixed droplets that can be accommodated by the plurality of first reaction electrodes is an integer multiple of the third volume.

21. The digital microfluidic chip according to claim 19, further comprising: The second reaction zone is configured to achieve mixing of the liquid within the second mixing volume and the second reaction, wherein the second reaction zone includes a plurality of second reaction electrodes, and the volume of droplets that can be accommodated and driven by each second reaction electrode is an integer multiple of the third volume.

22. The digital microfluidic chip according to claim 21, wherein the second reaction area and the third droplet driving module share the same electrode array. 23 . The digital microfluidic chip according to claim 21 , wherein the second reaction zone comprises a temperature control zone, and is configured to control the liquid in the temperature control zone to react at a temperature within a predetermined temperature range.

24. The digital microfluidic chip according to claim 23, wherein the temperature control zone comprises a first temperature control zone, a second temperature control zone and a third temperature control zone, which are respectively configured to control the liquid in the corresponding area to react at temperatures within a first temperature range, a second temperature range and a third temperature range that are different from each other.

25. The digital microfluidic chip according to claim 21, wherein the droplet generation structure and the interconnection module are located between the first reaction area and the second reaction area.

26. The digital microfluidic chip according to claim 19, wherein the first reaction zone serves as a common reaction zone and is configured to achieve mixing of liquids within the first mixing volume range and the first reaction and to achieve mixing of liquids within the second mixing volume range and the second reaction in different operation steps.

27. The digital microfluidic chip according to claim 2, wherein the droplet generation structure further comprises a droplet trimming module connected to the one or more droplet generation modules and configured to trim droplets from the one or more droplet generation modules into droplets having a target volume.

28. The digital microfluidic chip according to claim 27, wherein the droplet cutting module and the first droplet driving module in the interconnection module share the same electrode array, and the electrode array is directly connected to the first droplet generating module.

29. The digital microfluidic chip according to claim 27, wherein the droplet cutting module comprises a plurality of main cutting electrodes, and the volume of droplets that can be accommodated and driven by each main cutting electrode is substantially equal to the first volume.

30. The digital microfluidic chip according to claim 29, wherein the plurality of main cutting electrodes are arranged along a first direction, and the end main cutting electrodes of the plurality of main cutting electrodes located at the end of the droplet cutting module in the first direction are connected to the interconnection module.

31. The digital microfluidic chip according to claim 30, wherein one or more first droplet generation modules among the plurality of droplet generation modules for generating the first droplets are located on one side or opposite sides of the droplet cutting module in a second direction perpendicular to the first direction.

32. The digital microfluidic chip according to any one of claims 29-31, wherein the droplet cutting module further comprises an auxiliary cutting electrode, wherein the auxiliary cutting electrode is connected to the main cutting electrode close to the interconnection module and is connected to the interconnection module.

33. A digital microfluidic chip according to claim 32, wherein the plurality of main cutting electrodes have the same first width as each other, the auxiliary cutting electrode has a second width, and the second width is greater than the first width, and the first width and the second width are widths along a first direction.

34. The digital microfluidic chip according to claim 33, wherein the auxiliary cutting electrodes are located on opposite sides of the main cutting electrodes close to the interconnection module in a second direction perpendicular to the first direction. 35 . The digital microfluidic chip according to claim 3 , wherein the number of the second droplet driving modules is greater than the number of the first droplet driving modules and the number of the third droplet driving modules.

36. The digital microfluidic chip according to claim 35, wherein the droplet generation module for generating the second droplet and the third droplet in the one or more droplet generation modules is directly connected to one or more second droplet driving modules.

37. The digital microfluidic chip according to claim 1, comprising: The first substrate comprises: a first substrate base plate; A first conductive layer, located on one side of the first substrate; A second conductive layer is located on a side of the first conductive layer away from the substrate, and corresponding parts of the first conductive layer and the second conductive layer are electrically connected to each other; a dummy layer, located between the first conductive layer and the second conductive layer, and electrically floating or grounded; and The first hydrophobic layer is located on a side of the second conductive layer away from the first substrate.

38. The digital microfluidic chip according to claim 37, wherein the second conductive layer comprises a plurality of driving electrodes of the droplet generating structure, the droplet receiving module and the interconnection module, and the plurality of driving electrodes are spaced apart from each other; The dummy layer includes a plurality of dummy electrodes spaced apart from each other, and an outer contour of an orthographic projection of each dummy electrode on the first substrate coincides with an outer contour of an orthographic projection of a corresponding driving electrode among the plurality of driving electrodes on the substrate.

39. The digital microfluidic chip according to claim 38, wherein each of the plurality of dummy electrodes has an opening, and the plurality of driving electrodes are respectively connected to the first conductive layer through a plurality of conductive through holes, and the plurality of conductive through holes are respectively located in the openings corresponding to the plurality of dummy electrodes and spaced apart from the plurality of dummy electrodes. 40 . The digital microfluidic chip according to claim 38 , wherein the first conductive layer comprises a plurality of conductive traces, and the plurality of driving electrodes are connected to the binding electrodes through the plurality of conductive traces.

41. The digital microfluidic chip according to any one of claims 37 to 40, wherein the first conductive layer and the second conductive layer comprise transparent conductive materials, and the dummy layer comprises opaque conductive materials.

42. The digital microfluidic chip of claim 41, wherein the dummy layer comprises a metal material.

43. The digital microfluidic chip according to any one of claims 37 to 40, wherein the dummy layer further comprises additional dummy electrodes of one or more droplet generation modules, and the orthographic projection of the additional dummy electrodes on the first substrate does not overlap with the second conductive layer.

44. The digital microfluidic chip according to claim 43, further comprising: A second substrate is arranged opposite to the first substrate, wherein the second substrate comprises: a second substrate base plate; A third conductive layer is located on one side of the second substrate; as well as A second hydrophobic layer is located on a side of the third conductive layer away from the second substrate; as well as a droplet channel, located between the first hydrophobic layer of the first substrate and the second hydrophobic layer of the second substrate; The second substrate is provided with a plurality of sample inlets penetrating the second substrate, and the plurality of sample inlets are respectively arranged corresponding to the plurality of droplet generation modules; For the droplet generation module including the additional dummy electrode, the orthographic projection of the additional dummy electrode on the main surface of the first substrate facing the second substrate overlaps with the orthographic projection of the corresponding sample inlet on the main surface of the first substrate.

45. A digital microfluidic chip according to claim 44, wherein the one or more droplet generation modules include a droplet generation module without an additional dummy electrode, and the orthographic projection of the sample inlet corresponding to the droplet generation module on the main surface of the first substrate is close to the orthographic projection of the driving electrode of the droplet generation module on the main surface of the first substrate, and does not overlap with the orthographic projection of the driving electrode.

46. ​​A method for generating droplets with various volumes using the droplet generation structure in the digital microfluidic chip according to any one of claims 1 to 45, comprising at least one of the following: generating one or more first droplets each having a first volume using a droplet generation module; generating one or more second droplets each having a second volume using a droplet generation module; generating, using a droplet generation module, one or more third droplets each having a third volume, wherein the first volume is smaller than the second volume, and the second volume is smaller than the third volume; generating at least one of one or more fourth droplets each having a fourth volume and one or more fifth droplets each having a fifth volume using a droplet generation module and a droplet trimming module in combination, the fourth volume and the fifth volume being greater than the second volume and less than the third volume; as well as At least two of the one or more first droplets, the one or more second droplets, the one or more third droplets, the one or more fourth droplets, and the one or more fifth droplets are mixed to obtain a droplet having another target volume.

47. The method of claim 46, wherein generating the fourth droplet using the droplet generation module and the droplet tailoring module in combination comprises: Using a third droplet generation module to generate an initial droplet having the third volume; as well as The droplet trimming module trims two trimmed droplets each having the first volume from the initial droplet, and moves the two trimmed droplets to a waste area, The remaining droplets after the initial droplets are cut form the fourth droplets.

48. The method of claim 46, wherein generating the fifth droplet using the droplet generation module and the droplet tailoring module in combination comprises: Using a third droplet generation module to generate an initial droplet having the third volume; as well as A cut droplet having the first volume is cut from the initial droplet by the droplet cutter module, and the cut droplet is moved to a waste area, The remaining droplets after the initial droplets are cut form the fifth droplets.

49. A method for preparing a gene library using the digital microfluidic chip according to any one of claims 1 to 45, comprising: Adding deoxyribonucleotide sample solution, ligase buffer, linker solution, and ligase solution to corresponding droplet generation modules in the plurality of droplet generation modules, respectively, and generating sample droplets, ligase buffer droplets, linker droplets, and ligase droplets through the droplet generation structure; The sample droplet, the ligase buffer droplet, the adapter droplet, and the ligase droplet are moved to the droplet receiving module through the interconnection module, and the sample droplet, the ligase buffer droplet, the adapter droplet, and the ligase droplet are mixed and reacted in the first reaction area of ​​the droplet receiving module to form a sample mixed droplet; Adsorbing and eluting deoxyribonucleotides on the sample mixed liquid droplets to obtain a first deoxyribonucleotide chain; adding an amplification solution and a primer solution to corresponding droplet generation modules in the plurality of droplet generation modules respectively, and generating amplification droplets and primer droplets through the droplet generation structure; The amplification droplet and the primer droplet are moved to the droplet receiving module through the interconnection module, and the amplification droplet and the primer droplet are subjected to polymerase chain reaction with the first deoxyribonucleotide chain in the droplet receiving module to form amplification deoxyribonucleotide mixed droplets; as well as The amplified deoxyribonucleotide mixed droplets are subjected to deoxyribonucleotide adsorption and elution to obtain amplified deoxyribonucleotide chains.

50. The method of claim 49, wherein the sample droplet has a sixth volume, and generating the sample droplet comprises: A deoxyribonucleotide sample solution is added to the third droplet generation module, and a first sample droplet and a second sample droplet each having a third volume are generated by the third droplet generation module, the second sample droplet is moved to the droplet trimming module through the interconnection module, and the second sample droplet is trimmed into a trimmed sample droplet having the second volume and a third sample droplet having a remaining volume by the droplet trimming module, the first sample droplet and the third sample droplet constitute the sample droplet having the sixth volume.

51. The method of claim 50, wherein generating the ligase buffer droplet, the adapter droplet, and the ligase droplet comprises: adding a ligase buffer at a second droplet generation module, and generating a ligation buffer droplet having a second volume through the second droplet generation module; adding a ligase solution to a second droplet generation module, and generating two ligase sub-droplets each having a second volume through the second droplet generation module, wherein the two ligase sub-droplets constitute the ligase droplet; A linker solution is added to a first droplet generation module, and a linker droplet having a first volume is generated by the first droplet generation module.

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