Digital microfluidic chip, its driving method, and digital microfluidic device

Abstract

The embodiment of the present disclosure provides a digital microfluidic chip and a driving method thereof, and a digital microfluidic device. The digital microfluidic chip comprises a first substrate (1) and a second substrate (2) arranged opposite to each other, wherein the first substrate (1) is provided with a plurality of driving areas for driving droplet movement, at least one driving area comprises a driving transistor (50), a driving electrode (60) and a storage capacitor, wherein the driving electrode (60) is connected to the driving transistor (50) and the storage capacitor respectively, and the storage capacitor is configured to be charged when the driving transistor (50) is turned on and to maintain a voltage signal on the driving electrode (60) when the driving transistor (50) is turned off.

Description

Technical Field

[0001] The present disclosure relates to, but is not limited to, the field of micro-electromechanical technology, and in particular to a digital microfluidic chip and a driving method thereof, and a digital microfluidic device. Background Art

[0002] With the development of micro-electromechanical system technology, digital microfluidics technology has made breakthroughs in the driving and control of micro-droplets, and has been widely used in biology, chemistry, medicine and other fields based on its own advantages.

[0003] Digital microfluidics is an emerging interdisciplinary field encompassing chemistry, fluid physics, microelectronics, new materials, biology, and biomedical engineering. It enables precise control and manipulation of tiny droplets. Due to its miniaturization and integration, devices utilizing microfluidics are often referred to as digital microfluidic chips. They are a crucial component of laboratory-on-a-chip (LOC) systems. Various cell samples can be cultured, moved, detected, and analyzed within digital microfluidic chips, offering advantages such as low sample consumption, rapid detection, ease of operation, multifunctional integration, compact size, and portability. Summary of the Invention

[0004] The following is a summary of the subject matter described in detail herein. This summary is not intended to limit the scope of the claims.

[0005] On the one hand, an embodiment of the present disclosure provides a digital microfluidic chip, comprising a first substrate and a second substrate arranged relative to each other, wherein a plurality of driving areas are provided on the first substrate, at least one driving area comprises a driving transistor, a driving electrode and a storage capacitor, the driving electrode being connected to the driving transistor and the storage capacitor, respectively, and the storage capacitor being configured to be charged when the driving transistor is turned on and to maintain the voltage signal on the driving electrode when the driving transistor is turned off.

[0006] In an exemplary embodiment, a plurality of gate lines and a plurality of data lines are provided on the first substrate, and the plurality of gate lines and the plurality of data lines intersect with each other to define a plurality of driving areas. In at least one driving area, the driving transistor includes at least a first gate electrode, a second gate electrode, a first electrode, and a second electrode. The first gate electrode and the second gate electrode are connected to the gate line, the first electrode is connected to the data line, and the second electrode is connected to the driving electrode.

[0007] In an exemplary embodiment, at least one driving region further includes a capacitor electrode, an orthographic projection of the capacitor electrode on the first substrate at least partially overlaps with an orthographic projection of the driving electrode on the first substrate, and the capacitor electrode and the driving electrode constitute the storage capacitor.

[0008] In an exemplary embodiment, the capacitive electrode is connected to a system ground signal.

[0009] In an exemplary embodiment, in at least one driving region, the first substrate includes:

[0010] first base;

[0011] a first conductive layer disposed on the first substrate, the first conductive layer comprising at least a gate line, a first gate electrode and a second gate electrode, the first gate electrode and the second gate electrode being connected to the gate line respectively;

[0012] a first insulating layer covering the first conductive layer;

[0013] a semiconductor layer disposed on a side of the first insulating layer away from the first substrate, the semiconductor layer comprising at least a first active layer and a second active layer, an orthographic projection of the first active layer on the first substrate at least partially overlapping with an orthographic projection of the first gate electrode on the first substrate, and an orthographic projection of the second active layer on the first substrate at least partially overlapping with an orthographic projection of the second gate electrode on the first substrate;

[0014] a second conductive layer disposed on a side of the semiconductor layer away from the first substrate, the second conductive layer comprising at least a data line, a first electrode, a connecting electrode, and a second electrode, wherein a first end of the first electrode is connected to the data line, a second end of the first electrode and a first end of the connecting electrode are respectively disposed on the first active layer, and a second end of the connecting electrode and a first end of the second electrode are respectively disposed on the second active layer;

[0015] a second insulating layer covering the second conductive layer;

[0016] a third conductive layer provided on a side of the second insulating layer away from the first substrate, the third conductive layer comprising at least a capacitor electrode;

[0017] a third insulating layer covering the third conductive layer, wherein the third insulating layer is provided with a connecting via hole, and the connecting via hole exposes the second electrode;

[0018] A fourth conductive layer is arranged on a side of the third insulating layer away from the first substrate, the fourth conductive layer at least including a driving electrode, the driving electrode is connected to the second electrode through the connecting via, the orthographic projection of the driving electrode on the first substrate and the orthographic projection of the capacitor electrode on the first substrate at least partially overlap, and the capacitor electrode and the driving electrode constitute the storage capacitor.

[0019] In an exemplary embodiment, a plurality of counter electrodes are disposed on the second substrate, and the driving electrodes and the counter electrodes constitute a driving unit for driving the liquid droplets to move.

[0020] In an exemplary embodiment, the first substrate and the second substrate form a processing chamber through a sealant, and the processing chamber includes at least a screening area, a lysis area, a pre-amplification area, and a library preparation area. The screening area is configured to screen and enrich rare cells. The lysis area is arranged on one side of the screening area and is configured to single and lyse the rare cells after screening and enrichment. The pre-amplification area is arranged on a side of the lysis area away from the screening area and is configured to pre-amplify the nucleic acid of the rare single cell after cell lysis. The library preparation area is arranged on a side of the pre-amplification area away from the screening area and is configured to prepare a sample library after pre-amplification of the rare single cell.

[0021] In an exemplary embodiment, the screening area includes a plurality of driving units, and a first reagent port for the screening area, a second reagent port for the screening area, a third reagent port for the screening area, and a fourth reagent port for the screening area, respectively arranged in the corner areas of the screening area, and at least one of the first reagent port for the screening area, the second reagent port for the screening area, the third reagent port for the screening area, and the fourth reagent port for the screening area is configured to: receive a whole blood sample, or receive magnetic nanoparticles, or receive a buffer solution, or discharge waste liquid.

[0022] In an exemplary embodiment, the screening area includes a first magnetic field region including a plurality of regularly arranged first magnetic regions, and an orthographic projection of at least one first magnetic region on the first substrate includes an orthographic projection of at least one driving unit on the first substrate.

[0023] In an exemplary embodiment, the screening area includes a plurality of driving units, and a first reagent port in the lysis area, a second reagent port in the lysis area, a third reagent port in the lysis area, and a fourth reagent port in the lysis area, respectively arranged in the corner area of the screening area, and at least one of the first reagent port in the lysis area, the second reagent port in the lysis area, the third reagent port in the lysis area, and the fourth reagent port in the lysis area is configured to: receive a lysis solution, or receive a stop solution, or receive a buffer solution, or discharge waste liquid.

[0024] In an exemplary embodiment, the driving units in the screening area satisfy the following formula:

[0025]

[0026] Wherein, θ represents the initial contact angle between the droplet and the hydrophobic surface on the first substrate, H represents the thickness of the digital microfluidic chip, and L represents the size of the driving electrode.

[0027] In an exemplary embodiment, the cell thickness H of the digital microfluidic chip is ≤ 19.8 μm, and the size L of the driving electrode is ≤ 48.5 μm.

[0028] In an exemplary embodiment, the driving unit in the screening area is configured to detect impedance signals of single cell packages and vacuoles, where the impedance of the single cell packages includes resistance of the cytoplasm and capacitance of the cell membrane that packages the cytoplasm.

[0029] In an exemplary embodiment, the pre-amplification zone includes a plurality of driving units, and a first reagent port for the pre-amplification zone, a second reagent port for the pre-amplification zone, a third reagent port for the pre-amplification zone, and a fourth reagent port for the pre-amplification zone, respectively arranged in the corner areas of the pre-amplification zone, and at least one of the first reagent port for the pre-amplification zone, the second reagent port for the pre-amplification zone, the third reagent port for the pre-amplification zone, and the fourth reagent port for the pre-amplification zone is configured to: receive a fragmentation enzyme reagent, or receive a pre-amplification reagent, or receive a fragmentation buffer, or discharge waste liquid.

[0030] In an exemplary embodiment, the pre-amplification zone includes a plurality of amplification temperature zones having different temperatures, and a distance between adjacent amplification temperature zones is greater than or equal to 1 mm.

[0031] In an exemplary embodiment, the library preparation area includes a plurality of drive units, and a first reagent port for the preparation area, a second reagent port for the preparation area, a third reagent port for the preparation area, a fourth reagent port for the preparation area, a fifth reagent port for the preparation area, a sixth reagent port for the preparation area, a seventh reagent port for the preparation area, an eighth reagent port for the preparation area, a ninth reagent port for the preparation area, a tenth reagent port for the preparation area, and an eleventh reagent port for the preparation area, respectively arranged in an edge area of the library preparation area; the first reagent port for the preparation area, the second reagent port for the preparation area, the third reagent port for the preparation area, the fourth reagent port for the preparation area, and the fifth reagent port for the preparation area are arranged in an edge area on one side of the second direction of the library preparation area, and are arranged in sequence along the first direction, the sixth reagent port for the preparation area, the seventh reagent port for the preparation area, the eighth reagent port for the preparation area, the ninth reagent port for the preparation area, the tenth reagent port for the preparation area, and the fifth reagent port for the preparation area The reagent port, the eighth reagent port of the preparation area, the ninth reagent port of the preparation area and the tenth reagent port of the preparation area are arranged in the edge area on the side opposite to the second direction of the library preparation area, and are arranged sequentially along the first direction. The eleventh reagent port of the preparation area is arranged in the edge area on the side of the first direction of the library preparation area; at least one of the multiple preparation area reagent ports of the library preparation area is configured to: receive washing bead liquid, or receive end repair master mixed liquid, or receive size screening bead liquid, or receive eluent, or receive library amplification premix liquid, or receive A tracking master mixed liquid, or receive adapter liquid, or receive ligation master mixed liquid, or receive washing buffer, or receive primers, or discharge waste liquid.

[0032] In an exemplary embodiment, the library preparation zone includes a plurality of polymerization temperature zones having different temperatures, and the distance between adjacent polymerization temperature zones is greater than or equal to 0.5 mm.

[0033] In an exemplary embodiment, the library preparation area includes a second magnetic field region, the second magnetic field region includes a plurality of regularly arranged second magnetic regions, and the orthographic projection of at least one second magnetic region on the first substrate includes the orthographic projection of at least one driving unit on the first substrate.

[0034] On the other hand, an embodiment of the present disclosure also provides a digital microfluidic device, comprising the above-mentioned digital microfluidic chip, and also comprising a temperature control device, a magnetic control device and a detection device, wherein the temperature control device is configured to generate at least one temperature zone on the digital microfluidic chip, the magnetic control device is configured to generate at least one magnetic field zone on the digital microfluidic chip, the detection device is configured to identify and locate rare cells, and the digital microfluidic chip is configured to sequentially perform screening and enrichment of rare cells, singulation and cell lysis of rare cells, pre-amplification of nucleic acids of rare single cells, and sample library preparation.

[0035] In another aspect, the present disclosure further provides a method for driving a digital microfluidic chip, wherein the digital microfluidic chip includes a screening area, a lysis area, a pre-amplification area, and a library preparation area arranged in sequence, and the driving method includes:

[0036] Screening and enriching rare cells in the screening area;

[0037] Singulation and cell lysis of the rare cells after screening and enrichment are performed in the lysis zone;

[0038] Pre-amplification of nucleic acids of rare single cells after cell lysis is performed in the pre-amplification zone;

[0039] The library preparation area is used to prepare a sample library after rare single cell pre-amplification.

[0040] Still other aspects will become apparent upon reading and understanding the accompanying drawings and detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] The accompanying drawings are intended to provide a further understanding of the technical solutions of the present disclosure and constitute a part of the specification. Together with the embodiments of the present application, they are used to explain the technical solutions of the present disclosure and do not constitute a limitation of the technical solutions of the present disclosure. The shapes and sizes of the components in the accompanying drawings do not reflect the actual scale and are intended only to illustrate the contents of the present disclosure.

[0042] Figure 1 Schematic diagram of the structure of a digital microfluidic device according to an exemplary embodiment of the present disclosure;

[0043] Figure 2 Schematic diagram of the cross-sectional structure of a digital microfluidic chip according to an exemplary embodiment of the present disclosure;

[0044] Figure 3 Schematic diagram of a planar structure of a digital microfluidic chip according to an exemplary embodiment of the present disclosure;

[0045] Figure 4 This is a schematic planar structural diagram of a first substrate according to an exemplary embodiment of the present disclosure;

[0046] Figure 5 for Figure 4 Cross-sectional view along the AA axis;

[0047] Figure 6a and Figure 6b This is a schematic diagram after forming a first conductive layer pattern according to an embodiment of the present disclosure;

[0048] Figure 7a and Figure 7b This is a schematic diagram of a semiconductor layer pattern formed according to an embodiment of the present disclosure;

[0049] Figure 8a and Figure 8b This is a schematic diagram after forming a second conductive layer pattern according to an embodiment of the present disclosure;

[0050] Figure 9a and Figure 9b This is a schematic diagram of an embodiment of the present disclosure after forming a third conductive layer pattern;

[0051] Figure 10a and Figure 10b This is a schematic diagram after forming a second insulating layer pattern according to an embodiment of the present disclosure;

[0052] Figure 11a and Figure 11b This is a schematic diagram of an embodiment of the present disclosure after forming a fourth conductive layer pattern;

[0053] Figure 12 This is a schematic diagram of a planar structure of a screening area according to an exemplary embodiment of the present disclosure;

[0054] Figures 13a to 13c This is a schematic diagram of a rare cell screening and enrichment process disclosed herein;

[0055] Figure 14 This is a schematic diagram of a planar structure of a cracking zone according to an exemplary embodiment of the present disclosure;

[0056] Figures 15a to 15c A schematic diagram of a rare cell lysis process disclosed herein;

[0057] Figure 16 A schematic diagram of a droplet in a digital microfluidic chip;

[0058] Figure 17 and Figure 18 A schematic diagram of the principle of an impedance analysis method;

[0059] Figure 19 This is a schematic diagram of a planar structure of a pre-amplification zone according to an exemplary embodiment of the present disclosure;

[0060] Figures 20a to 20c This is a schematic diagram of a rare single cell pre-amplification process disclosed herein;

[0061] Figure 21 This is a schematic diagram of the planar structure of a library preparation area according to an exemplary embodiment of the present disclosure;

[0062] Figures 22a to 22c A schematic diagram of a rare single-cell library preparation process disclosed herein.

[0063] Description of the accompanying drawings:

[0064] 1—first substrate; 2—second substrate; 10—digital microfluidic chip;

[0065] 11—first substrate; 12—first structural layer; 13—first liquid-repellent layer;

[0066] 20—Temperature control device; 20-1—First temperature control device; 20-2—Second temperature control device;

[0067] 21—second substrate; 22—second structural layer; 23—second liquid-repellent layer;

[0068] 30—Magnetic control device; 30-1—First magnetic control device; 30-2—Second magnetic control device;

[0069] 31—first gate electrode; 32—second gate electrode; 33—first active layer;

[0070] 34—second active layer; 35—first electrode; 36—connecting electrode;

[0071] 37—second electrode; 38—capacitive electrode; 40—detection device;

[0072] 50—driving transistor; 51—gate line; 52—data line;

[0073] 60—driving electrode; 61—first insulating layer; 62—second insulating layer;

[0074] 63—third insulating layer; 64—fourth insulating layer; 70—counter electrode;

[0075] 100—screening area; 110—first magnetic field area; 111—first magnetic area;

[0076] 200—lysis zone; 210—detection zone; 300—pre-amplification zone;

[0077] 310—first amplification temperature zone; 320—second amplification temperature zone; 400—library preparation zone;

[0078] 420—first polymerization temperature zone; 430—second polymerization temperature zone; 440—third polymerization temperature zone;

[0079] 450—second magnetic field region; 451—second magnetic zone. DETAILED DESCRIPTION

[0080] The specific embodiments of the present disclosure are further described in detail below in conjunction with the accompanying drawings and examples. The following examples are used to illustrate the present disclosure but are not intended to limit the scope of the present disclosure. It should be noted that, unless there is a conflict, the embodiments in this application and the features in the embodiments can be combined with each other in any manner.

[0081] In order to make the purpose, technical solutions and advantages of the present disclosure clearer, the embodiments of the present disclosure will be described in detail below with reference to the accompanying drawings. Note that the embodiments can be implemented in a variety of different forms. A person of ordinary skill in the art can easily understand the fact that the methods and contents can be transformed into various forms without departing from the purpose and scope of the present disclosure. Therefore, the present disclosure should not be interpreted as being limited to the contents described in the following embodiments. In the absence of conflict, the embodiments in the present disclosure and the features in the embodiments can be arbitrarily combined with each other.

[0082] The scales of the figures in this disclosure can be used as a reference for actual processes, but are not limited to such. For example, the width-to-length ratio of the channel, the thickness and spacing of the various film layers, and the width and spacing of the various signal lines can be adjusted according to actual needs. The number of pixels in the display substrate and the number of sub-pixels in each pixel are not limited to the numbers shown in the figures. The figures described in this disclosure are merely schematic structural diagrams, and one embodiment of this disclosure is not limited to the shapes or values shown in the figures.

[0083] In this specification, ordinal numbers such as “first”, “second” and “third” are provided to avoid confusion among constituent elements, and are not intended to limit the number.

[0084] In this specification, for convenience, words and phrases indicating orientation or positional relationships, such as "middle," "upper," "lower," "front," "back," "vertical," "horizontal," "top," "bottom," "inside," and "outside," are used to illustrate the positional relationships of constituent elements with reference to the accompanying drawings. This is merely for the purpose of facilitating the description of this specification and simplifying the description, and does not indicate or imply that the device or element referred to must have a specific orientation, be constructed, or operate in a specific orientation. Therefore, they should not be construed as limiting the present disclosure. The positional relationships of constituent elements may be appropriately changed depending on the direction in which each constituent element is described. Therefore, the present disclosure is not limited to the words and phrases described in the specification and may be appropriately replaced according to the circumstances.

[0085] In this specification, unless otherwise specified or limited, the terms "mounted," "connected," and "connected" should be understood broadly. For example, they can refer to fixed, removable, or integral connections; mechanical or electrical connections; direct connections, indirect connections through intermediaries, or internal communication between two components. Those skilled in the art will understand the specific meanings of these terms in this disclosure.

[0086] In this specification, a transistor refers to a device that includes at least three terminals: a gate electrode, a drain electrode, and a source electrode. A transistor has a channel region between a drain electrode (drain electrode terminal, drain region, or drain electrode) and a source electrode (source electrode terminal, source region, or source electrode), and current can flow through the drain electrode, the channel region, and the source electrode. Note that in this specification, the channel region refers to the region through which current primarily flows.

[0087] In this specification, "electrically connected" includes components connected together via an element having some electrical function. There are no particular limitations on the "element having some electrical function" as long as it enables the transfer of electrical signals between the connected components. Examples of "element having some electrical function" include not only electrodes and wiring, but also switching elements such as transistors, resistors, inductors, capacitors, and other components with various functions.

[0088] In this specification, "parallel" refers to a state where the angle formed by two straight lines is greater than -10° and less than 10°, and thus also includes a state where the angle is greater than -5° and less than 5°. Furthermore, "perpendicular" refers to a state where the angle formed by two straight lines is greater than 80° and less than 100°, and thus also includes a state where the angle is greater than 85° and less than 95°.

[0089] The triangles, rectangles, trapezoids, pentagons or hexagons in this specification are not in the strict sense, but may be approximate triangles, rectangles, trapezoids, pentagons or hexagons, etc. There may be some small deformations caused by tolerances, and there may be chamfers, arc edges and deformations.

[0090] The term "about" in the present disclosure refers to a numerical value that is not strictly defined and allows for process and measurement errors.

[0091] Since the launch of the Human Genome Project, high-throughput sequencing (HTS) technology has rapidly advanced. However, conventional sequencing involves combining tissue samples containing thousands of cells to generate whole-genome sequence information. Consequently, the final sequencing result reflects the average of all gene signals within a population of cells, or the genetic information of cells that are dominant in number. This makes it difficult to discern heterogeneity within a population. To address this limitation, single-cell sequencing (SCS) has emerged. SCS involves sequencing the genetic information contained in individual cells, aiming to obtain molecular-level information on the gene sequence, transcripts, proteins, and epigenetic expression profiles of a specific cell type. By sequencing the deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) of rare single cells, it is possible to accurately understand cellular mutations at the single-cell level. SCS has been widely used in a variety of research areas, including tumor heterogeneity, embryonic stem cell differentiation, and microbial community diversity.

[0092] The single-cell sequencing process for rare samples mainly includes three steps: (1) obtaining a single cell sample; (2) lysing the obtained single cell and preparing the library; (3) performing high-throughput sequencing analysis. In order to perform single-cell sequencing of rare samples, the rare single cells of interest must first be isolated. At present, traditional single-cell isolation technology relies more on manual operation, which not only easily causes the loss and destruction of rare samples during the operation, but also is complicated, tedious, time-consuming, and has a high probability of error in library construction. For example, although the traditional gradient dilution method has the characteristics of simple operation and low cost, this method is prone to operational errors and has poor specificity. For another example, although the traditional flow cytometry sorting technology has high specificity, this method requires a large amount of samples and may cause mechanical damage to cells. For another example, although the laser capture microdissection technology has the characteristics of accuracy, speed and visualization, this method requires manual operation and is easy to destroy cell integrity. Therefore, traditional single-cell isolation technologies are difficult to avoid the loss and destruction of rare samples. Even if single-cell samples are obtained, optimizing library construction quality at the rare single-cell level is extremely difficult. Manually generated libraries are difficult to meet the requirements of deep sequencing. These problems affect the quality of sample processing before rare single-cell sequencing and hinder the clinical application and promotion of rare single-cell sequencing technology. Therefore, an integrated solution for rare single-cell sample capture, isolation, and library preparation is urgently needed.

[0093] Digital microfluidic chips use the principle of electrowetting on dielectric (EWOD) to place droplets on a surface with a hydrophobic layer. With the help of the electrowetting effect, voltage is applied to the droplets, changing the wettability between the droplets and the hydrophobic layer, causing a pressure difference and asymmetric deformation inside the droplets, thereby achieving directional movement of the droplets. Droplets can be moved, mixed, and separated at the micron scale. It has the ability to miniaturize the basic functions of biological, chemical, and other laboratories onto a chip of a few square centimeters. It has the advantages of small size, portability, flexible functional combinations, and high integration.

[0094] Digital microfluidics is categorized into active and passive digital microfluidics. The primary difference between the two is that active digital microfluidics employs arrayed droplet actuation, enabling precise control of individual droplet movement at a specific location, while passive digital microfluidics involves simultaneous movement or stopping of droplets at all locations. Active digital microfluidics utilizes thin-film transistors (TFTs) to control drive electrodes, enabling independent control of the drive electrodes and precise droplet control. Compared to passive digital microfluidics, passive digital microfluidics requires M×N control signals for M×N drive electrodes, while active digital microfluidics, with its row- and column-addressed drive scheme, requires only M+N control signals, where M and N are positive integers greater than 1. Therefore, active digital microfluidics is more suitable for high-throughput sample manipulation, enabling arbitrary programmable motion paths for individual or multiple droplets and simultaneous manipulation of multiple samples. The process flow of active digital microfluidics technology is compatible with the production of electrical and optical sensors. It can integrate electrical detection, optical detection and other means into the chip to form a multifunctional active digital microfluidics chip laboratory.

[0095] The exemplary embodiments of the present disclosure provide an automated, integrated digital microfluidic device for rare single cell capture, separation, and library preparation based on an active digital microfluidic chip.

[0096] Figure 1 FIG. 1 is a schematic diagram of a digital microfluidic device according to an exemplary embodiment of the present disclosure. Figure 1As shown, the digital microfluidic device may include a digital microfluidic chip 10, a temperature control device 20, a magnetic control device 30 and a detection device 40. The temperature control device 20 is configured to generate at least one temperature zone on the digital microfluidic chip 10, the magnetic control device 30 is configured to generate at least one magnetic field zone on the digital microfluidic chip 10, the detection device 40 is configured to identify and locate rare cells, and the digital microfluidic chip 10 is configured to sequentially perform rare cell screening and enrichment, rare cell singulation and cell lysis, rare single cell nucleic acid pre-amplification, and sample library preparation, thereby realizing automated and integrated rare single cell capture, separation and library preparation.

[0097] Figure 2 FIG1 is a schematic cross-sectional structure diagram of a digital microfluidic chip according to an exemplary embodiment of the present disclosure. Figure 3 for Figure 2 The schematic diagram of the planar structure of the digital microfluidic chip. Figure 2 and Figure 3 As shown, in an exemplary embodiment, the digital microfluidic chip 10 may include a first substrate 1 and a second substrate 2 arranged relatively to each other, the first substrate 1 may include a first base 11, a first structural layer 12 arranged on the side of the first substrate 11 facing the second substrate 2, and a first liquid-repellent layer 13 arranged on the side of the first structural layer 12 facing the second substrate 2, the second substrate 2 may include a second base 21, a second structural layer 22 arranged on the side of the second substrate 21 facing the first substrate 1, and a second liquid-repellent layer 23 arranged on the side of the second structural layer 22 facing the first substrate.

[0098] In an exemplary embodiment, the first substrate 1 and the second substrate 2 disposed opposite each other can be packaged in a box using a sealant. The first substrate 1, the second substrate 2, and the sealant together form a closed processing chamber, and the sample to be processed can be placed in the processing chamber. In an exemplary embodiment, the processing chamber can be divided into a plurality of functional zones disposed in sequence, and the plurality of functional zones can include at least a screening zone 100, a lysis zone 200, a pre-amplification zone 300, and a library preparation zone 400. The lysis zone 200 is disposed on one side of the screening zone 100, the pre-amplification zone 300 is disposed on a side of the lysis zone 200 away from the screening zone 100, and the library preparation zone 400 is disposed on a side of the pre-amplification zone 300 away from the screening zone 100. In an exemplary embodiment, the screening area 100 is configured to screen and enrich rare cells, the lysis area 200 is configured to single and lyse the rare cells after screening and enrichment, the pre-amplification area 300 is configured to pre-amplify the nucleic acid of the rare single cells after cell lysis, and the library preparation area 400 is configured to prepare a sample library after the pre-amplification of the rare single cells.

[0099] In an exemplary embodiment, the detection device 40 can be arranged on a side of the first substrate 1 away from the second substrate 2, or on a side of the second substrate 2 away from the first substrate 1, and the position corresponds to the area where the lysis area 200 is located. The detection device 40 is configured to form a detection area 210 in the lysis area 200, and identify and locate droplets containing rare cells in the detection area 210.

[0100] In an exemplary embodiment, the temperature-control device 20 may include at least a first temperature-control device 20 - 1 and a second temperature-control device 20 - 2 .

[0101] In an exemplary embodiment, the first temperature control device 20-1 can be disposed on a side of the first substrate 1 away from the second substrate 2, or on a side of the second substrate 2 away from the first substrate 1, corresponding to the area where the pre-amplification zone 300 is located. The first temperature control device 20-1 is configured to generate multiple amplification temperature zones having different temperatures in the pre-amplification zone 300. For example, the first temperature control device 20-1 can generate a first amplification temperature zone 310 and a second amplification temperature zone 320 in the pre-amplification zone 300. In an exemplary embodiment, the first amplification temperature zone 310 and the second amplification temperature zone 320 are configured to implement pre-amplification processing of rare single cells.

[0102] In an exemplary embodiment, the second temperature control device 20-2 can be disposed on a side of the first substrate 1 away from the second substrate 2, or on a side of the second substrate 2 away from the first substrate 1, in a position corresponding to the area where the library preparation area 400 is located. The second temperature control device 20-2 is configured to generate a plurality of polymerization temperature zones with different temperatures in the library preparation area 400. For example, the second temperature control device 20-2 can generate a first polymerization temperature zone 420, a second polymerization temperature zone 430, and a third polymerization temperature zone 440 in the library preparation area 400. In an exemplary embodiment, the first polymerization temperature zone 420, the second polymerization temperature zone 430, and the third polymerization temperature zone 440 are configured to implement a polymerase chain reaction (PCR) thermal cycling process.

[0103] In an exemplary embodiment, the first temperature control device 20 - 1 and the second temperature control device 20 - 2 may include a heater, a temperature sensor, a controller, etc. The heater, the temperature sensor, and the controller form a closed-loop control to accurately and effectively control the temperature of the hot zone.

[0104] In an exemplary embodiment, the magnetron device 30 may include at least a first magnetron device 30 - 1 and a second magnetron device 30 - 2 .

[0105] In an exemplary embodiment, the first magnetic control device 30-1 can be disposed on a side of the first substrate 1 away from the second substrate 2, or on a side of the second substrate 2 away from the first substrate 1, in a position corresponding to the area where the screening area 100 is located. The first magnetic control device 30-1 is configured to generate at least one first magnetic field region 110 in the screening area 100. In an exemplary embodiment, the at least one first magnetic field region 110 is configured to achieve a capture process for rare cells, and the first magnetic field region 110 can include a plurality of regularly arranged first magnetic regions.

[0106] In an exemplary embodiment, the second magnetic control device 30-2 can be disposed on a side of the first substrate 1 away from the second substrate 2, or on a side of the second substrate 2 away from the first substrate 1, corresponding to the area where the library preparation area 400 is located. The second magnetic control device 30-2 is configured to generate at least one second magnetic field region 450 in the library preparation area 400. In an exemplary embodiment, the at least one second magnetic field region 450 is configured to implement a sample purification process, and the second magnetic field region 450 can include a plurality of regularly arranged second magnetic regions.

[0107] In an exemplary embodiment, the first magnetic control device 30-1 and the second magnetic control device 30-2 may include a permanent magnet or an electromagnet, a controller, etc. The controller controls the magnetic field area formed and the strength of the magnetic field by adjusting the distance between the permanent magnet and the first substrate or the second substrate or by turning the electromagnet on and off.

[0108] In an exemplary embodiment, the temperature control device 20 and the magnetic control device 30 may be provided separately, or may be combined into a temperature control and magnetic control integrated device.

[0109] Figure 4 FIG1 is a schematic diagram of a planar structure of a first substrate according to an exemplary embodiment of the present disclosure, illustrating the structure of a driving unit. Figure 5 for Figure 4 In an exemplary embodiment, the driving array of the digital microfluidic chip adopts an active driving implementation mode, which can accurately control the individual movement of each droplet. The first substrate may include a first substrate, a first structural layer arranged on the side of the first substrate facing the second substrate, and a first liquid-repellent layer arranged on the side of the first structural layer facing the second substrate. The first structural layer may include at least gate lines, data lines, driving transistors, and driving electrodes. Figure 4 and Figure 5As shown, on a plane parallel to the first substrate, the first substrate may include a plurality of gate lines 51 extending along a first direction D1 and a plurality of data lines 52 extending along a second direction D2. The plurality of gate lines 51 and the plurality of data lines 52 intersect with each other to form a plurality of drive regions arranged in an array, with the first direction D1 and the second direction D2 intersecting. A drive transistor 50 and a drive electrode 60 are provided in at least one drive region. A drive electrode array is formed on the first substrate. The drive transistors 50 are respectively connected to the gate lines 51, data lines 52, and drive electrodes 60 in their respective drive regions. The gate lines 51 are configured to provide scan signals to corresponding drive transistors 50. In response to the gate line scan signals, the drive transistors 50 are turned on, applying the data voltages from the data lines 52 to the drive electrodes 60.

[0110] In an exemplary embodiment, on a plane perpendicular to the first substrate, the first substrate may include:

[0111] a first substrate 11;

[0112] A first conductive layer is provided on the first substrate 11, and the first conductive layer may include at least a gate line 51 and a first gate electrode 31 and a second gate electrode 32 located in each driving unit, wherein the first gate electrode 31 and the second gate electrode 32 are respectively connected to the gate line 51;

[0113] a first insulating layer 61 covering the first conductive layer;

[0114] A semiconductor layer disposed on a side of the first insulating layer 61 away from the first substrate, the semiconductor layer may include at least a first active layer 33 and a second active layer 34 located in each driving unit, wherein an orthographic projection of the first active layer 33 on the first substrate at least partially overlaps with an orthographic projection of the first gate electrode 31 on the first substrate, and an orthographic projection of the second active layer 34 on the first substrate at least partially overlaps with an orthographic projection of the second gate electrode 32 on the first substrate;

[0115] A second conductive layer is provided on a side of the semiconductor layer away from the first substrate. The second conductive layer may include at least a data line 52, and a first electrode 35, a connecting electrode 36, and a second electrode 37 located in each driving unit. A first end of the first electrode 35 is connected to the data line 52, and a second end of the first electrode 35 is provided on a side of the first active layer 33 closer to the data line 52. A first end of the connecting electrode 36 is provided on a side of the first active layer 33 closer to the data line 52, and a second end of the connecting electrode 36 is provided on a side of the second active layer 34 closer to the data line 52. A first end of the second electrode 37 is provided on a side of the second active layer 34 closer to the data line 52, and a second end of the second electrode 37 is provided on the first insulating layer 61. A first channel is formed between the second end of the first electrode 35 and the first end of the connecting electrode 36, and a second channel is formed between the second end of the connecting electrode 36 and the first end of the second electrode 37.

[0116] a second insulating layer 62 covering the second conductive layer;

[0117] A third conductive layer is provided on a side of the second insulating layer 62 away from the first substrate, the third conductive layer may include at least a capacitor electrode 38 located in each driving unit, and an orthographic projection of the capacitor electrode 38 on the first substrate includes an orthographic projection of the first channel and a second channel on the first substrate;

[0118] a third insulating layer 63 covering the third conductive layer, wherein the third insulating layer 63 is provided with a connection via hole, and the third insulating layer 63 and the second insulating layer 62 in the connection via hole are removed to expose the surface of the second electrode 37;

[0119] A fourth conductive layer is provided on a side of the third insulating layer 63 away from the first substrate. The fourth conductive layer may include at least a driving electrode 60 located in each driving unit. The driving electrode 60 is connected to the second electrode 37 through a connecting via. The orthographic projection of the driving electrode 60 on the first substrate at least partially overlaps with the orthographic projection of the capacitor electrode 38 on the first substrate.

[0120] a fourth insulating layer 64 covering the fourth conductive layer, where the fourth insulating layer 64 may be referred to as a dielectric layer;

[0121] The first lyophobic layer 13 is disposed on a side of the fourth insulating layer 64 away from the first substrate.

[0122] In an exemplary embodiment, the first direction D1 may be a horizontal direction, and the second direction D2 may be a vertical direction, with the first direction D1 and the second direction D2 being perpendicular to each other.

[0123] In an exemplary embodiment, the first gate electrode 31, the second gate electrode 32, the first active layer 33, the second active layer 34, the first electrode 35, the connecting electrode 36 and the second electrode 37 constitute a dual-gate structure driving transistor 50, and the driving transistor 50 is respectively connected to the gate line 51, the data line 52 and the driving electrode 60, that is, the first gate electrode 31 and the second gate electrode 32 in the driving transistor 50 are connected to the gate line 51, the first electrode 35 in the driving transistor 50 is connected to the data line 52, and the second electrode 37 in the driving transistor 50 is connected to the driving electrode 60, thereby realizing independent control and addressing of the driving electrode 60 in each driving unit.

[0124] In an exemplary embodiment, the first and second gate electrodes 31 and 32 and the gate line 51 may be an integral structure connected to each other, and the first electrode 35 and the data line 52 may be an integral structure connected to each other.

[0125] In example embodiments, the first electrode may be a drain electrode and the second electrode may be a source electrode, or the first electrode may be a source electrode and the second electrode may be a drain electrode.

[0126] In an exemplary embodiment, the capacitor electrode 38 and the driving electrode 60 may constitute a storage capacitor C st , storage capacitor C st It is configured to maintain the voltage of the driving electrode 60 for a certain period of time. In an exemplary embodiment, the driving electrode 60 is connected to the second electrode 37 in the driving transistor 50, and the capacitor electrode 38 can be connected to the ground signal (GND) of the system. When the driving transistor 50 is turned on, the data voltage (such as 20V) transmitted by the data line 52 is output to the driving electrode 60 through the driving transistor 50. Due to the voltage difference between the driving electrode 60 and the capacitor electrode 38, the storage capacitor C st After the driving transistor 50 is turned off, the storage capacitor C st It can be done within a certain time T (T is greater than or equal to the droplet dielectric wetting reaction time T drop ) to maintain the voltage on the driving electrode 60 at the set holding voltage V hold , to ensure the smooth deformation of the droplets and achieve effective control of the droplets.

[0127] In an exemplary embodiment, in order to ensure effective manipulation of the droplet, the holding voltage V hold Usually greater than or equal to the threshold voltage V for droplet actuation drop-th , that is, V hold ≥V drop-th Since the holding voltage V hold and the storage capacitor C st The capacitance value of the storage capacitor C st The capacitance value is used to obtain the appropriate setting holding voltage V hold .

[0128] In an exemplary embodiment, the storage capacitor C st The capacitance value is proportional to the facing area of the capacitor electrode 38 and the driving electrode 60, the vacuum dielectric constant and the dielectric constant of the third insulating layer, and is inversely proportional to the thickness of the third insulating layer of the capacitor electrode 38 and the driving electrode 60 (i.e., the distance between the capacitor electrode 38 and the driving electrode 60). Therefore, a suitable storage capacitor C can be obtained by adjusting the facing area of the capacitor electrode 38 and the driving electrode 60 and the distance between the capacitor electrode 38 and the driving electrode 60. st capacitance value.

[0129] In an exemplary embodiment, since the orthographic projection of the capacitor electrode 38 on the first substrate includes the orthographic projections of the first channel and the second channel on the first substrate, the capacitor electrode 38 can serve as a shielding layer to block natural light from the external environment, preventing natural light from directly irradiating the channel of the driving transistor and avoiding affecting the electrical performance of the driving transistor.

[0130] The study found that traditional digital microfluidic chips have problems such as uncontrollable droplet deformation and droplet manipulation failure. Further research found that the reason why traditional digital microfluidic chips have problems such as uncontrollable droplet deformation and droplet manipulation failure is due to changes in the characteristics of the driving transistor. In order to achieve controllable droplet deformation, especially the fine manipulation (such as generation, splitting, mixing, etc.) of tiny droplets (such as pL-level droplets), the driving electrode usually uses a larger driving voltage. For example, the driving voltage is usually greater than or equal to 20V. Since traditional digital microfluidic chips use a single-gate driving transistor, and the single-gate driving transistor is prone to electrical performance deterioration when conducting high voltage, such as a large threshold voltage offset, a large leakage current, or even breakdown, etc., resulting in problems such as uncontrollable droplet deformation and droplet manipulation failure, and it is impossible to achieve precise parallel control of multiple droplets. The exemplary embodiment of the present disclosure adopts a dual-gate structure driving transistor, which has the characteristics of high voltage resistance, low leakage current and stable performance, effectively reduces the threshold voltage offset and leakage current, and effectively avoids problems such as uncontrollable droplet deformation and droplet manipulation failure. It can realize the fine manipulation of tiny droplets and can realize the precise control of multiple droplets in parallel.

[0131] The following is an illustrative explanation through the preparation process of the first substrate. The "patterning process" mentioned in the present disclosure includes processes such as coating photoresist, mask exposure, development, etching, and stripping photoresist for metal materials, inorganic materials or transparent conductive materials, and includes processes such as coating organic materials, mask exposure and development for organic materials. Deposition can be carried out by any one or more of sputtering, evaporation, and chemical vapor deposition, coating can be carried out by any one or more of spraying, spin coating and inkjet printing, and etching can be carried out by any one or more of dry etching and wet etching, and the present disclosure does not limit this. "Thin film" refers to a thin film made by deposition, coating or other processes on a substrate of a certain material. If the "thin film" does not require a patterning process during the entire production process, the "thin film" can also be called a "layer". If the "thin film" requires a patterning process during the entire production process, it is called a "thin film" before the patterning process and a "layer" after the patterning process. The "layer" after the patterning process contains at least one "pattern". As used in this disclosure, "A and B are disposed in the same layer" means that A and B are formed simultaneously through the same patterning process, and the "thickness" of the film layer refers to the dimension of the film layer in a direction perpendicular to the display substrate. In exemplary embodiments of this disclosure, "the orthographic projection of B is within the range of the orthographic projection of A" means that the boundary of the orthographic projection of B falls within the boundary of the orthographic projection of A, or that the boundary of the orthographic projection of A overlaps with the boundary of the orthographic projection of B.

[0132] In an exemplary embodiment, the preparation process of the first substrate in the digital microfluidic chip of the embodiment of the present disclosure may include the following operations.

[0133] (1) Forming a first conductive layer pattern on the first substrate. In an exemplary embodiment, forming the first conductive layer pattern on the first substrate may include: depositing a first conductive film on the first substrate, patterning the first conductive film through a patterning process, and forming the first conductive layer pattern on the first substrate 11. The first conductive layer pattern may include at least a gate line 51, a first gate electrode 31, and a second gate electrode 32. The first gate electrode 31 and the second gate electrode 32 are both connected to the gate line 51, such as Figure 6a and Figure 6b As shown, Figure 6b for Figure 6a Cross-sectional view along the AA axis.

[0134] (2) Forming a semiconductor layer pattern. In an exemplary embodiment, forming the semiconductor layer pattern may include: sequentially depositing a first insulating film and a semiconductor film on a first substrate having the aforementioned pattern formed thereon, patterning the semiconductor film through a patterning process to form a first insulating layer 61 covering the first conductive layer pattern and a semiconductor layer pattern disposed on the first insulating layer 61, wherein the semiconductor layer pattern includes at least a first active layer 33 and a second active layer 34, wherein an orthographic projection of the first active layer 33 on the first substrate at least partially overlaps with an orthographic projection of the first gate electrode 31 on the first substrate, and an orthographic projection of the second active layer 34 on the first substrate at least partially overlaps with an orthographic projection of the second gate electrode 32 on the first substrate, as shown in FIG. Figure 7a and Figure 7b As shown, Figure 7b for Figure 7a Cross-sectional view along the AA axis.

[0135] (3) Forming a second conductive layer pattern. In an exemplary embodiment, forming the second conductive layer pattern may include: depositing a second conductive film on the first substrate on which the aforementioned pattern is formed, patterning the second conductive film through a patterning process to form a second conductive layer pattern, wherein the second conductive layer pattern may include at least a data line 52, a first electrode 35, a connecting electrode 36, and a second electrode 37, wherein a first end of the first electrode 35 is connected to the data line 52, a second end of the first electrode 35 is arranged on a side of the first active layer 33 close to the data line 52, a first end of the connecting electrode 36 is arranged on a side of the first active layer 33 away from the data line 52, a second end of the connecting electrode 36 is arranged on a side of the second active layer 34 close to the data line 52, a first end of the second electrode 37 is arranged on a side of the second active layer 34 away from the data line 52, a second end of the second electrode 37 is arranged on the first insulating layer, a first channel is formed between the second end of the first electrode 35 and the first end of the connecting electrode 36, and a second channel is formed between the second end of the connecting electrode 36 and the first end of the second electrode 37, as shown in FIG. Figure 8a and Figure 8b As shown, Figure 8b for Figure 8a Cross-sectional view along the AA axis.

[0136] (4) Forming a third conductive layer pattern. In an exemplary embodiment, forming the third conductive layer pattern may include: sequentially depositing a second insulating film and a third conductive film on the first substrate on which the aforementioned pattern is formed, patterning the third conductive film through a patterning process to form a second insulating layer 62 covering the second conductive layer pattern and a third conductive layer pattern disposed on the second insulating layer 62, wherein the third conductive layer pattern may include at least a capacitor electrode 38, and the positive projection of the capacitor electrode 38 on the first substrate may include the positive projection of the first channel and the second channel on the first substrate, as shown in FIG. Figure 9a and Figure 9b As shown, Figure 9b for Figure 9a Cross-sectional view along the AA axis.

[0137] In an exemplary embodiment, the capacitive electrodes 38 of the plurality of driving units may be an integrated structure connected to each other and connected to a ground signal (GND) of the system.

[0138] (5) Forming a second insulating layer pattern. In an exemplary embodiment, forming a third insulating layer pattern may include: depositing a third insulating film on the first substrate on which the aforementioned pattern is formed, patterning the third insulating film through a patterning process to form a third insulating layer 63 pattern covering the third conductive layer pattern, forming a connecting via K1 on the third insulating layer 63, and removing the third insulating layer and the second insulating layer in the connecting via K1 to expose the surface of the second electrode 37, as shown in FIG. Figure 10a and Figure 10b As shown, Figure 10b for Figure 10a Cross-sectional view along the AA axis.

[0139] (6) Forming a fourth conductive layer pattern. In an exemplary embodiment, forming the fourth conductive layer pattern may include: depositing a fourth conductive film on the first substrate on which the aforementioned pattern is formed, patterning the fourth conductive film through a patterning process, and forming a fourth conductive layer pattern on the third insulating layer 63. The fourth conductive layer pattern may include at least a driving electrode 60, wherein the orthographic projection of the driving electrode 60 on the first substrate at least partially overlaps with the orthographic projection of the capacitor electrode 38 on the first substrate, and the driving electrode 60 is connected to the second electrode 37 through a connecting via K1, as shown in FIG. Figure 11a and Figure 11b As shown, Figure 11b for Figure 11a Cross-sectional view along the AA axis.

[0140] (7) Forming a dielectric layer and a first hydrophobic layer pattern. In an exemplary embodiment, forming a dielectric layer and a first hydrophobic layer pattern may include: sequentially forming a fourth insulating layer 64 and a first hydrophobic layer 13 on the first substrate having the aforementioned pattern formed thereon, such as Figure 5shown.

[0141] In an exemplary embodiment, the substrate may be a rigid substrate or a flexible substrate. In an exemplary embodiment, the rigid substrate may be made of a material such as glass or quartz, and the flexible substrate may be made of a material such as polyimide (PI). The flexible substrate may have a single-layer structure or a laminated structure composed of an inorganic material layer and a flexible material layer, which is not limited in this disclosure.

[0142] In an exemplary embodiment, the first insulating layer, the second insulating layer and the third insulating layer may be made of inorganic materials, and the fourth insulating layer and the first lyophobic layer may be made of organic materials. The inorganic material may be any one or more of silicon oxide (SiOx), silicon nitride (SiNx) and silicon oxynitride (SiON), and may be a single layer, a multilayer or a composite layer. The first insulating layer may be referred to as a gate insulating (GI) layer, and the second insulating layer and the third insulating layer may be referred to as a passivation (PVX) layer. The first conductive layer, the second conductive layer and the third conductive layer may be made of metal materials, such as any one or more of silver (Ag), copper (Cu), aluminum (Al), titanium (Ti) and molybdenum (Mo), or alloy materials of the above metals, such as aluminum neodymium alloy (AlNd) or molybdenum niobium alloy (MoNb), and may be a single layer structure or a multilayer composite structure. The fourth conductive layer may be made of a transparent conductive material, such as indium tin oxide (ITO) or indium zinc oxide (IZO). The semiconductor layer can be made of various materials such as amorphous indium gallium zinc oxide (a-IGZO), zinc oxynitride (ZnON), indium zinc tin oxide (IZTO), amorphous silicon (a-Si), polycrystalline silicon (p-Si), sexithiophene, polythiophene, etc., that is, the present disclosure is applicable to transistors manufactured based on oxide technology, silicon technology and organic technology.

[0143] It should be noted that the aforementioned structure and its preparation process are merely exemplary. In the exemplary embodiment, the corresponding structure can be changed and the patterning process can be increased or decreased according to actual needs, and the present disclosure does not limit this.

[0144] In an exemplary embodiment, the second substrate may include a second substrate, a second structural layer arranged on the side of the second substrate facing the first substrate, and a second liquid-repellent layer arranged on the side of the second structural layer facing the first substrate. In an exemplary embodiment, the second structural layer may include at least a plurality of counter electrodes, and the positions and sizes of the plurality of counter electrodes may correspond to the positions and sizes of the driving units on the first substrate, forming a counter electrode array on the second substrate, and the driving electrode array of the first substrate and the counter electrode array of the second substrate together constitute a driving unit array for driving the droplets, and each driving unit includes at least a driving electrode and a counter electrode. In some possible exemplary embodiments, the second structural layer may include a counter electrode of a full-surface structure, which is not limited in the present disclosure.

[0145] Figure 12 FIG1 is a schematic diagram of a planar structure of a screening area according to an exemplary embodiment of the present disclosure. As shown in FIG12 , in an exemplary embodiment, the screening area 100 may include a plurality of drive units and a plurality of screening area reagent ports arranged in a matrix, wherein the plurality of screening area reagent ports may include at least a first screening area reagent port 101, a second screening area reagent port 102, a third screening area reagent port 103, and a fourth screening area reagent port 104 provided on the second substrate.

[0146] In an exemplary embodiment, the screening area 100 can be rectangular, and multiple driving units can be arranged in a matrix manner. The first reagent port 101, the second reagent port 102, the third reagent port 103 and the fourth reagent port 104 in the screening area can be respectively set in the four corner areas of the screening area 100 to avoid the liquid entering the screening area 100 from the reagent port from contaminating the cells and affecting cell processing.

[0147] In an exemplary embodiment, the first reagent port 101 of the screening area can be configured to receive a whole blood sample injected by an external device, the second reagent port 102 of the screening area can be configured to receive magnetic nanoparticles injected by an external device, the third reagent port 103 of the screening area can be configured to receive a first buffer solution injected by an external device, and the fourth reagent port 104 of the screening area can be configured to discharge a first waste liquid using an external device.

[0148] In an exemplary embodiment, the number, location, and size of the screening area reagent ports in the screening area 100, as well as the type of reagent injected into each screening area reagent port, can be set according to actual needs. For example, the first screening area reagent port 101 can be configured to receive magnetic nanoparticles, and the second screening area reagent port 102 can be configured to receive a whole blood sample, although this disclosure is not limited thereto.

[0149] In an exemplary embodiment, the first magnetic control device can be arranged on a side of the first substrate away from the second substrate, or on a side of the second substrate away from the first substrate, and the position corresponds to the area where the screening area 100 is located. The first magnetic control device is configured to generate a first magnetic field region 110 in the screening area 100, and the first magnetic field region 110 is configured to achieve capture processing of rare cells.

[0150] In an exemplary embodiment, the first magnetic field region 110 may include a plurality of regularly arranged first magnetic regions 111. To achieve effective magnetic capture, the orthographic projection of at least one first magnetic region 111 (magnetic capture point) on the first substrate includes the orthographic projection of at least one driving unit in the screening region 100 on the first substrate. For example, the driving unit may be rectangular, having a first long side and a first wide side, and the first magnetic region 111 may be rectangular, having a second long side and a second wide side. The length of the second long side may be greater than or equal to the length of the first long side, and the width of the second wide side may be greater than or equal to the width of the first wide side. The orthographic projection of the driving unit on the first substrate is within the range of the orthographic projection of the first magnetic region 111 on the first substrate.

[0151] In an exemplary embodiment, the first magnetic region 111 may be block-shaped, and the plurality of first magnetic regions 111 may be arranged in a regular manner, such as a square, a nine-square shape, a herringbone shape, a diamond shape, etc., and each block-shaped first magnetic region 111 may cover one drive unit. In another exemplary embodiment, each block-shaped first magnetic region 111 may cover multiple drive units. In yet another exemplary embodiment, the first magnetic region 111 may be strip-shaped extending along a first direction, and the plurality of first magnetic regions 111 may be arranged in sequence along a second direction, and each strip-shaped first magnetic region 111 may cover multiple drive units arranged along the first direction, and the first direction and the second direction intersect. In yet another exemplary embodiment, the shapes and sizes of the plurality of first magnetic regions 111 may be the same, or may be different, and the present disclosure is not limited thereto.

[0152] In an exemplary embodiment, the multiple drive units in the screening area 100 are configured to screen and enrich rare cells. The multiple drive units in the screening area 100 are configured to: uniformly mix the sample, magnetic nanoparticles, and the first buffer into a mixed droplet; disperse the mixed droplet into a plurality of sub-droplets; move the sub-droplets not captured by the first magnetic field region to the first waste port for discharge; and mix the sub-droplets captured by the first magnetic field region into an enriched droplet containing a rare cell-magnetic nanoparticle complex.

[0153] Figures 13a to 13c Schematic diagram of a screening area for rare cell screening and enrichment according to an exemplary embodiment of the present disclosure. In an exemplary embodiment, the screening area for rare cell screening and enrichment includes the following steps.

[0154] (11) Cell-magnetic bead mixing and incubation step. First, a drop of blood sample, a drop of magnetic particle droplet and several drops of first buffer solution are injected into the screening area 100 from the first reagent port 101, the second reagent port 102 and the third reagent port 103 of the screening area respectively. The blood sample contains red blood cells, white blood cells and rare cells, and the magnetic particle droplet contains several magnetic nanoparticles coupled with special antibodies. Then, the driving unit drives the blood sample, magnetic particle droplet and buffer solution to mix to form mixed droplets. By driving the mixed droplet to move back and forth, the mixed droplet is shaken and mixed several times to mix evenly. The immunomagnetic nanoparticles coated with specific antibodies are fully in contact with the rare cells in the sample, so that the antibodies coated with the magnetic nanoparticles specifically bind to the rare cell surface antigens. The rare cells are surrounded by multiple magnetic nanoparticles to form a rare cell (target cell)-magnetic nanoparticle complex, such as Figure 13a shown.

[0155] (12) Rare cell capture step. Several drops of the first buffer solution are injected into the screening area 100 from the third reagent port 103 of the screening area, and the driving unit uses the first buffer solution to disperse the evenly mixed mixed droplets into several sub-droplets of equal volume. A first magnetic field region 110 including a plurality of first magnetic regions 111 is formed by a first magnetic control device. When the sub-droplets containing the rare cell-magnetic nanoparticle complex move to the position of the first magnetic region 111, the rare cell-magnetic nanoparticle complex is adsorbed on the surface of the first substrate under the action of the magnetic field, thereby achieving the capture of the sub-droplets containing the rare cell-magnetic nanoparticle complex. The driving unit drives the sub-droplets that are not captured by the first magnetic region 111 to move to the fourth reagent port 104 of the screening area for discharge, thereby achieving the separation of rare cells from red blood cells and white blood cells, such as Figure 13b shown.

[0156] (13) Rare cell enrichment step. Several drops of the first buffer solution are injected into the screening area 100 from the third reagent port 103 of the screening area. The first magnetic control device stops working, the first magnetic field area is canceled, and the driving unit drives the first buffer solution to mix with the captured sub-droplets into an enriched droplet. The rare cell-magnetic nanoparticle complex is suspended in the enriched droplet, as shown in FIG. Figure 13c shown.

[0157] Figure 14 FIG14 is a schematic diagram of a planar structure of a cleavage zone according to an exemplary embodiment of the present disclosure. As shown in FIG14 , in an exemplary embodiment, the cleavage zone 200 may include a plurality of drive units and a plurality of cleavage zone reagent ports arranged in a matrix, wherein the plurality of cleavage zone reagent ports may include at least a first cleavage zone reagent port 201, a second cleavage zone reagent port 202, a third cleavage zone reagent port 203, and a fourth cleavage zone reagent port 204 provided on the second substrate.

[0158] In an exemplary embodiment, the lysis zone 200 can be rectangular, and multiple driving units can be arranged in a matrix manner. The first reagent port 201 of the lysis zone, the second reagent port 202 of the lysis zone, the third reagent port 203 of the lysis zone, and the fourth reagent port 204 of the lysis zone can be respectively set in the four corner areas of the lysis zone 200 to avoid the liquid entering the lysis zone 200 from the reagent port from contaminating the cells and affecting cell processing.

[0159] In an exemplary embodiment, the first reagent port 201 in the lysis zone can be configured to receive a lysis solution injected from an external device, the second reagent port 202 in the lysis zone can be configured to receive a stop solution injected from an external device, the third reagent port 203 in the lysis zone can be configured to receive a second buffer solution injected from an external device, and the fourth reagent port 204 in the lysis zone can be configured to discharge a second waste liquid using an external device.

[0160] In an exemplary embodiment, the number, location, and size of the lysis zone reagent ports in the lysis zone 200, as well as the type of reagent injected into each lysis zone reagent port, can be configured according to actual needs. For example, the first lysis zone reagent port 201 is configured to receive a stop solution injected from an external device, and the second lysis zone reagent port 202 is configured to receive a lysis solution injected from an external device, although this disclosure is not limited thereto.

[0161] In an exemplary embodiment, the multiple drive units in the lysis zone 200 are configured to perform rare cell singulation and cell lysis. The multiple drive units in the lysis zone 200 are configured to disperse the enriched droplet into a plurality of daughter droplets, place the plurality of daughter droplets on different drive units, reuse the drive units as detection units, identify and locate daughter droplets containing rare cell-magnetic nanoparticle complexes, and then mix the daughter droplets containing the rare cell-magnetic nanoparticle complexes to form lysis droplets, thereby obtaining a single-cell nucleic acid sample.

[0162] In an exemplary embodiment, the detection device can be connected to multiple driving units of the lysis area 200, so that the multiple driving units of the lysis area 200 are multiplexed into multiple detection units, and the multiple detection units identify and locate sub-droplets containing rare cell-magnetic nanoparticle complexes in the lysis area 200, thereby realizing the identification and positioning of rare cells.

[0163] Figures 15a to 15c FIG2 is a schematic diagram of a lysis zone for singulation and cell lysis of rare cells according to an exemplary embodiment of the present disclosure. In an exemplary embodiment, the lysis zone for singulation and cell lysis of rare cells includes the following steps.

[0164] (21) Rare cell singulation step. After the enriched droplets obtained in the screening area 100 are moved to the lysis area 200, several drops of the second buffer solution are injected into the lysis area 200 from the third reagent port 203 of the lysis area. The driving unit uses the second buffer solution to disperse the enriched droplets into several sub-droplets of equal volume, so that each sub-droplet contains only one rare cell-magnetic nanoparticle complex (single cell package) or no rare cell-magnetic nanoparticle complex (vacuoles), as shown in FIG. Figure 15a shown.

[0165] (22) Rare cell impedance detection step. The driving unit drives multiple sub-droplets to be located in multiple driving units within the detection area 210, forming a single cell / vacuole array. The sub-droplets containing the rare cell-magnetic nanoparticle complex are identified and located by the detection device, such as Figure 15b shown.

[0166] (23) Rare cell lysis step. After obtaining the position information of the sub-droplets containing the rare cell-magnetic nanoparticle complex, the driving unit drives the cavitation bubble to move to the second waste liquid port 204 for discharge, thereby realizing the separation of the sub-droplets containing the rare cell-magnetic nanoparticle complex and the cavitation bubble. Subsequently, several drops of lysis solution are injected into the lysis zone 200 from the first reagent port 201 of the lysis zone, and the driving unit drives the lysis solution to mix with the sub-droplets containing the rare cell-magnetic nanoparticle complex to form lysis droplets. By driving the lysis droplets to move back and forth, the lysis droplets are shaken and mixed several times, so that the lysis solution is fully in contact with the rare cells to lyse the cell membrane and completely expose the nucleic acid in the rare cells. Subsequently, a drop of stop solution is injected into the lysis zone 200 from the second reagent port 202 of the lysis zone, and the driving unit drives the lysis droplets to mix with the stop solution to terminate the lysis reaction and form a single-cell nucleic acid sample, such as Figure 15c shown.

[0167] In an exemplary embodiment, to ensure that each daughter droplet contains only one rare cell-magnetic nanoparticle complex (single-cell encapsulation) or no rare cell-magnetic nanoparticle complex (i.e., vacuoles), the size of the driving electrodes in the digital microfluidic chip needs to be matched to the size of the daughter droplets. This disclosure uses a large-volume averaging method to calculate the size of the single-cell encapsulation, assuming that the distribution of cells in the droplet follows the Poisson distribution law, and its function is shown below:

[0168]

[0169] Where λ is the theoretical average number of cells in each droplet, n is the number of cells in the droplet, and f(λ;n) represents the probability of encapsulating n cells, that is, the percentage of droplets encapsulating n cells in the total number of droplets.

[0170] When the droplets are diluted to a certain extent, taking tumor cells as an example, the diameter D of tumor cells is generally between 10μm and 20μm, and the concentration of tumor cells in the blood sample is about 1cells / mL to 10cells / mL. According to the Poisson distribution formula, when λ = 1.98, the theoretical single cell encapsulation rate is f(1) = 27.3%, and the characteristic diameter of the droplet D drop Close to 19.8μm, droplet volume V drop Close to the picoliter (pL) level, single-cell encapsulation can be achieved. The volume of a single-cell encapsulation droplet can be expressed as follows:

[0171]

[0172]

[0173] Wherein, θ represents the initial contact angle between the droplet and the hydrophobic surface on the first substrate, which is generally close to 120°, H represents the thickness of the digital microfluidic chip, and L represents the size of a single driving electrode.

[0174] Figure 16 Schematic diagram of a droplet in a digital microfluidic chip. Figure 16 As shown, the cell thickness H of the digital microfluidic chip refers to the distance between the first lyophobic layer 13 in the first substrate 1 and the second lyophobic layer 23 in the second substrate 2, and the size L of the driving electrode refers to the length of the driving electrode along the moving direction of the droplet. In order to achieve single cell encapsulation, that is, the volume of a single sub-droplet V drop Close to the pL level, according to the above formula, when the box thickness H of the digital microfluidic chip is ≤ 19.8 μm, the size of a single driving electrode L is ≤ 48.5 μm.

[0175] Figure 17 A schematic diagram of the principle of an impedance analysis method. Figure 18 for Figure 17 Schematic diagram of equivalent impedance of cavitation bubble and single cell package. Figure 17 As shown, in an exemplary embodiment, the detection unit can identify and locate the sub-droplets containing rare cell-magnetic nanoparticle complexes by using impedance analysis, applying an AC signal ( ) between the driving electrode 60 and the counter electrode 70 in the area where each sub-droplet is located. Figure 17 The dashed lines in the middle represent electric field lines) and detect the impedance signal of each sub-droplet. By comparing the impedance signals of adjacent sub-droplets and performing differential operations, the presence and location of rare cells can be determined. Figure 17As shown, for cavitation, the impedance between the driving electrode 60 and the counter electrode 70 may include the impedance of the dielectric layer 64 (the resistance R1 of the dielectric layer and the capacitance C1 of the dielectric layer), the impedance of the first lyophobic layer 13 (the resistance R2 of the first lyophobic layer and the capacitance C2 of the first lyophobic layer), the impedance of the droplet (the resistance R3 of the droplet and the capacitance C3 of the droplet), and the impedance of the second lyophobic layer 23 (the resistance R4 of the second lyophobic layer and the capacitance C4 of the second lyophobic layer). For sub-droplets containing rare cell-magnetic nanoparticle complexes, the impedance between the driving electrode 60 and the counter electrode 70 may include the impedance of the dielectric layer 64 (R1 and C1), the impedance of the first lyophobic layer 13 (R2 and C2), the impedance of the droplet (R3 and C3), the impedance of the single-cell package (the resistance R5 of the single-cell package, the capacitance C5 and C6 of the single-cell package), and the impedance of the second lyophobic layer 23 (R4 and C4). In an exemplary embodiment, the resistance R5 of the single cell wrapper may be the resistance of the cytoplasm, and the capacitances C5 and C6 of the complex may be the capacitance of the cell membrane wrapping the cytoplasm.

[0176] Figure 19 FIG19 is a schematic diagram of a planar structure of a pre-amplification zone according to an exemplary embodiment of the present disclosure. As shown in FIG19 , in an exemplary embodiment, the pre-amplification zone 300 may include a plurality of drive units and a plurality of pre-amplification zone reagent ports arranged in a matrix. The plurality of pre-amplification zone reagent ports may include at least a first pre-amplification zone reagent port 301, a second pre-amplification zone reagent port 302, a third pre-amplification zone reagent port 303, and a fourth pre-amplification zone reagent port 304, disposed on the second substrate.

[0177] In an exemplary embodiment, the pre-amplification zone 300 may be rectangular in shape, a plurality of drive units may be arranged in a matrix, and the first reagent port 301, the second reagent port 302, the third reagent port 303, and the fourth reagent port 304 of the pre-amplification zone may be respectively disposed at the four corner regions of the pre-amplification zone 300 to prevent liquid entering the pre-amplification zone 300 from contaminating cells and affecting cell processing.

[0178] In an exemplary embodiment, the first reagent port 301 of the pre-amplification zone can be configured to receive a fragmentation enzyme reagent injected from an external device, the second reagent port 302 of the pre-amplification zone can be configured to receive a pre-amplification reagent injected from an external device, the third reagent port 303 of the pre-amplification zone can be configured to receive a fragmentation buffer injected from an external device, and the fourth reagent port 304 of the pre-amplification zone can be configured to discharge a third waste liquid using an external device.

[0179] In an exemplary embodiment, the number, location, and size of the pre-amplification zone reagent ports in the pre-amplification zone 300, as well as the type of reagent injected into each pre-amplification zone reagent port, can be configured according to actual needs. For example, the first pre-amplification zone reagent port 301 is configured to receive a pre-amplification reagent injected from an external device, and the second pre-amplification zone reagent port 302 is configured to receive a fragmentation enzyme reagent injected from an external device, although this disclosure is not limited thereto.

[0180] In an exemplary embodiment, the first temperature control device can be disposed on a side of the first substrate away from the second substrate, or on a side of the second substrate away from the first substrate, corresponding to the area where the pre-amplification zone 300 is located. The first temperature control device is configured to generate a first amplification temperature zone 310 and a second amplification temperature zone 320 in the pre-amplification zone 300. The two amplification temperature zones have different temperatures. The first amplification temperature zone 310 and the second amplification temperature zone 320 are configured to perform pre-amplification processing for rare single cells. For example, the temperature of the first amplification temperature zone 310 can be approximately 30°C, and the temperature of the second amplification temperature zone 320 can be approximately 105°C.

[0181] In an exemplary embodiment, the multiple drive units in the pre-amplification zone 300 are configured to perform rare single-cell pre-amplification. The multiple drive units in the pre-amplification zone 300 are configured to: process the single-cell nucleic acid sample into a fragmented DNA sample, mix it with pre-amplification reagents to form amplification droplets, and drive the amplification droplets to move between the first amplification temperature zone and the second amplification temperature zone to ultimately obtain a pre-amplified nucleic acid sample of the rare single cell.

[0182] Figures 20a to 20c FIG2 is a schematic diagram of a rare single cell pre-amplification process performed in a pre-amplification zone according to an exemplary embodiment of the present disclosure. In an exemplary embodiment, the rare single cell pre-amplification process performed in the pre-amplification zone may include the following steps.

[0183] (31) Nucleic acid fragmentation step. After the single-cell nucleic acid sample obtained in the lysis zone 200 is moved to the pre-amplification zone 300, the fragmentation enzyme reagent and the fragmentation buffer are injected into the pre-amplification zone 300 from the first reagent port 301 and the third reagent port 303 of the pre-amplification zone, respectively. The driving unit drives the fragmentation enzyme reagent and the fragmentation buffer to mix with the single-cell nucleic acid sample for fragmentation treatment, and the long-chain DNA sample is evenly cut into a certain length to form a fragmented DNA sample, such as Figure 20a shown.

[0184] (32) Nucleic acid pre-amplification step. The pre-amplification reagent is injected into the pre-amplification zone 300 from the second reagent port 302 of the pre-amplification zone, and the driving unit drives the pre-amplification reagent to mix with the fragmented DNA sample to form an amplification droplet. The first amplification temperature zone 310 and the second amplification temperature zone 320 are formed in the pre-amplification zone 300 by the first temperature control device. The driving unit drives the amplification droplet to move quickly between the first amplification temperature zone 310 and the second amplification temperature zone 320, so that the amplification droplet is quickly heated and cooled, and the fragmented DNA sample is pre-amplified to achieve whole genome pre-amplification at the single cell level, and finally a rare single cell pre-amplified nucleic acid sample is obtained, such as Figure 20b and Figure 20c shown.

[0185] In an exemplary embodiment, the whole genome pre-amplification technology may be a multiple strand displacement amplification (MDA) technology or a multiple annealing loop amplification cycle (MALBAC) technology, which is not limited in the present disclosure.

[0186] In an exemplary embodiment, the first amplification temperature zone 310 and the second amplification temperature zone 320 can be strips extending along the first direction D1, and the first amplification temperature zone 310 and the second amplification temperature zone 320 can be arranged in sequence along the second direction D2. In an exemplary embodiment, the heat conduction rate of the amplification droplet is proportional to the heat transfer area and the temperature difference between the amplification temperature zones, and is inversely proportional to the spacing between the amplification temperature zones. In order to avoid temperature crosstalk between the first amplification temperature zone (low temperature zone) and the second amplification temperature zone (high temperature zone), the minimum first distance L1 between the first amplification temperature zone and the second amplification temperature zone can be greater than or equal to 0.1*B1, where B1 is the width of the first amplification temperature zone or the first width of the second amplification temperature zone, and the first distance L1 and the first width are both dimensions in the second direction D2. For example, in a typical PCR application, the first width B1 of the medium temperature zone can be about 10 mm, and the minimum first distance L1 between the first amplification temperature zone and the second amplification temperature zone is greater than or equal to about 1 mm.

[0187] In an exemplary embodiment, the pre-amplification region 300 may include multiple temperature zones with different temperatures. The temperature, arrangement, shape, and size of the temperature zones may be set according to actual needs, and are not limited in this disclosure.

[0188] Figure 2121, in an exemplary embodiment, the library preparation area 400 may include a plurality of drive units and a plurality of preparation area reagent ports arranged in a matrix, and the plurality of preparation area reagent ports may include at least a first preparation area reagent port 401, a second preparation area reagent port 402, a third preparation area reagent port 403, a fourth preparation area reagent port 404, a fifth preparation area reagent port 405, a sixth preparation area reagent port 406, a seventh preparation area reagent port 407, an eighth preparation area reagent port 408, a ninth preparation area reagent port 409, a tenth preparation area reagent port 410, and an eleventh preparation area reagent port 411, provided on the second substrate.

[0189] In an exemplary embodiment, the library preparation area 400 can be rectangular, and the multiple drive units can be arranged in a matrix manner. The first reagent port 401, the second reagent port 402, the third reagent port 403, the fourth reagent port 404, and the fifth reagent port 405 of the preparation area can be set in the edge area of the library preparation area 400 on the second direction D2 side, and can be arranged sequentially along the first direction D1. The sixth reagent port 406, the seventh reagent port 407, the eighth reagent port 408, the ninth reagent port 409, and the tenth reagent port 410 of the preparation area can be set in the edge area of the library preparation area 400 on the opposite side of the second direction D2, and can be arranged sequentially along the first direction D1. The eleventh reagent port 411 of the preparation area can be set in the edge area of the library preparation area 400 on the first direction D1 side, and can be located in the middle area of the library preparation area 400 in the second direction D2.

[0190] In an exemplary embodiment, the preparation area first reagent port 401 , the preparation area fifth reagent port 405 , the preparation area sixth reagent port 406 , and the preparation area tenth reagent port 410 may be respectively disposed at four corner regions of the library preparation area 400 .

[0191] In an exemplary embodiment, the first reagent port 401 of the preparation area can be configured to receive a wash bead solution injected from an external device, the second reagent port 402 of the preparation area can be configured to receive an end repair master mix solution injected from an external device, the third reagent port 403 of the preparation area can be configured to receive a size selection bead solution injected from an external device, the fourth reagent port 404 of the preparation area can be configured to receive an elution buffer injected from an external device, the fifth reagent port 405 of the preparation area can be configured to receive a library amplification master mix solution injected from an external device, the sixth reagent port 406 of the preparation area can be configured to receive an A-tailing master mix solution injected from an external device, the seventh reagent port 407 of the preparation area can be configured to receive an adapter solution injected from an external device, and the eighth reagent port 408 of the preparation area can be configured to receive a ligation master mix solution injected from an external device. mastermix), the ninth reagent port 409 of the preparation area can be configured to receive a wash buffer (Washbuffer) injected by an external device, the tenth reagent port 410 of the preparation area can be configured to receive a primer (Primer) injected by an external device, and the eleventh reagent port 411 of the preparation area can be configured to discharge a fourth waste liquid using an external device.

[0192] In an exemplary embodiment, the number, location, and size of the preparation area reagent ports in the library preparation area 400, as well as the type of reagents injected into each preparation area reagent port, can be configured according to actual needs. For example, the first preparation area reagent port 401 can be configured to receive a terminal repair master mix solution injected from an external device, and the second preparation area reagent port 402 can be configured to receive wash beads injected from an external device, although this disclosure is not limited thereto.

[0193] In an exemplary embodiment, the second temperature control device and the second magnetic control device can be disposed on a side of the first substrate away from the second substrate, or on a side of the second substrate away from the first substrate, in a location corresponding to the area where the library preparation area 400 is located. The second temperature control device is configured to sequentially form a first polymerization temperature zone 420, a second polymerization temperature zone 430, and a third polymerization temperature zone 440 in the library preparation area 400. The three polymerization temperature zones have different temperatures. The first polymerization temperature zone 420, the second polymerization temperature zone 430, and the third polymerization temperature zone 440 are configured to implement PCR thermal cycling. For example, the temperature of the first polymerization temperature zone 420 can be approximately 98°C, the temperature of the second polymerization temperature zone 430 can be approximately 72°C, and the temperature of the third polymerization temperature zone 440 can be approximately 60°C. The second magnetic control device is configured to generate a second magnetic field region 450 in the library preparation area 400. The second magnetic field region 450 may include a plurality of regularly arranged second magnetic regions 451. The orthographic projection of at least one second magnetic region 451 on the first substrate includes the orthographic projection of at least one drive unit in the library preparation area 400 on the first substrate.

[0194] In an exemplary embodiment, the first polymerization temperature zone 420, the second polymerization temperature zone 430, and the third polymerization temperature zone 440 may be strip-shaped and extend along the second direction D2. The first polymerization temperature zone 420, the second polymerization temperature zone 430, and the third polymerization temperature zone 440 may be arranged sequentially along the first direction D1. To prevent temperature crosstalk between adjacent polymerization temperature zones, the minimum second distance L2 between adjacent polymerization temperature zones may be greater than or equal to 0.05*B2, where B2 is the second width of the first polymerization temperature zone, the second width of the second polymerization temperature zone, or the second width of the third polymerization temperature zone. The second distance L2 and the second width are both dimensions along the first direction D1. For example, in a typical PCR application, the second width B2 of the intermediate temperature zone may be approximately 10 mm. In this case, the minimum second distance L2 between adjacent polymerization temperature zones may be greater than or equal to approximately 0.5 mm.

[0195] In an exemplary embodiment, the second magnetic field region 450 may be located on a side of the third polymerization temperature zone 440 away from the first polymerization temperature zone 420, and the second magnetic field region 450 is configured to implement sample purification processing. In an exemplary embodiment, the second magnetic region 451 may be block-shaped, and multiple second magnetic regions 451 may be arranged in sequence along the second direction D2, and each block-shaped second magnetic region 451 may cover one drive unit. In another exemplary embodiment, the second magnetic region 451 may be strip-shaped extending along the second direction D2, and the strip-shaped second magnetic region 451 may cover multiple drive units. In yet another exemplary embodiment, the shapes and sizes of the multiple second magnetic regions 451 may be the same, or may be different, and the present disclosure is not limited thereto.

[0196] In an exemplary embodiment, the multiple drive units in library preparation area 400 are configured to prepare a rare single-cell library. The multiple drive units in library preparation area 400 are configured to perform end-repair on pre-amplified nucleic acid samples, screen for DNA fragments of the desired length, add A bases, adapters, and target inserts to the DNA fragments, perform PCR enrichment, and purify the DNA fragments to ultimately generate a library.

[0197] Figures 22a to 22c Schematic diagram of a library preparation area performing rare single cell library preparation processing according to an exemplary embodiment of the present disclosure. In an exemplary embodiment, the library preparation area performing rare single cell library preparation processing may include the following steps.

[0198] (41) End repair and fragment screening steps. After the rare single cell pre-amplified nucleic acid sample obtained in the pre-amplification area is moved to the library preparation area 400, the washing bead liquid is injected into the library preparation area 400 from the first reagent port 401 of the preparation area. The washing bead liquid contains a number of washing beads. After the driving unit drives the washing beads to mix with the pre-amplified nucleic acid sample, it drives the mixed droplets to move to the second magnetic field area 450, and performs magnetic bead purification under the magnetic field environment. The eluent is injected into the library preparation area 400 from the fourth reagent port 404 of the preparation area. The driving unit drives the eluent to elute the pre-amplified nucleic acid sample. The end repair master mixed material liquid is injected into the library preparation area 400 from the second reagent port 402 of the preparation area. The driving unit drives the end repair master mixed material liquid to mix with the pre-amplified nucleic acid sample, and performs end repair on the pre-amplified nucleic acid sample to make it a consistent form that meets the requirements of connector connection. The size screening bead liquid is injected into the library preparation area 400 from the third reagent port 403 of the preparation area. The size screening bead liquid contains a number of size screening beads. The driving unit drives the size screening beads to mix with the pre-amplified nucleic acid sample, and drives the mixed droplets to move to the second magnetic field region 450, where fragment screening is performed. By controlling the volume of the added size screening beads, DNA fragments of a desired length can be selectively screened out, such as Figure 22a shown.

[0199] (42) Sample plus A plus adapter step. The A tracking master mix is injected into the library preparation area 400 from the sixth reagent port 406 of the preparation area. The driving unit drives the A tracking master mix to mix with the nucleic acid sample, and adds the A base to the 3'-end of all blunt-end DNA. Subsequently, the droplet is driven to move to the second magnetic field area 450, and the end-repaired sample is purified in the second magnetic field area 450 using size screening beads. Subsequently, the adapter liquid and the ligation master mix are injected into the library preparation area 400 from the seventh reagent port 407 of the preparation area and the eighth reagent port 408 of the preparation area respectively. The driving unit drives the adapter liquid and the ligation master mix to mix with the sample after adding A. Under the action of the ligase, the adapter and the target insert are connected to the sample. Subsequently, the droplet is driven to move to the second magnetic field area 450, and the sample is purified in the second magnetic field area 450 using size screening beads to remove by-products in the sample to obtain a purified connection product, such as Figure 22b In an exemplary embodiment, the byproducts may include free linkers, one end with one linker and none at the other end, neither end with one linker, an empty linker self-ligation, etc.

[0200] (43) Sample PCR enrichment and purification step. The library amplification premix and primers are injected into the library preparation area 400 from the fifth reagent port 405 and the tenth reagent port 410 of the preparation area respectively. The driving unit drives the library amplification premix and primers to mix with the purified connection products, and drives the droplets to move back and forth between the first polymerization temperature zone 420, the second polymerization temperature zone 430 and the third polymerization temperature zone 440, so that the droplets undergo several PCR thermal cycles (for example, about 5 to 13 cycles) in different temperature zones, selectively amplifying DNA fragments that are successfully connected to the linker at both ends to increase the total amount of the DNA library. The size screening bead liquid is injected into the library preparation area 400 from the third reagent port 403 of the preparation area. The driving unit drives the PCR amplified products to mix with the size screening beads and move to the second magnetic field area 450, and the sample is purified in the second magnetic field area 450. The washing buffer is injected into the library preparation area 400 from the ninth reagent port 409 of the preparation area. The driving unit drives the washing buffer to elute the purified sample to obtain the final library. The library quality inspection is performed under the chip and then the library is sequenced. Figure 22c shown.

[0201] In an exemplary embodiment, PCR thermal cycling can employ the following protocol.

[0202]

[0203] An exemplary embodiment of the present disclosure provides a digital microfluidic device. By setting a screening area, a lysis area, a pre-amplification area, and a library preparation area on an active digital microfluidic chip, the screening area is used to screen and enrich rare cells, the lysis area is used to single and lyse rare cells, the pre-amplification area is used to pre-amplify the nucleic acids of rare single cells, and the library preparation area is used to prepare sample libraries after pre-amplification of rare single cells. This device realizes an integrated process of capturing, isolating, and preparing libraries for rare single cells. This process is fully automated and does not require manual operation. This effectively avoids errors introduced by manual operation in the process of building libraries for trace samples, ensures the repeatability and stability of the output library quality, and provides a strong guarantee for subsequent single-cell sequencing.

[0204] Compared with the traditional manual operation library output technology, the digital microfluidic device proposed in the present disclosure, through the cooperation of the digital microfluidic chip with the temperature control device, the magnetic control device and the detection device, does not need to transfer samples between different chambers, avoids the trace loss and rare sample loss caused by the transfer of samples between different chambers, and can realize the integrated process of lossless automatic separation of rare cells and single cell sample library preparation in the digital microfluidic chip. The present disclosure uses the active droplet manipulation function of the active digital microfluidic chip to realize the automatic movement, mixing and separation of samples and reagents, with low sample consumption, fast speed, less manual operation and low cost, and no need for peripheral micropumps, valves and complex pipelines, thereby improving the integration of the system; the active digital microfluidic chip is used to realize the uniform arrangement of single cell droplets, and the detection device uses impedance information to identify and locate rare single cells, with high accuracy of identification and positioning. The present disclosure does not require large-scale detection equipment, and has the characteristics of compact structure, small size, low power consumption and low cost. The present disclosure does not require cumbersome sample pretreatment outside the chip, saving samples and reagents and shortening processing time. The disclosed library preparation process requires no manual operation and is fully automated, avoiding the tedious and error-prone manual library construction process. It directly outputs a library that can be sequenced on a machine, and has good application prospects in early diagnosis of cancer, cancer heterogeneity, embryonic development, and other aspects.

[0205] The present invention utilizes magnetic nanoparticles coupled with specific antibodies to combine with specific antigens on the surface of rare cells to form a rare cell (target cell)-magnetic nanoparticle complex, and utilizes a first magnetic field region formed by a first magnetic control device to adsorb the magnetic nanoparticles on the chip surface, thereby achieving the separation of rare cells from other cells. Compared with traditional single-cell separation technology, it can not only quickly and accurately obtain rare cells, but also will not lose rare cells, will not cause damage to rare cells, and ensure the integrity of rare cells, and does not require manual operation. The separation process is simple, convenient, rapid, and highly specific, with the characteristics of simple operation, short time consumption, and low cost.

[0206] The present disclosure also provides a digital microfluidic driving method, which can utilize the aforementioned digital microfluidic chip, wherein the digital microfluidic chip includes a screening area, a lysis area, a pre-amplification area, and a library preparation area arranged in sequence. In an exemplary embodiment, the driving method may include:

[0207] S1. Screening and enriching rare cells in the screening area;

[0208] S2, singulation and cell lysis of the rare cells after screening and enrichment in the lysis zone;

[0209] S3, pre-amplifying nucleic acids of rare single cells after cell lysis in the pre-amplification zone;

[0210] S4. Preparing a sample library after rare single cell pre-amplification in the library preparation area.

[0211] In an exemplary embodiment, step S1 may include:

[0212] driving the blood sample, magnetic particle droplets, and buffer to mix to form mixed droplets, wherein the mixed droplets contain rare cell-magnetic nanoparticle complexes;

[0213] dispersing the mixed droplet into a plurality of sub-droplets, and capturing the sub-droplets containing the cell-magnetic nanoparticle complexes using a magnetic field;

[0214] The captured daughter droplets are mixed into one enriched droplet.

[0215] In an exemplary embodiment, step S2 may include:

[0216] After the enriched droplets obtained in the screening zone are moved to the lysis zone, the enriched droplets are dispersed into a plurality of sub-droplets, each of which contains only one rare cell-magnetic nanoparticle complex or no rare cell-magnetic nanoparticle complex;

[0217] Multiple daughter droplets are formed into single cell / vacuole arrays, and daughter droplets containing rare cell-magnetic nanoparticle complexes are identified and located;

[0218] The daughter droplets containing the rare cell-magnetic nanoparticle complexes are subjected to a lysis reaction to form single-cell nucleic acid samples.

[0219] In an exemplary embodiment, step S3 may include:

[0220] After the single-cell nucleic acid sample obtained in the lysis zone is moved to the pre-amplification zone, the single-cell nucleic acid sample is fragmented to form a fragmented DNA sample;

[0221] The fragmented DNA sample is pre-amplified to form a rare single-cell pre-amplified nucleic acid sample.

[0222] In an exemplary embodiment, step S4 may include:

[0223] After the rare single cell pre-amplified nucleic acid sample obtained in the pre-amplification zone is moved to the library preparation zone, the rare single cell pre-amplified nucleic acid sample is sequentially end-repaired, and a magnetic field is used to selectively screen out DNA fragments of a desired length;

[0224] The DNA fragments of the required length are sequentially treated with A and adapters to obtain purified ligation products;

[0225] The ligation product is subjected to polymerase chain reaction thermal cycling treatment, followed by purification and elution treatment to obtain a library.

[0226] Although the embodiments disclosed in this disclosure are as described above, the contents described are merely embodiments adopted to facilitate understanding of the disclosure and are not intended to limit the disclosure. Any person skilled in the art to which the disclosure belongs may make any modifications and changes in the form and details of the implementation without departing from the spirit and scope of the disclosure. However, the scope of patent protection of the disclosure shall still be based on the scope defined by the attached claims.

Claims

1. A digital microfluidic chip, comprising a first substrate and a second substrate disposed opposite each other, the first substrate being provided with a plurality of drive regions, at least one of which comprises a drive transistor, a drive electrode, and a storage capacitor, the drive electrode being connected to the drive transistor and the storage capacitor, respectively, the storage capacitor being configured to charge when the drive transistor is on and to maintain a voltage signal on the drive electrode when the drive transistor is off; A plurality of gate lines and a plurality of data lines are provided on the first substrate, and the plurality of gate lines and the plurality of data lines intersect with each other to define a plurality of driving areas, and in at least one driving area, the driving transistor includes at least a first gate electrode, a second gate electrode, a first electrode, and a second electrode, wherein the first gate electrode and the second gate electrode are connected to the gate line, the first electrode is connected to the data line, and the second electrode is connected to the driving electrode; In at least one driving region, the first substrate includes: first base; a first conductive layer disposed on the first substrate, the first conductive layer comprising at least a gate line, a first gate electrode and a second gate electrode, the first gate electrode and the second gate electrode being connected to the gate line respectively; a first insulating layer covering the first conductive layer; a semiconductor layer disposed on a side of the first insulating layer away from the first substrate, the semiconductor layer comprising at least a first active layer and a second active layer, an orthographic projection of the first active layer on the first substrate at least partially overlapping with an orthographic projection of the first gate electrode on the first substrate, and an orthographic projection of the second active layer on the first substrate at least partially overlapping with an orthographic projection of the second gate electrode on the first substrate; a second conductive layer disposed on a side of the semiconductor layer away from the first substrate, the second conductive layer comprising at least a data line, a first electrode, a connecting electrode, and a second electrode, wherein a first end of the first electrode is connected to the data line, a second end of the first electrode and a first end of the connecting electrode are respectively disposed on the first active layer, and a second end of the connecting electrode and a first end of the second electrode are respectively disposed on the second active layer; a second insulating layer covering the second conductive layer; a third conductive layer provided on a side of the second insulating layer away from the first substrate, the third conductive layer comprising at least a capacitor electrode; a third insulating layer covering the third conductive layer, wherein the third insulating layer is provided with a connecting via hole, and the connecting via hole exposes the second electrode; A fourth conductive layer is arranged on a side of the third insulating layer away from the first substrate, the fourth conductive layer at least including a driving electrode, the driving electrode is connected to the second electrode through the connecting via, the orthographic projection of the driving electrode on the first substrate and the orthographic projection of the capacitor electrode on the first substrate at least partially overlap, and the capacitor electrode and the driving electrode constitute the storage capacitor.

2. The digital microfluidic chip according to claim 1, wherein: The capacitor electrode is connected to a system ground signal.

3. The digital microfluidic chip according to claim 1, wherein: A plurality of counter electrodes are provided on the second substrate, and the driving electrodes and the counter electrodes constitute a driving unit for driving the liquid droplets to move.

4. The digital microfluidic chip according to any one of claims 1 to 3, wherein: The first substrate and the second substrate form a processing chamber through a sealant. The processing chamber includes at least a screening area, a lysis area, a pre-amplification area and a library preparation area. The screening area is configured to screen and enrich rare cells. The lysis area is arranged on one side of the screening area and is configured to single and lyse the rare cells after screening and enrichment. The pre-amplification area is arranged on a side of the lysis area away from the screening area and is configured to pre-amplify the nucleic acid of the rare single cell after cell lysis. The library preparation area is arranged on a side of the pre-amplification area away from the screening area and is configured to prepare a sample library after pre-amplification of the rare single cell.

5. The digital microfluidic chip according to claim 4, wherein: The screening area includes multiple driving units, and a first reagent port, a second reagent port, a third reagent port and a fourth reagent port in the screening area respectively arranged in the corner area of the screening area. At least one of the first reagent port, the second reagent port, the third reagent port and the fourth reagent port in the screening area is configured to: receive a whole blood sample, or receive magnetic nanoparticles, or receive a buffer solution, or discharge waste liquid.

6. The digital microfluidic chip according to claim 4, wherein: The screening area includes a first magnetic field region, which includes a plurality of regularly arranged first magnetic regions, and an orthographic projection of at least one first magnetic region on the first substrate includes an orthographic projection of at least one driving unit on the first substrate.

7. The digital microfluidic chip according to claim 4, wherein: The screening area includes multiple driving units, and a first reagent port in the lysis area, a second reagent port in the lysis area, a third reagent port in the lysis area, and a fourth reagent port in the lysis area, respectively arranged in the corner area of the screening area. At least one of the first reagent port in the lysis area, the second reagent port in the lysis area, the third reagent port in the lysis area, and the fourth reagent port in the lysis area is configured to: receive a lysis solution, or receive a stop solution, or receive a buffer solution, or discharge waste liquid.

8. The digital microfluidic chip according to claim 7, wherein: The driving units in the screening area satisfy the following formula: Wherein, θ represents the initial contact angle between the droplet and the hydrophobic surface on the first substrate, H represents the thickness of the digital microfluidic chip, and L represents the size of the driving electrode.

9. The digital microfluidic chip according to claim 8, wherein: The box thickness H of the digital microfluidic chip is ≤19.8 μm, and the size L of the driving electrode is ≤48.5 μm.

10. The digital microfluidic chip according to claim 7, wherein: The driving unit in the screening area is configured to detect impedance signals of single cell packages and vacuoles, where the impedance of the single cell packages includes the resistance of the cytoplasm and the capacitance of the cell membrane that packages the cytoplasm.

11. The digital microfluidic chip according to claim 4, wherein: The pre-amplification zone includes a plurality of drive units, and a first reagent port for the pre-amplification zone, a second reagent port for the pre-amplification zone, a third reagent port for the pre-amplification zone, and a fourth reagent port for the pre-amplification zone, respectively arranged in the corner areas of the pre-amplification zone. At least one of the first reagent port for the pre-amplification zone, the second reagent port for the pre-amplification zone, the third reagent port for the pre-amplification zone, and the fourth reagent port for the pre-amplification zone is configured to: receive a fragmentation enzyme reagent, or receive a pre-amplification reagent, or receive a fragmentation buffer, or discharge waste liquid.

12. The digital microfluidic chip according to claim 4, wherein: The pre-amplification zone includes a plurality of amplification temperature zones with different temperatures, and the distance between adjacent amplification temperature zones is greater than or equal to 1 mm.

13. The digital microfluidic chip according to claim 4, wherein: The library preparation area includes a plurality of driving units, and a first reagent port for the preparation area, a second reagent port for the preparation area, a third reagent port for the preparation area, a fourth reagent port for the preparation area, a fifth reagent port for the preparation area, a sixth reagent port for the preparation area, a seventh reagent port for the preparation area, an eighth reagent port for the preparation area, a ninth reagent port for the preparation area, a tenth reagent port for the preparation area and an eleventh reagent port for the preparation area, respectively arranged in an edge area of the library preparation area; the first reagent port for the preparation area, the second reagent port for the preparation area, the third reagent port for the preparation area, the fourth reagent port for the preparation area and the fifth reagent port for the preparation area are arranged in an edge area on one side of the second direction of the library preparation area, and are arranged in sequence along the first direction, the sixth reagent port for the preparation area, the seventh reagent port for the preparation area, the eighth reagent port for the preparation area, the ninth reagent port for the preparation area, the tenth reagent port for the preparation area and the eleventh reagent port for the preparation area The eighth reagent port of the preparation area, the ninth reagent port of the preparation area, and the tenth reagent port of the preparation area are arranged in the edge area on the side opposite to the second direction of the library preparation area, and are arranged sequentially along the first direction. The eleventh reagent port of the preparation area is arranged in the edge area on the side of the first direction of the library preparation area; at least one of the multiple preparation area reagent ports of the library preparation area is configured to: receive washing bead liquid, or receive end repair master mixed liquid, or receive size screening bead liquid, or receive eluent, or receive library amplification premix liquid, or receive A tracking master mixed liquid, or receive adapter liquid, or receive ligation master mixed liquid, or receive washing buffer, or receive primers, or discharge waste liquid.

14. The digital microfluidic chip according to claim 4, wherein: The library preparation area includes a plurality of polymerization temperature zones with different temperatures, and the distance between adjacent polymerization temperature zones is greater than or equal to 0.5 mm.

15. The digital microfluidic chip according to claim 4, wherein: The library preparation area includes a second magnetic field region, which includes a plurality of regularly arranged second magnetic regions, and the orthographic projection of at least one second magnetic region on the first substrate includes the orthographic projection of at least one driving unit on the first substrate.

16. A digital microfluidic device, wherein: The digital microfluidic chip comprises the digital microfluidic chip according to any one of claims 1 to 15, further comprising a temperature control device, a magnetic control device and a detection device, wherein the temperature control device is configured to generate at least one temperature zone on the digital microfluidic chip, the magnetic control device is configured to generate at least one magnetic field zone on the digital microfluidic chip, the detection device is configured to identify and locate rare cells, and the digital microfluidic chip is configured to sequentially perform screening and enrichment of rare cells, singulation and cell lysis of rare cells, pre-amplification of nucleic acids of rare single cells, and sample library preparation.

17. A driving method for a digital microfluidic chip, applied to the digital microfluidic chip according to any one of claims 1 to 15; the digital microfluidic chip comprises a screening area, a lysis area, a pre-amplification area, and a library preparation area arranged in sequence, the driving method comprising: Screening and enriching rare cells in the screening area; Singulation and cell lysis of the rare cells after screening and enrichment are performed in the lysis zone; Pre-amplification of nucleic acids of rare single cells after cell lysis is performed in the pre-amplification zone; The library preparation area is used to prepare a sample library after rare single cell pre-amplification.

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