Digital micro-fluidic chip and application

Abstract

The invention provides a digital micro-fluidic chip and application, and relates to the technical field of chips. The method comprises the steps that if a driving instruction including address information of an electrode is received through a signal line in a signal line cluster corresponding to a functional area, the electrode indicated by the address information is gated in the functional area according to the address information, and the address information includes a row number and a column number of an operation position in the functional area; in the area positions in the functional area array, one operation position corresponds to at least one electrode; and executing corresponding operation at the operation position of the electrode through the electrode. According to the invention, multi-dimensional addresses can be set for the operation positions in the functional area, so that the same functional area can be reused, the number of the operation positions in the functional area is increased, and the chip has the capability of controlling a huge amount of micro-droplets.

Description

Technical Field

[0001] The present disclosure relates to the field of chip technology, and in particular to a digital microfluidic chip and its application. Background Art

[0002] In many current biomedical testing applications, there is an urgent need for high-throughput, flexible control, integrated and miniaturized droplet manipulation and detection platforms.

[0003] For example, in droplet digital PCR, tens of thousands of target molecule droplets must be analyzed for absolute quantification to obtain statistically significant data. Furthermore, single-cell sequencing requires massively parallel DNA analysis of a large number of single cells. Therefore, many applications require microfluidic platforms capable of controlling massive numbers of droplets. Summary of the Invention

[0004] In view of this, the purpose of the present disclosure is to propose a digital microfluidic chip and its application, which can specifically solve the existing problems.

[0005] Based on the above-mentioned purpose, in the first aspect, the present disclosure proposes a digital microfluidic chip, including a sample operation area, which is a functional area array; the sample operation area is used to receive a driving instruction including address information of an electrode through a signal line in a signal line cluster corresponding to the functional area, and then, according to the address information, select the electrode indicated by the address information in the functional area, the address information including the row number and column number of the operation position in the functional area, and the area position in the functional area array, one operation position corresponds to at least one electrode; through the electrode, the corresponding operation is performed at the operation position where the electrode is located.

[0006] In a second aspect, a DNA amplification method is also provided, which adopts the digital microfluidic chip of the first aspect, wherein the digital microfluidic chip includes a functional area array, and the functional area array includes a sample import and export area, a sample operation area and an interconnection communication area; the method includes: introducing a DNA template sample into the sample import and export area through a sample liquid inlet of the sample import and export area, receiving a driving instruction through a signal line in a signal line cluster in the sample import and export area to drive the electrodes on the sample transport route in the sample import and export area to distribute the DNA template sample to the corresponding operation position; transporting auxiliary materials required for DNA amplification to the DNA amplification area in the sample operation area, mixing the auxiliary materials and the DNA template sample at each operation position in the DNA amplification area, and performing DNA amplification.

[0007] In a third aspect, a single-cell sequencing method is also provided, using the digital microfluidic chip as described in claim 1, the digital microfluidic chip comprising a functional area array, the functional area array comprising a sample import and export area, a sample operation area and an interconnection communication area; the method comprising: introducing a sample for single-cell lysis into the sample import and export area through a sample inlet of the sample import and export area, receiving a drive instruction through a signal line in a signal line cluster of the sample import and export area to drive electrodes on a sample transport route in the sample import and export area to distribute the sample to a corresponding operation position, wherein the sample comprises a cell suspension, a solution containing a lysis reagent and a solution containing an amplification reagent; transporting the sample to the sample operation area, generating single-cell droplets in a first sub-area of ​​the sample operation area, mixing the single-cell droplets and the solution containing the lysis reagent in a second sub-area of ​​the sample operation area to lyse the single-cell droplets, mixing the lysed droplets with a solution containing the amplification reagent in the second sub-area to perform DNA amplification, and sequencing the amplification results in a third sub-area of ​​the sample operation area.

[0008] In a fourth aspect, an electronic device is also provided, comprising a memory, a processor, and a computer program stored in the memory and executable on the processor, wherein the processor executes the computer program to implement the method described in the first aspect.

[0009] In a fifth aspect, a computer-readable storage medium is provided, on which a computer program is stored, and the program is executed by a processor to implement any method described in the first aspect.

[0010] In general, the present disclosure has at least the following beneficial effects: multi-dimensional addresses can be set for the operating positions in the functional area, so that the same functional area can be reused to achieve an increase in the number of operating positions in the functional area, thereby enabling the chip to have the ability to control a large number of microdroplets. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] In the accompanying drawings, unless otherwise specified, the same reference numerals throughout the multiple drawings represent the same or similar components or elements. These drawings are not necessarily drawn to scale. It should be understood that these drawings only depict some embodiments according to the present disclosure and should not be regarded as limiting the scope of the present disclosure.

[0012] Figure 1a shows an overall framework diagram of chip electrode array control according to an embodiment of the present disclosure;

[0013] Figure 1b Another chip electrode array control overall framework diagram according to an embodiment of the present disclosure is shown;

[0014] Figure 1c Schematic diagram showing various regions in a digital microfluidic chip according to an embodiment of the present disclosure;

[0015] Figure 1d A schematic diagram of signal line control for each functional area of ​​a dielectric wetting chip according to an embodiment of the present disclosure is shown;

[0016] Figure 2 A flow chart of a DNA amplification method according to an embodiment of the present disclosure is shown;

[0017] Figure 3a A chip structure diagram based on the overall chip architecture according to an embodiment of the present disclosure is shown;

[0018] Figure 3b A chip structure diagram based on the overall chip architecture according to an embodiment of the present disclosure is shown;

[0019] Figure 3c Another schematic diagram showing various regions in a digital microfluidic chip according to an embodiment of the present disclosure;

[0020] Figure 4 A schematic diagram of a DNA amplification device according to an embodiment of the present disclosure is shown;

[0021] Figure 5 A schematic structural diagram of an electronic device provided by an embodiment of the present disclosure is shown;

[0022] Figure 6 A schematic diagram of a storage medium provided by an embodiment of the present disclosure is shown. DETAILED DESCRIPTION

[0023] The present disclosure will be further described in detail below with reference to the accompanying drawings and examples. It should be understood that the specific embodiments described herein are intended only to illustrate the relevant invention and are not intended to limit the invention. It should also be noted that, for ease of description, only portions relevant to the relevant invention are shown in the accompanying drawings.

[0024] It should be noted that, in the absence of conflict, the embodiments and features of the embodiments in the present disclosure may be combined with each other. The present disclosure will be described in detail below with reference to the accompanying drawings and in combination with the embodiments.

[0025] Figure 1a The working flow diagram of the digital microfluidic chip of the present disclosure is shown. In the embodiment of the present disclosure, the chip includes a sample operation area, which is a functional area array.

[0026] Step S101: If a driving instruction including the address information of the electrode is received through the signal line in the signal line cluster corresponding to the functional area, the electrode indicated by the address information is selected in the functional area according to the address information. The address information includes the row number and column number of the electrode in the functional area, and the regional position in the functional area array. One operating position corresponds to at least one electrode.

[0027] In this embodiment, the digital microfluidic chip can, upon receiving a driving instruction of address information via a signal line in a signal line cluster corresponding to a functional area, select an electrode indicated by the address information in the functional area according to the address information. The driving instruction can be an instruction for a trigger operation.

[0028] The address information indicates the row and column number of the electrodes. Each operating position corresponds to at least one electrode. Each operating position includes at least one pixel area, the smallest operating unit. There is a one-to-one relationship between pixel areas and electrodes: one pixel area corresponds to one electrode.

[0029] Electrode states include on and off. When an electrode is on, it can execute drive instructions to perform the corresponding operation at the electrode's corresponding operating position. When an electrode is off, it does not execute any instructions, meaning the chip will not perform any operation at that operating position.

[0030] The address information may include not only the row number and column number inside the functional area, but also the position of the functional area in the functional area array.

[0031] The address information includes the row number and column number of the operation position in the functional area, as well as the area position. Therefore, the address information is at least three-dimensional information.

[0032] The sample manipulation area is also known as the sample operation area. This area can perform various operations, such as reaction operations, on samples in the operation positions within the sample manipulation area. For example, the operation can be heating, mixing, reaction operations, etc. For example, the reaction here can refer to cell lysis, DNA amplification reaction, etc.

[0033] In the present application, samples can be introduced into the sample operation area and waste liquid can be discharged manually or in other ways.

[0034] Step S102: performing a corresponding operation at the operating position of the electrode via the electrode.

[0035] In this embodiment, the digital microfluidic chip can perform operations corresponding to the operating positions at the operating positions of the electrodes through the electrodes.

[0036] Each operating position has a corresponding operation. For example, driving the electrode at the operating position can perform the operation. In the sample operation area, the density of the electrodes is relatively high, specifically, for example, greater than a preset density.

[0037] This embodiment can set multi-dimensional addresses for the operating positions in the functional area, so that the same functional area can be reused to achieve an increase in the number of operating positions in the functional area, thereby enabling the chip to have the ability to control a large number of microdroplets.

[0038] like Figure 1a As shown in the figure, the overall framework of the chip electrode array control is shown. The electrode arrays in all areas are mounted on the bus (row signal lines, i.e., scan lines, column signal lines, i.e., data lines). The sample import and export areas can be set to the same area or different areas according to application requirements. The sample operation area is divided into several areas. The interconnection and communication area is considered as a functional area as a whole. Electrode areas with similar functional types reuse the same bus array. It is worth noting that Figure 1a The number of row signal lines and column signal lines in each area, such as the sample import and export area, may be equal or unequal. i and A h , line a j and a k The relationship between the subscripts i and h, j and k is not unique. The line B in the sample operation area m and B x , line b n and b y The size relationship between the subscript m and x, n and y is also not unique, which means that the size and number of individual electrodes in different electrode array areas are not unique. Figure 1a The most general case is shown in the figure, i≠h, j≠k, m≠x, n≠y. This case means that the number of single electrodes in the sample introduction electrode area and the sample extraction electrode area is different, and the number of single electrodes in each area of ​​the sample operation area is also different. Figure 1b Another special case is shown in the figure, i = h, j = k, m = x, n = y. This case means that the number of single electrodes in the sample input electrode area and the sample output electrode area is the same, and the number of single electrodes in each area of ​​the sample operation area is the same. When designing a bus array, the signal lines with the same number of single electrodes in the reused area are serially multiplexed, and signal lines are reserved to connect the extra electrodes compared to other areas. For example Figure 1a and Figure 1b There are N sample operation areas (the 1st to the Nth), and each area has M electrodes (x columns and y rows, and the first electrode in each area shares the same row signal line and column signal line). Only x+y signal lines are needed to drive N*M electrodes, and so on. The same is true for other functional areas, so that the number of electrodes can be increased while saving signal lines.

[0039] In some optional implementations of any embodiment of the present disclosure, if the address information is three-dimensional information, the area position is the element number of the functional area in the functional area array; if the address information is four-dimensional information, the area position is the row number and column number of the functional area in the functional area array.

[0040] In these optional implementations, in the sample operation area, the address information can be three-dimensional information, in which case the area location is the element number of the functional area in the functional area array. That is, the three-dimensional information includes the element number, as well as the row and column numbers of the operation position within the functional area. That is, the functional area array is a functional area matrix, and the element number is the position number of the functional area within the functional area matrix. Alternatively, the address information can be four-dimensional information, in which case the area location is the row and column number of the functional area in the functional area array. That is, the four-dimensional information includes the row and column numbers of the functional area, as well as the row and column numbers of the operation position within the functional area.

[0041] These implementation methods can expand the number of operating positions within the functional area through three-dimensional addresses and four-dimensional addresses.

[0042] In some optional implementations of any embodiment of the present disclosure, the digital microfluidic chip also includes a sample import and export area and an interconnection and communication area; the sample import and export area is used to import samples into the operating position or export waste liquid from the operating position; the interconnection and communication area is used to transport the sample to the destination operating position through a preset transport path and a preset destination operating position for the sample, and the interconnection and communication area is located between different sample operating areas.

[0043] In these optional implementations, the sample import / export area can be used to import samples into the operating position or to export waste liquid from the operating position. Specifically, the sample import / export area can include a sample import sub-area and a sample export sub-area. The sample import sub-area can be used to inject samples, that is, to introduce samples from the sample inlet into the sample import sub-area. The sample export sub-area can be used to export waste liquid from the operating position.

[0044] The interconnected communication area is used to transport samples to the target operation location via a preset transport path and a preset target operation location. The interconnected communication area is located between different sample operation areas. The target operation location refers to the operation location where the operation is performed. The interconnected communication area exists between different functional areas in the sample operation area, and these areas can serve as physical isolation.

[0045] These implementation methods can import and export samples to and from the chip through the sample import and export area, and can transport the samples to the target operation position through the interconnected communication area, and achieve physical isolation between different functional areas.

[0046] In some optional application scenarios of these implementations, the method includes: each operating position in the functional area includes at least one pixel area, each pixel area contains a switching device and an electrode, and each pixel area is obtained by arranging row signal lines and column signal lines through insulation intersection.

[0047] In these application scenarios, each operating location in the functional area array is a pixel area. This pixel area is the smallest unit within the functional area. A pixel area is controlled by switches and electrodes within it. A pixel area is electrically connected to a row signal line via one terminal of a switching device and to a column signal line via the other terminal of the switching device.

[0048] The pixel area is formed by the row signal lines and the column signal lines being arranged through insulation intersection. Specifically, a plurality of row signal lines and a plurality of column signal lines are defined by insulation intersection to form a plurality of pixel units arranged in an array.

[0049] These application scenarios can form multiple pixel areas in the sample operation area through row signal lines and column signal lines, thereby helping to multiplex the same functional area and further expand the number of operating positions in the functional area.

[0050] In some optional application scenarios of these implementations, when the digital microfluidic chip is used for DNA amplification, the sample operation area includes a PCR primer storage area, a DNA polymerase storage area, a dNTPs storage area, a fluorescent probe storage area, a DNA amplification area, and a fluorescence measurement area; when the digital microfluidic chip is used for single-cell sequencing, the sample operation area includes a single-cell droplet generation area, a reagent storage multiplexing area, a single-cell lysis area, a single-cell library preparation area, and a pooled sequencing area.

[0051] In these application scenarios, the PCR primer storage area in the sample operation area can be used to store PCR primers. The DNA polymerase storage area can be used to store DNA polymerase. The dNTPs storage area can be used to store dNTPs for PCR. The fluorescent probe storage area is used to quickly identify specific nucleic acid fragments using the fluorescent probe method. The DNA amplification area can be used to amplify DNA. Specifically, the DNA amplification area may include: a sample storage area, a denaturation area, and an annealing / extension area. The fluorescence measurement area can be used to measure fluorescence. In this area, each sample to be tested, that is, a droplet, can be tested one by one. If the test result is a fluorescent signal, the droplet is determined to be a positive droplet, otherwise it is determined to be a negative droplet.

[0052] like Figure 1cAs shown, the figure shows the sample introduction sub-area A, PCR primer storage area B, DNA polymerase storage area C, dNTPs storage area D, fluorescent probe storage area E, DNA amplification area including sample liquid storage area F, denaturation area G, annealing / extension area H, fluorescence measurement area I, and sample derivation sub-area J.

[0053] like Figure 1d As shown, the figure shows a schematic diagram of signal line control of each functional area of ​​the dielectric wetting chip of this patent, in which the address line is the signal line.

[0054] Figure 2 The method for amplifying DNA according to an embodiment of the present disclosure is shown. Figure 2 As shown, the method uses the above-mentioned digital microfluidic chip, which includes a functional area array, and the functional area array includes a sample import and export area, a sample operation area and an interconnection communication area. The method includes: step S201, introducing a DNA template sample into the sample import and export area through a sample liquid inlet of the sample import and export area, receiving a driving instruction through a signal line in a signal line cluster of the sample import and export area to drive electrodes on a sample transport route in the sample import and export area to distribute the DNA template sample to a corresponding operation position; step S202, transporting auxiliary materials required for DNA amplification to a DNA amplification area in the sample operation area, mixing the auxiliary materials and the DNA template sample at each operation position in the DNA amplification area, and performing DNA amplification.

[0055] In this embodiment, the DNA amplification method may include the following steps: (1) loading and storing each sample droplet: in the serial control mode of the chip, each sample droplet is loaded into the sample introduction sub-area through the sample inlet, and then the electrodes on the pre-set sample droplet transport route are driven in sequence, thereby transporting each sample droplet to the designated position corresponding to the sample droplet in the sample storage area. The sample droplet includes a DNA template. (2) preparing microdroplets: in the parallel control mode of the chip, the sample operation area where each sample droplet is located is driven at multiple levels, thereby dividing the large droplet containing the DNA template into several microdroplets containing the DNA template that are suitable for the size of the electrode. (3) performing PCR reaction: each microdroplet containing the DNA template is equivalent to an independent PCR microreaction chamber. In the serial mode of the chip, the electrodes in the sample storage area corresponding to each sample and the electrodes on the droplet designated movement route are driven in sequence, thereby transporting the auxiliary materials required for PCR amplification: primers, DNA polymerase and dNTPS droplets, to the PCR amplification area. In the chip's parallel mode, the electrodes in the droplet mixing area of ​​the PCR amplification zone are driven, causing the aforementioned droplets to merge and mix sequentially with microdroplets containing the DNA template. The mixed droplets are then heated and denatured in the denaturation zone and cooled, annealed, and extended in the annealing / extension zone to amplify the DNA template and accumulate fluorescence signals. The final step is detection and analysis. After the PCR amplification reaction is complete, each droplet is individually detected in the fluorescence measurement area of ​​the chip. Droplets that emit a fluorescent signal are determined to be 1 (marked as positive droplets), while those that do not are determined to be 0 (marked as negative droplets). The starting copy number or concentration of the gene to be tested can then be determined based on the Poisson distribution principle and the number and proportion of positive droplets.

[0056] like Figure 3a and 3b As shown in the figure, the EWOD-DMF chip structure diagram based on the overall chip architecture is shown. Figure 3a Area ① is the sample introduction sub-area, area ② is the sample export sub-area, area ③ is the 12 sample operation areas, and area ④ is the interconnection communication area. The backs of the electrodes in each major electrode area are connected to the switch device. The electrode array area on the chip is connected to the switch device through the row signal line and the column signal line to lead out each electrode, and is electrically connected to the peripheral hardware drive circuit through the interface set by the chip. The main control circuit is programmed and the corresponding control algorithm is used to customize different working modes for the digital microfluidic chip. Figure 3a The size, number and position of the three electrode regions can be designed according to actual needs. According to different application requirements and scenarios, the layout design of the EWOD-DMF chip based on the present invention is not unique. Figure 3b Another alternative is shown, Figure 3bArea ① is the sample import and export area, area ③ is the 12 sample operation area, and area ④ is the interconnection communication area.

[0057] as follows Figure 3c The chip structure shown is divided into the following major areas: sample introduction electrode area A, single-cell droplet generation area B, reagent reservoir multiplexing area C, single-cell lysis area D, single-cell library preparation area (DNA culture and amplification area E, DNA fragmentation and labeling area F), and pooled sequencing area G. The reagents mentioned above are lysis reagents and amplification reagents. The single-cell library preparation area includes the DNA culture and amplification area E and the DNA fragmentation and labeling area F.

[0058] After the digital microfluidic system based on dielectric wetting is assembled, the first step is to load and store each sample droplet. Under the serial control mode of the chip, each sample droplet is loaded into the sample introduction sub-area through the sample inlet, and then the electrodes on the pre-set sample droplet transport route are driven in sequence, thereby transporting each sample droplet to the designated position of each sample storage area in the sample operation area. The second step is to prepare microdroplets. Under the parallel control mode of the chip, the single-cell droplet generation area B (i.e., the first sub-area) in the sample operation area where each sample droplet is located is driven at multiple levels to process the cell suspension into single-cell droplets. In the single-cell lysis area D (i.e., the second sub-area), the single-cell droplets, the solution containing the lysis reagent, and the solution containing the amplification reagent are split into lysis reagent droplets and amplification reagent droplets of appropriate sizes respectively. The third step is to prepare the single-cell library (i.e., the third sub-area) under the serial mode of the chip. The electrodes in the droplet storage area of ​​the corresponding sample and the electrodes on the designated droplet movement route are driven in sequence to transport the lysis reagent droplets and single-cell droplets to the single-cell lysis area in sequence, and the electrodes in the single-cell lysis area are driven in the parallel control mode of the chip. In the single-cell library preparation area, droplets containing single cells are mixed with droplets containing lysis reagent in the single-cell lysis area by operating the droplet matrix. Each mixed micro-droplet on the chip is equivalent to a micro-reaction chamber. These droplets are cultured to lyse the single cells, and the DNA of each cell is retained in its own micro-droplet. In the same area, the droplets after cell lysis are merged with the amplification reagent droplets and cultured for DNA amplification; similarly, the micro-droplets after DNA amplification are transported to the DNA fragmentation and labeling area and merged with the droplets containing the reagent to fragment the DNA and add adapters (Nextera). The final step is pooled sequencing. Under the chip's serial control mode, the droplets from the previous step are transported to the next area and merged with the droplets containing barcodes. The barcodes are then attached to the fragmented DNA molecules within each microdroplet using polymerase chain reaction (PCR). Finally, the droplets are broken, sequencing adapters are added, and sequencing and data analysis are performed. By integrating a complete set of single-cell sequencing workflows into a digital microfluidic chip based on this patent, similar, non-interfering steps can be reused in the same area at different time intervals, significantly compressing the detection space. Furthermore, the different granularity of the workflow provides refined control of large-scale electrode arrays, which not only increases the throughput of cells to be tested but also shortens the operation time, providing a new solution for the development of single-cell sequencing technology based on digital microfluidics.

[0059] An embodiment of the present disclosure provides a DNA amplification device, which is used to perform the DNA amplification method described in the above embodiment and adopts the above-mentioned digital microfluidic chip, wherein the digital microfluidic chip includes a functional area array, and the functional area array includes a sample import and export area, a sample operation area, and an interconnection and communication area. The above-mentioned device includes: a first unit 401, which is used to introduce a DNA template sample into the sample import and export area through a sample liquid inlet of the sample import and export area, receive a drive instruction through a signal line in a signal line cluster in the sample import and export area, and drive electrodes on the sample transport route in the sample import and export area to distribute the DNA template sample to a corresponding operation position; a second unit 402, which is used to transport auxiliary materials required for DNA amplification to the DNA amplification area in the sample operation area, mix the auxiliary materials and the DNA template sample at each operation position in the DNA amplification area, and perform DNA amplification.

[0060] The present disclosure also provides a single-cell sequencing method, which uses the above-mentioned digital microfluidic chip, wherein the digital microfluidic chip includes a functional area array, and the functional area array includes a sample import and export area, a sample operation area and an interconnection and communication area; the above-mentioned method includes: introducing a sample for single-cell lysis into the sample import and export area through a sample inlet of the sample import and export area, receiving a driving instruction through a signal line in a signal line cluster of the sample import and export area to drive electrodes on a sample transport route in the sample import and export area to distribute the sample to a corresponding operation position, wherein the sample includes a cell suspension, a solution containing a lysis reagent and a solution containing an amplification reagent; transporting the sample to the sample operation area, generating single-cell droplets in a first sub-area of ​​the sample operation area, mixing the single-cell droplets with a solution containing a lysis reagent in a second sub-area of ​​the sample operation area to lyse the single-cell droplets, mixing the lysed droplets with a solution containing an amplification reagent in the second sub-area to perform DNA amplification, and sequencing the amplification results in a third sub-area of ​​the sample operation area.

[0061] In this embodiment, the digital microfluidic chip can be specifically designed to achieve efficient, high-throughput single-cell sequencing technology by utilizing dedicated and isolated areas. It integrates single-cell sorting and subsequent operations, parallel automated operation of picoliter droplets, and optical detection functions, providing a solution for efficient single-cell sorting and operation. The steps for implementing single-cell sequencing technology on a digital microfluidic chip are as follows: Figure 3cAs shown, the chip includes single-cell sample droplet sorting and packaging, single-cell lysis and library preparation, and pooled sequencing. A designated area of ​​the chip is dedicated to reagent and sample loading, using multiple independent inlets to prevent reagent contamination. A "one-to-two" approach is used to generate individual droplets in the single-cell droplet generation zone. The resulting droplet consists of a single droplet controlled by two electrodes. For example, 64 droplets can be automatically generated from a cell-containing suspension in the single-cell droplet generation zone through six steps consisting of a splitting operation. This "one-to-two" approach efficiently and quickly splits a large droplet into multiple smaller droplets, allowing the number of individual droplets to grow exponentially, forming a regularly arranged droplet matrix. This approach is ideal for high-throughput, large-scale single-cell droplet generation and sorting. Droplets containing single cells are identified from fluorescence microscopy images, and target droplets containing single cells are selected based on research needs. Droplets containing single cells are then freely moved in a directed manner. The cells can then be moved to a free electrode area where additional single-cell-based manipulations can be performed, or the entire chip system can be placed in a cell culture incubator to achieve cell expansion in single-cell droplets.

[0062] An embodiment of the present disclosure provides a single-cell sequencing device, which uses the digital microfluidic chip as described above, wherein the digital microfluidic chip includes a functional area array, the functional area array including a sample import and export area, a sample operation area, and an interconnection and communication area; the device includes: a third unit, for introducing a sample for single-cell lysis into the sample import and export area through a sample inlet of the sample import and export area, receiving a drive instruction through a signal line in a signal line cluster of the sample import and export area to drive electrodes on a sample transport route in the sample import and export area to distribute the sample to a corresponding operation position, wherein the sample includes a cell suspension, a solution containing a lysis reagent, and a solution containing an amplification reagent; a fourth unit, for transporting the sample to the sample operation area, generating single-cell droplets in a first sub-area of ​​the sample operation area, mixing the single-cell droplets with a solution containing a lysis reagent in a second sub-area of ​​the sample operation area to lyse the single-cell droplets, mixing the lysed droplets with a solution containing an amplification reagent in the second sub-area to perform DNA amplification, and sequencing the amplification results in a third sub-area of ​​the sample operation area.

[0063] The DNA amplification device provided in the above-mentioned embodiment of the present disclosure and the DNA amplification method provided in the embodiment of the present disclosure are based on the same inventive concept and have the same beneficial effects as the methods adopted, run or implemented by the application programs stored therein.

[0064] The present disclosure also provides an electronic device corresponding to the DNA amplification method provided in the above embodiment to perform the above DNA amplification method.

[0065] Please refer to Figure 5 , which shows a schematic diagram of an electronic device provided by some embodiments of the present disclosure. Figure 5 As shown, the electronic device 50 includes: a processor 500, a memory 501, a bus 502 and a communication interface 503, and the processor 500, the communication interface 503 and the memory 501 are connected via the bus 502; the memory 501 stores a computer program that can be run on the processor 500, and when the processor 500 runs the computer program, it executes the method provided in any of the aforementioned embodiments of the present disclosure.

[0066] The memory 501 may include high-speed random access memory (RAM) and may also include non-volatile memory, such as at least one disk storage. The system network element and at least one other network element are connected via at least one communication interface 503 (which may be wired or wireless), and may use the Internet, a wide area network, a local area network, a metropolitan area network, or the like.

[0067] Bus 502 can be an ISA bus, a PCI bus, or an EISA bus. The bus can be divided into an address bus, a data bus, a control bus, etc. Memory 501 is used to store programs, and processor 500 executes the programs upon receiving execution instructions. The DNA amplification method disclosed in any of the aforementioned embodiments of the present disclosure can be applied to or implemented by processor 500.

[0068] The processor 500 may be an integrated circuit chip with signal processing capabilities. During implementation, each step of the above method may be completed by hardware integrated logic circuits or software instructions in the processor 500. The processor 500 may be a general-purpose processor, including a central processing unit (CPU), a network processor (NP), etc.; it may also be a digital signal processor (DSP), an application-specific integrated circuit (ASIC), a field-programmable gate array (FPGA), or other programmable logic devices, discrete gate or transistor logic devices, or discrete hardware components. It may implement or execute the various methods, steps, and logic block diagrams disclosed in the embodiments of the present disclosure. The general-purpose processor may be a microprocessor or any conventional processor. The steps of the method disclosed in conjunction with the embodiments of the present disclosure may be directly implemented and executed by a hardware decoding processor, or by a combination of hardware and software modules in the decoding processor. The software module may be located in a storage medium mature in the art, such as random access memory, flash memory, read-only memory, programmable read-only memory, electrically erasable programmable memory, registers, etc. The storage medium is located in the memory 501 , and the processor 500 reads the information in the memory 501 and completes the steps of the above method in combination with its hardware.

[0069] The electronic device provided by the embodiment of the present disclosure and the DNA amplification method provided by the embodiment of the present disclosure are based on the same inventive concept and have the same beneficial effects as the methods adopted, operated or implemented by them.

[0070] The single-cell sequencing device provided in the above-mentioned embodiment of the present disclosure and the single-cell sequencing method provided in the embodiment of the present disclosure are based on the same inventive concept and have the same beneficial effects as the methods adopted, run or implemented by the application programs stored therein.

[0071] The present disclosure also provides an electronic device corresponding to the single-cell sequencing method provided in the above embodiment to perform the above single-cell sequencing method. This disclosure is not limited to the embodiment.

[0072] The present disclosure also provides a computer-readable storage medium corresponding to the DNA amplification method provided in the above embodiment. Figure 6 The computer-readable storage medium shown is a CD 60 on which a computer program (ie, a program product) is stored. When the computer program is run by a processor, the DNA amplification method provided by any of the aforementioned embodiments is executed.

[0073] It should be noted that examples of the computer-readable storage medium may also include, but are not limited to, phase change memory (PRAM), static random access memory (SRAM), dynamic random access memory (DRAM), other types of random access memory (RAM), read-only memory (ROM), electrically erasable programmable read-only memory (EEPROM), flash memory or other optical or magnetic storage media, which are not listed here one by one.

[0074] The computer-readable storage medium provided by the above-mentioned embodiment of the present disclosure and the DNA amplification method provided by the embodiment of the present disclosure are based on the same inventive concept and have the same beneficial effects as the method adopted, run or implemented by the application program stored therein.

[0075] The presently disclosed embodiments also provide a computer-readable storage medium corresponding to the single-cell sequencing method provided in the aforementioned embodiments. The computer-readable storage medium is a CD on which a computer program (i.e., a program product) is stored. When the computer program is executed by a processor, it executes the single-cell sequencing method provided in any of the aforementioned embodiments.

[0076] It should be noted that:

[0077] In the above text, the terms "comprises", "comprising" or any other variants thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device comprising a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or device. In the absence of further restrictions, an element defined by the statement "comprising a ..." does not exclude the presence of other identical elements in the process, method, article or device comprising the element. In addition, it should be noted that the scope of the methods and devices in the embodiments of the present disclosure is not limited to performing functions in the order shown or discussed, and may also include performing functions in a substantially simultaneous manner or in the opposite order according to the functions involved. For example, the described method may be performed in an order different from that described, and various steps may also be added, omitted, or combined. In addition, the features described with reference to certain examples may be combined in other examples.

[0078] Through the description of the above embodiments, those skilled in the art can clearly understand that the above embodiment methods can be implemented by means of software plus the necessary general hardware platform, and of course can also be implemented by hardware, but in many cases the former is a better embodiment. Based on this understanding, the technical solution of the present disclosure, or the part that contributes to the prior art, can be embodied in the form of a software product, which is stored in a storage medium (such as ROM / RAM, magnetic disk, optical disk), and includes a number of instructions for enabling a terminal (which can be a mobile phone, computer, server, air conditioner, or network device, etc.) to execute the methods described in each embodiment of the present disclosure.

[0079] The embodiments of the present disclosure are described above in conjunction with the accompanying drawings, which are only specific implementation methods of the present disclosure. However, the present disclosure is not limited to the above-mentioned specific implementation methods. The above-mentioned specific implementation methods are merely illustrative and not restrictive. Under the guidance of the present disclosure, ordinary technicians in this field can also make many forms without departing from the scope of protection of the purpose of the present disclosure and the claims, which are all within the protection of the present disclosure.

Claims

1. A digital microfluidic chip, characterized in that: It includes a sample operation area, which is a functional area array; The sample operation area is used to select the electrode indicated by the address information in the functional area according to the address information if a driving instruction including the address information of the electrode is received through the signal line in the signal line cluster corresponding to the functional area. The address information includes the row number and column number of the operation position in the functional area, and the area position in the functional area array. One operation position corresponds to at least one electrode; through the electrode, the corresponding operation is performed at the operation position where the electrode is located.

2. The chip according to claim 1, characterized in that If the address information is three-dimensional information, the region position is the element number of the functional area in the functional area array; If the address information is four-dimensional information, the region position is the row number and column number of the functional area in the functional area array.

3. The chip according to claim 1, characterized in that The digital microfluidic chip also includes a sample import and export area and an interconnection communication area; The sample import and export area is used to import samples into the operating position or export waste liquid from the operating position; The interconnected communication area is used to transport the sample to the target operation position through a preset transportation path and a preset target operation position for the sample. The interconnected communication area is located between different sample operation areas.

4. The chip according to claim 1, characterized in that A first cluster of signal lines and a second cluster of signal lines are provided, wherein the first cluster of signal lines is used to gate the sample introduction and export areas, and the second cluster of signal lines is used to gate the electrodes in the sample introduction and export areas; The interconnection communication area is provided with a third cluster of signal lines and a fourth cluster of signal lines. The third cluster of signal lines is used for selecting different functional areas, and the fourth cluster of signal lines is used for selecting different electrodes in the functional areas.

5. The chip according to claim 4, characterized in that The method comprises: Each operating position in the functional area includes at least one pixel area, each pixel area includes a switch device and an electrode, and each pixel area is obtained by arranging row signal lines and column signal lines through insulation intersection.

6. The chip according to claim 4, characterized in that In the case where the digital microfluidic chip is used for DNA amplification, the sample operation area includes a PCR primer storage area, a DNA polymerase storage area, a dNTPs storage area, a fluorescent probe storage area, a DNA amplification area, and a fluorescence measurement area; When the digital microfluidic chip is used for single-cell sequencing, the sample operation area includes a single-cell droplet generation area, a reagent storage multiplexing area, a single-cell lysis area, a single-cell library preparation area, and a pooled sequencing area.

7. A method for amplifying DNA, characterized in that: The digital microfluidic chip according to claim 1 is used, wherein the digital microfluidic chip comprises a functional area array, wherein the functional area array comprises a sample import and export area, a sample operation area, and an interconnection communication area; The method comprises: A DNA template sample is introduced into the sample import / export area through a sample liquid inlet of the sample import / export area, and a driving instruction is received through a signal line in a signal line cluster of the sample import / export area to drive electrodes on a sample transport route in the sample import / export area to distribute the DNA template sample to a corresponding operation position; The auxiliary materials required for DNA amplification are transported to the DNA amplification area in the sample operation area, and the auxiliary materials and the DNA template sample are mixed at each operation position in the DNA amplification area, and DNA amplification is performed.

8. A single-cell sequencing method, characterized in that: The digital microfluidic chip according to claim 1 is used, wherein the digital microfluidic chip comprises a functional area array, wherein the functional area array comprises a sample import and export area, a sample operation area, and an interconnection communication area; The method comprises: A sample for single cell lysis is introduced into the sample import / export area through a sample liquid inlet of the sample import / export area, and a driving instruction is received through a signal line in a signal line cluster of the sample import / export area to drive electrodes on a sample transport route in the sample import / export area to distribute the sample to a corresponding operating position, wherein the sample includes a cell suspension, a solution containing a lysis reagent, and a solution containing an amplification reagent; The sample is transported to the sample operation area, single-cell droplets are generated in a first sub-area of ​​the sample operation area, and the single-cell droplets are mixed with a solution containing a lysis reagent in a second sub-area of ​​the sample operation area to lyse the single-cell droplets. In the second sub-area, the lysed droplets are mixed with a solution containing an amplification reagent to perform DNA amplification, and the amplification results are sequenced in a third sub-area of ​​the sample operation area.

9. An electronic device comprising a memory, a processor, and a computer program stored in the memory and executable on the processor, wherein: The processor runs the computer program to implement the method according to any one of claims 7 to 8.

10. A computer-readable storage medium having a computer program stored thereon, characterized in that: The program is executed by a processor to implement the method according to any one of claims 7 to 8.

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