Digital micro-fluidic chip and application thereof

Abstract

The invention relates to a digital micro-fluidic chip and application thereof, and the digital micro-fluidic chip comprises a first substrate (10) which comprises a first substrate (11), and the first substrate (11) is provided with a sample introduction hole (12); one side of the intermediate layer (20) is connected with the first substrate (10), the intermediate layer (20) comprises a flow channel communicated with the sample introduction hole (12), and the flow channel sequentially comprises a liquid drop generation area (21), a liquid drop deceleration area (22) and a digital micro-fluidic area (23); the droplet generation area (21) is configured to generate micro-droplets; the droplet deceleration region (22) is configured to decelerate the microdroplets; the second substrate (30) is connected with one side, far away from the first substrate (10), of the interposer (20); the second substrate (30) comprises a driving electrode (31), and the driving electrode (31) is configured to drive the micro-droplets in the interposer (20) to move.

Description

Digital microfluidic chip and its application Technical Field

[0001] The embodiments of the present application relate to, but are not limited to, the field of biomedical technology, and in particular to a digital microfluidic chip and its use. Background Art

[0002] In recent years, digital microfluidic chip technology has been widely used in biology, chemistry, medicine and other fields due to its small size, low power consumption, low cost, small amount of samples and reagents required, ability to achieve individual and precise control of droplets, short detection time, high sensitivity, and easy integration with other devices.

[0003] With the advancement of medical technology, monoclonal antibody drugs have become an effective treatment for a wide range of diseases. For example, they have become a key drug type in tumor immunotherapy. Science magazine's 2021 Top Ten Scientific Breakthroughs recognized monoclonal antibody drugs as demonstrating remarkable effectiveness against the novel coronavirus and other life-threatening pathogens, including HIV and malaria parasites. Global sales of monoclonal antibody drugs exceeded $200 billion in 2021, maintaining growth rates exceeding 10% for eight consecutive years.

[0004] Based on this, some countries have developed fully integrated instrument platforms for screening monoclonal antibodies, such as Berkeley Light's Beacon platform and Molecular Devices' ClonePix platform. These platforms can be used for the discovery and development of targets for cancer and the novel coronavirus, increasing the success rate of positive cell cloning and shortening the R&D cycle. Therefore, fully integrated single-cell cloning platforms have become a key core instrument platform for innovative biopharmaceutical R&D. However, the high price of such instruments, often reaching tens of millions of yuan, is a major factor hindering the promotion and development of monoclonal antibody technology.

[0005] Summary of the Invention

[0006] The following is an overview of the subject matter described in detail herein. This overview is not intended to limit the scope of protection of this application.

[0007] The present invention provides a digital microfluidic chip, comprising:

[0008] A first substrate, comprising a first base having an injection hole;

[0009] an interposer, one side of the interposer being connected to the first substrate, the interposer comprising a flow channel communicating with the injection hole, the flow channel sequentially comprising a droplet generation region, a droplet deceleration region, and a digital microfluidics region; the droplet generation region being configured to generate microdroplets, and the droplet deceleration region being configured to decelerate the microdroplets;

[0010] a second substrate connected to a side of the intermediary layer away from the first substrate; the second substrate comprising a driving electrode; the driving electrode being configured to drive the micro-droplets in the intermediary layer to move;

[0011] The droplet deceleration region is configured to reduce the velocity of the micro-droplets to a value equal to L×f;

[0012] Wherein, L is the length of the driving electrode;

[0013] f is the driving frequency of the driving electrode.

[0014] In an exemplary embodiment of the present application, the droplet deceleration region is configured to reduce the velocity of the micro-droplet from v1 to v2, where v2 = L×f;

[0015] v1 and v2 satisfy:

[0016] Wherein, v1 is the average flow velocity of all fluids in the flow channel at the inlet of the droplet deceleration region, v2 is the average flow velocity of all fluids in the flow channel at the outlet of the droplet deceleration region, and v2 is equal to the average flow velocity of all fluids in the flow channel in the digital microfluidic region;

[0017] p1 is the pressure of all fluids in the flow channel at a certain point at the inlet of the droplet deceleration area, and p2 is the pressure at a certain point at the outlet of the droplet deceleration area;

[0018] h′ w is the total energy loss per unit weight of the fluid in the flow channel in the droplet deceleration area;

[0019] ρ1 is the average density of all fluids in the flow channel at the outlet of the droplet deceleration region;

[0020] If the flow channel in the droplet deceleration area is a horizontal flow channel, then p1=p2, h′ w =0.

[0021] In an exemplary embodiment of the present application, the digital microfluidic chip may include one injection hole for adding an aqueous phase and at least one injection hole for adding an oil phase; wherein,

[0022] Wherein, a, b, ... z are used to distinguish different injection holes for adding oil phase;

[0023] v0 is the flow rate of the water phase at the injection hole, v a 、v b 、……v z are the flow rates of the oil phase at different injection holes. If there is only one injection hole for adding the oil phase, then v b、 ……v z All are zero;

[0024] ρ0 is the density of the aqueous phase entering the flow channel through the injection hole, ρ a , ρ b 、……ρ z are the densities of the oil phase entering the flow channel through different injection holes, ρ a , ρ b 、……ρ z are the same or different. If there is only one injection hole for adding oil phase, then ρ b 、……ρ z All are zero;

[0025] D0 is the aperture of the injection hole for adding the aqueous phase, D a 、D b ,……D z are the apertures of different injection holes for adding the oil phase, and D1 is the diameter of the flow channel at the outlet of the droplet generation area.

[0026] In an exemplary embodiment of the present application, the droplet deceleration area includes any one or both of a pipe diameter enlargement section and a bend pipe, and the flow channel of the pipe diameter enlargement section is a horizontal flow channel;

[0027] The tube diameter enlarged section has an area with a tube diameter of D2, where D1<D2.

[0028] In an exemplary embodiment of the present application, the droplet generation region may be configured to have a flow focusing effect, so that the water phase and the oil phase entering the droplet generation region can generate water-in-oil micro droplets.

[0029] In an exemplary embodiment of the present application, the flow channel of the droplet generation area has a cross structure, and the cross structure is a "cross"-shaped cross, a "T"-shaped cross, or a "Y"-shaped cross.

[0030] In an exemplary embodiment of the present application, the second substrate includes an array of driving devices and an array of photodetection devices, the driving devices include the driving electrodes and a first transistor for controlling the driving electrodes, and the photodetection devices include a photosensor and a second transistor for controlling the photosensor; the photosensor is configured to detect optical signals of micro-droplets in the interposer and convert the optical signals into electrical signals;

[0031] The photodetection device is configured to have a detection speed that is the same as a driving speed of the driving electrode.

[0032] In an exemplary embodiment of the present application, both the first transistor and the second transistor may be thin film transistors, and the photosensor may be a photodiode having a PIN junction.

[0033] In an exemplary embodiment of the present application, the first substrate may further include a transparent first conductive layer and a transparent hydrophobic layer, the first conductive layer is located on one side of the first base, the hydrophobic layer is located between the first conductive layer and the intermediate layer, and the injection hole passes through the first conductive layer and the hydrophobic layer.

[0034] In an exemplary embodiment of the present application, the driving electrode is electrically connected to the first transistor; the photosensor is electrically connected to the second transistor, the first transistor, the photosensor and the second transistor are horizontally distributed on the second substrate, and the orthographic projection of the driving electrode on the second substrate covers the orthographic projections of the first transistor, the photosensor and the second transistor on the second substrate.

[0035] The present application also provides a use of the digital microfluidic chip described above for screening positive single cells, wherein the positive single cells are single cells that can secrete target antibodies, and the use includes:

[0036] Injecting an oil phase and an aqueous solution containing the single cell into the digital microfluidic chip, wherein the aqueous solution generates microdroplets in the droplet generation area, and at least part of the microdroplets contain the single cell;

[0037] The driving electrode drives the microdroplets containing the single cell to move to the digital microfluidic area, and drives the microdroplets not containing the single cell to move to the waste liquid area;

[0038] injecting an antibody detection reagent into the digital microfluidic chip to detect whether the single cell secretes antibodies;

[0039] Immunoluminescence detection is performed in the digital microfluidic area, and the driving electrode drives the microdroplets that do not contain the target antibody to move to the waste liquid area, and drives the microdroplets containing the target antibody to be screened out and remain in the digital microfluidic area. After screening, the single cell located in the microdroplet in the digital microfluidic area is the positive single cell.

[0040] In an exemplary embodiment of the present application, injecting an oil phase and an aqueous solution containing the single cell into the digital microfluidic chip, wherein the aqueous solution generates microdroplets in the droplet generation area may include:

[0041] injecting the oil phase and the aqueous solution containing the single cells into the droplet generation area through the injection hole;

[0042] The aqueous solution generates water-in-oil microdroplets in the droplet generation area, and at least part of the microdroplets contain the single cells.

[0043] In an exemplary embodiment of the present application, the use may further include: after the micro-droplets are generated, the driving electrode drives the micro-droplets to move to the digital microfluidic area or the waste liquid area,

[0044] The micro-droplets are decelerated when passing through the droplet deceleration area. The speed of the micro-droplets before entering the droplet deceleration area is v1, and the speed of the micro-droplets after passing through the droplet deceleration area is v2, where v1>v2.

[0045] In an exemplary embodiment of the present application, the driving electrode driving the microdroplet containing the single cell to move to the digital microfluidics area, and driving the microdroplet not containing the single cell to move to the waste liquid area may include:

[0046] After being decelerated in the droplet deceleration area, the microdroplets arrive at the droplet deceleration area and the digital microfluidic area, and a photoelectric sensor is used to detect light signals of different microdroplets and convert the detected light signals into electrical signals;

[0047] The first transistor is used to control the driving electrode to transport the microdroplets containing the single cell to the digital microfluidic area, and to control the driving electrode to transport the microdroplets not containing the single cell to the waste liquid area.

[0048] In an exemplary embodiment of the present application, injecting an antibody detection reagent into the digital microfluidic chip to detect whether the single cell secretes antibodies may include:

[0049] injecting the target antigen and horseradish peroxidase / alkaline phosphatase labeled antibody into the droplet generation area through the injection hole;

[0050] The target antigen and horseradish peroxidase / alkaline phosphatase labeled antibody move to the digital microfluidic area and form a target antigen array and a horseradish peroxidase / alkaline phosphatase labeled antibody array that match the number of microdroplets located in the digital microfluidic area, respectively;

[0051] The target antigen and the horseradish peroxidase / alkaline phosphatase-labeled antibody enter the microdroplet located in the digital microfluidic area and mix with the single cell in the microdroplet to form a double antibody sandwich complex. The target antigen and the horseradish peroxidase / alkaline phosphatase-labeled antibody detect whether the single cell in the microdroplet secretes the target antibody.

[0052] In an exemplary embodiment of the present application, the use may also include: injecting an enhancer and a luminescent agent into the digital microfluidic chip, wherein the enhancer and the luminescent agent enter the microdroplets located in the digital microfluidic area and produce a chemiluminescence effect with the double antibody sandwich complex in the microdroplets to enhance the intensity of the light signal of the microdroplets.

[0053] In an exemplary embodiment of the present application, the driving electrode driving the microdroplets not containing the target antibody to move to the waste liquid area, and driving the microdroplets containing the target antibody to be screened out and remain in the digital microfluidic area may include:

[0054] Using a photoelectric sensor to detect light signals of different micro-droplets and converting the detected light signals into electrical signals;

[0055] The first transistor is used to control the driving electrode to transport the microdroplets containing the target antibody to the digital microfluidic area, and to control the driving electrode to transport the microdroplets not containing the target antibody to the waste liquid area.

[0056] In an exemplary embodiment of the present application, the use may further include: after screening out the positive single cells by the immunoluminescence detection,

[0057] The immunoluminescence detection is repeated to screen out positive single cells with desired secretion characteristics.

[0058] In an exemplary embodiment of the present application, repeatedly performing the immunoluminescence detection to screen out positive single cells with desired antibody secretion performance may include:

[0059] The first transistor is used to control the driving electrode to transport the positive single cells with desired antibody secretion performance to the digital microfluidic area, and to transport the microdroplets containing the positive single cells that do not reach the desired antibody secretion performance to the waste liquid area.

[0060] In an exemplary embodiment of the present application, the single cell may be a hybridoma single cell.

[0061] Other features and advantages of the present application will be described in the following description, and in part will become more apparent from the description, or understood by practicing the present application. Other advantages of the present application can be realized and obtained by the solutions described in the description and the drawings.

[0062] Still other aspects will become apparent upon reading and understanding the accompanying drawings and detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[0063] The accompanying drawings are used to provide an understanding of the technical solution of the present application and constitute a part of the specification. Together with the embodiments of the present application, they are used to explain the technical solution of the present application and do not constitute a limitation on the technical solution of the present application.

[0064] FIG1 is a schematic structural diagram of a digital microfluidic chip provided by an exemplary embodiment of the present application;

[0065] FIG2 is a schematic structural diagram of an interposer of a digital microfluidic chip provided by an exemplary embodiment of the present application;

[0066] FIG3 is a partial enlarged view of FIG2;

[0067] FIG4 is a diagram showing the velocity change of a microdroplet when passing through a droplet deceleration area of ​​a digital microfluidic chip of an exemplary embodiment of the present application;

[0068] FIG5 is a schematic structural diagram of a second substrate of a digital microfluidic chip according to an exemplary embodiment of the present application;

[0069] FIG6 is a top view of the pixel structure of the second substrate shown in FIG5 ;

[0070] FIG7 is a flow chart of a digital microfluidic chip for cloning single cells according to an exemplary embodiment of the present application;

[0071] FIG8 is a flow chart of a digital microfluidic chip for screening monoclonal antibodies according to an exemplary embodiment of the present application;

[0072] FIG9 is a flowchart of micro-droplet sorting in a process of screening monoclonal antibodies using a digital microfluidic chip according to an exemplary embodiment of the present application;

[0073] FIG10 is a schematic diagram of a digital microfluidic chip according to an exemplary embodiment of the present application for forming a double antibody sandwich complex when used to screen monoclonal antibodies;

[0074] FIG11 is a schematic diagram of a chemiluminescent reaction occurring when a digital microfluidic chip according to an exemplary embodiment of the present application is used to screen monoclonal antibodies;

[0075] FIG12 is a top view of the double antibody sandwich complex in the microdroplet when chemiluminescence effect occurs;

[0076] FIG13 is a schematic diagram of the detection principle of a photoelectric detection device;

[0077] FIG14 is a schematic diagram of the process of screening positive hybridoma cells before separation of positive hybridoma cells and negative hybridoma cells;

[0078] FIG15 is a schematic diagram showing the separation of positive hybridoma cells from negative hybridoma cells during the screening process of positive hybridoma cells;

[0079] FIG16 is a graph showing the relationship between the number of positive hybridoma cells and time.

[0080] The meanings of the symbols in the drawings are as follows: 10 - first substrate; 11 - first base; 12 - injection hole; 13 - first conductive layer; 14 - hydrophobic layer; 20 - intermediary layer; 21 - Droplet generation area; 22-droplet deceleration area; 23-digital microfluidic area; 30-second substrate; 31-driving electrode; 32-first transistor; 33-photoelectric sensor; 34-second transistor; 40-hybridoma cell; 50-aqueous phase; 60-oil phase; 70-microdroplet; 80-target antigen; 90-horseradish peroxidase / alkaline phosphatase labeled antibody; 100-double antibody sandwich complex; 110-enhancer; 120-luminescent agent; 01-second substrate; 02-first gate insulating layer; 03-first electrode layer; 04-bottom electrode; 041-second semiconductor layer; 042-top electrode; 05-first insulating layer; 06-encapsulation layer; 07-dielectric layer; 08-second insulating layer; 09-top metal layer; 15-blocking wall; 16-second conductive layer; G1-first gate electrode; G2-second gate electrode; P1-first channel region; P2-second channel region. DETAILED DESCRIPTION

[0081] To make the purpose, technical solutions and advantages of this application more clear, the embodiments of this application will be described in detail below with reference to the accompanying drawings. It should be noted that, unless there is a conflict, the embodiments and features in the embodiments of this application can be combined with each other in any way.

[0082] The embodiments herein can be implemented in a variety of different forms. A person skilled in the art can easily understand that the implementation and content can be transformed into various forms without departing from the purpose and scope of this application. Therefore, this application should not be interpreted as being limited to the contents described in the following embodiments. In the absence of conflict, the embodiments in this application and the features in the embodiments can be combined with each other in any manner.

[0083] The scales of the figures in this application 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 application are merely schematic diagrams of the structures, and one embodiment of this application is not limited to the shapes or values ​​shown in the figures.

[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 convenience of describing 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 limitations on this application. The positional relationships of constituent elements may be appropriately changed depending on the direction in which each constituent element is described. Therefore, the words and phrases are not limited to those described in the specification and may be appropriately replaced according to the circumstances.

[0085] In this specification, unless otherwise specified or limited, the terms "disposed" 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 an intermediary, or internal communication between two components. Those skilled in the art will understand the specific meanings of these terms in this application.

[0086] In the description of this application, ordinal numbers such as "first" and "second" are provided to avoid confusion of constituent elements, rather than to limit the quantity.

[0087] In this specification, "film" and "layer" can be interchanged. For example, "hydrophobic layer" can sometimes be replaced with "hydrophobic film".

[0088] Currently, the development of monoclonal antibody drugs primarily relies on the traditional "limiting dilution method," which involves dispersing individual antibody-secreting cells into microporous cell culture plates through solution dilution, and then cloning and culturing individual cells in each microwell. However, the "limiting dilution method" cannot meet the "time-sensitive" and "high-throughput screening" requirements of monoclonal antibody drug development. Furthermore, the method is characterized by long development times, high costs, low yields, and significant labor requirements. These factors contribute to high prices and low penetration rates for monoclonal antibody drugs, and the development of monoclonal antibody drugs continues to face significant challenges.

[0089] The key technology for the development of monoclonal antibody drugs lies in the screening and breeding of hybridoma B cells and Chinese Hamster Ovary (CHO) cells. How to realize automated, high-throughput single-cell clone screening technology is crucial for shortening the development cycle of monoclonal antibody drugs, increasing production, reducing costs, and improving the popularity of monoclonal antibody drugs.

[0090] An embodiment of the present application provides a digital microfluidic chip.

[0091] Figure 1 is a schematic diagram of the structure of a digital microfluidic chip provided by an exemplary embodiment of the present application; Figure 2 is a schematic diagram of the structure of an interposer of a digital microfluidic chip provided by an exemplary embodiment of the present application. As shown in Figures 1 and 2, the digital microfluidic chip may include: a first substrate 10, an interposer 20, and a second substrate 30; the first substrate 10 and the second substrate 30 may be located on either side of the interposer 20, forming a box-like structure;

[0092] The first substrate 10 includes a first base 11, and the first base has a sampling hole 12. The sampling hole 12 can be multiple, for example, it can include one water phase sampling hole and two oil phase sampling holes;

[0093] The intermediary layer 20 includes a flow channel connected to the injection hole 12, and the flow channel includes a droplet generation area 21, a droplet deceleration area 22, and a digital microfluidic area 23; the two ends of the droplet deceleration area 22 are respectively connected to the droplet generation area 21 and the digital microfluidic area 23; the droplet generation area 21 is configured to generate microdroplets, for example, microdroplets containing single cells; the droplet deceleration area 22 is configured to reduce the velocity of the microdroplets from v1 to v2, and the digital microfluidic area 23 is configured to allow the desired microdroplets to enter therein;

[0094] The second substrate 30 includes a driving electrode; the driving electrode is configured to drive the micro-droplet in the intermediary layer 20 to move; v2 = L×f;

[0095] Wherein, v2 is the average flow velocity of all fluids in the flow channel at the outlet of the droplet deceleration region, and v2 is equal to the average flow velocity of all fluids in the flow channel in the digital microfluidic region;

[0096] L is the length of the driving electrode;

[0097] f is the driving frequency of the driving electrode;

[0098] v1 is the average flow velocity of all fluids in the flow channel at the entrance of the droplet deceleration zone.

[0099] The digital microfluidic chip of the embodiment of the present application is based on digital microfluidic technology. It uses the droplet generation area of ​​the intermediary layer to generate microdroplets, which can realize the encapsulation of high-throughput single cells with microdroplets; the driving electrode is used to control the movement of the microdroplets, thereby screening out the target single cells, automatically forming a single cell array, single cell cloning culture, and screening positive single cells and positive single cells with high antibody secretion performance. For example, it can automatically realize the generation of high-throughput single-cell microdroplets, microdroplet screening and single cell culture, and for example, it can automatically realize the high-throughput generation of hybridoma single-cell microdroplets in the monoclonal antibody screening process, sorting and detection of hybridoma single-cell microdroplets, single cell cloning culture, positive hybridoma cell screening and screening of positive hybridoma cells with high antibody secretion performance. Therefore, the digital microfluidic chip of the embodiment of the present application can realize high-throughput, large-scale single-cell sorting, single-cell cloning culture, accurate determination of antibody specificity and screening and extraction of target cell lines, and is expected to be used for screening hybridoma cells that can secrete monoclonal antibodies and shorten the monoclonal antibody drug development cycle.

[0100] The injection hole 12 on the first substrate 10 is used to inject required raw materials into the digital microfluidic chip, for example, including but not limited to silicone oil, cell suspension containing single cells, cell culture medium, etc.

[0101] In an exemplary embodiment of the present application, the injection hole 12 on the first substrate 10 can be formed by laser or mechanical drilling.

[0102] As shown in FIG1 , in an exemplary embodiment of the present application, the first substrate 10 may further include a transparent first conductive layer 13 and a transparent hydrophobic layer 14. The first conductive layer 13 is located on one side of the first base 11, and the hydrophobic layer 14 is located between the first conductive layer 13 and the intermediary layer 20. The injection hole 12 extends through the first conductive layer 13 and the hydrophobic layer 14. The first conductive layer 13 may be made of a conductive material such as ITO or Pedot:PSS. The hydrophobic layer 14 may be made of a material such as CYTOP or Teflon.

[0103] The digital microfluidic chip of the embodiment of the present application can rapidly analyze thousands of single cells by encapsulating single cells in microdroplets for parallel analysis.

[0104] The generated microdroplets have a relatively high velocity, typically less than 10 m / s. The drive frequency of the drive electrodes is typically 1 Hz to 50 Hz, and the electrode size is 50 μm to 200 μm. To ensure that the microdroplet velocity matches the drive speed of the drive electrodes (typically on a mm / s scale, such as 1 mm / s to 10 mm / s), the microdroplets require a period of deceleration. The droplet deceleration region 22 reduces the microdroplet velocity from v1 to v2. When v2 = L × f, the microdroplet velocity matches the drive speed of the drive electrodes, improving detection accuracy.

[0105] In an exemplary embodiment of the present application, v1 and v2 satisfy:

[0106] Wherein, v1 is the average flow velocity of all fluids in the flow channel at the inlet of the droplet deceleration region, v2 is the average flow velocity of all fluids in the flow channel at the outlet of the droplet deceleration region, and v2 is equal to the average flow velocity of all fluids in the flow channel in the digital microfluidic region;

[0107] p1 is the pressure of all fluids in the flow channel at a certain point at the inlet of the droplet deceleration area, and p2 is the pressure at a certain point at the outlet of the droplet deceleration area;

[0108] h′ w is the total energy loss per unit weight of the fluid in the flow channel in the droplet deceleration area;

[0109] ρ1 is the average density of all fluids in the flow channel at the outlet of the droplet deceleration region;

[0110] If the flow channel in the droplet deceleration area is a horizontal flow channel, then p1=p2, h′ w =0.

[0111] In an exemplary embodiment of the present application, the first substrate includes one injection hole for adding an aqueous phase and at least one injection hole for adding an oil phase; wherein,

[0112] Wherein, a, b, ... z are used to distinguish different injection holes for adding oil phase;

[0113] v0 is the flow rate of the water phase at the injection hole, v a 、v b 、……v z are the flow rates of the oil phase at different injection holes. If there is only one injection hole for adding the oil phase, then v b 、……v z All are zero;

[0114] ρ0 is the density of the aqueous phase entering the flow channel through the injection hole, ρa , ρ b 、……ρ z are the densities of the oil phase entering the flow channel through different injection holes, ρ a , ρ b 、……ρ z are the same or different. If there is only one injection hole for adding oil phase, then ρ b 、……ρ z All are zero;

[0115] D0 is the aperture of the injection hole for adding the aqueous phase, D a 、D b ,……D z are the apertures of different injection holes for adding the oil phase, and D1 is the diameter of the flow channel at the outlet of the droplet generation area.

[0116] In an exemplary embodiment of the present application, the micro-droplets can be decelerated by providing a pipe diameter enlargement section, a bend pipe, etc. in the droplet deceleration area;

[0117] The tube diameter enlarged section has an area with a tube diameter of D2, where D1<D2.

[0118] For example, the tube diameter enlargement section can be a liquid reservoir with a diameter larger than that of the droplet generation area, such as a circular, polygonal (e.g., hexagonal) or irregularly shaped liquid reservoir; the bend can be a 180° bend, etc.

[0119] Figure 3 is a partial enlarged view of Figure 2. In the intermediary layer shown in Figures 1 to 3, the droplet deceleration region 22 includes a hexagonal liquid reservoir and multiple 180° elbows. When the fluid passes through these components, the flow direction of the fluid changes dramatically due to energy loss caused by viscous friction between the fluid and the wall and within the fluid along the flow path. This causes a rapid change in the velocity field, increases friction between the fluids, collisions, and the formation of vortices, thereby reducing the flow rate of the fluid. At the inlet of the droplet deceleration region 22, the average flow velocity of all fluids in the flow channel is v1; at the outlet of the hexagonal liquid reservoir, the average flow velocity of all fluids in the flow channel is v3; and at the outlet of the 180° elbow, the average flow velocity of all fluids in the flow channel is v2. In other words, the droplet deceleration region 22 shown in Figure 3 reduces the velocity of the microdroplets from v1 to v3 and then to v2.

[0120] After the aqueous solution containing the single cells enters the flow channel, it is squeezed by the oil-phase sheath fluid, gradually squeezing the single cells from the wider inlet state to a thinner single-cell stream. The law of conservation of mass states that in two dimensions, the mass of the fluid passing through the central channel should remain equal to the mass of the fluid after focusing.

[0121] The principles involved are as follows:

[0122] Since the flow rate Q is constant, Q = v × A, v is the flow velocity of the fluid at that point, A is the longitudinal cross-sectional area of ​​the flow channel, v1 × A1 = v3 × A3,

[0123] so,

[0124] Specifically, when the diameter of the microchannel doubles, the velocity will drop to 1 / 4 of the initial velocity. Considering the flow rate and density of the oil-phase sheath fluid, the following formula is obtained:

[0125] Wherein, a, b, ... z are used to distinguish different injection holes for adding oil phase;

[0126] v0 is the flow rate of the water phase at the injection hole, v a 、v b 、……v z are the flow rates of the oil phase at different injection holes. If there is only one injection hole for adding the oil phase, then v b 、……v z are all zero; v1 is the average flow velocity of all fluids in the flow channel at the entrance of the droplet deceleration region;

[0127] ρ0 is the density of the aqueous phase entering the flow channel through the injection hole, ρ a , ρ b 、……ρ z are the densities of the oil phase entering the flow channel through different injection holes, ρ a , ρ b 、……ρ z are the same or different. If there is only one injection hole for adding oil phase, then ρ b 、……ρ z All are zero;

[0128] D0 is the aperture of the injection hole for adding the aqueous phase, D a 、D b ,……D z are the apertures of different injection holes for adding the oil phase, and D1 is the diameter of the flow channel at the outlet of the droplet generation area.

[0129] Then, when the liquid passes through the 180° elbow, the movement of the fluid conforms to the Bernoulli equation for viscous fluid:

[0130] Where h′ w is the total energy loss per unit weight of fluid in the flow channel in the droplet deceleration area. However, since the flow channel of the pipe diameter enlargement section such as the reservoir is a horizontal flow channel, the energy loss in the pipe diameter enlargement section can be ignored, so h′w Equal to the energy loss of the fluid in the elbow;

[0131] p2 is the pressure of all fluids in the flow channel at a certain point at the outlet of the droplet deceleration area, and p3 is the pressure of all fluids in the flow channel at a certain point at the junction of the pipe diameter enlargement section and the elbow;

[0132] v2 is the average flow velocity of all fluids in the flow channel at the outlet of the droplet deceleration zone, and v3 is the average flow velocity of all fluids in the flow channel at the junction of the pipe diameter enlargement section and the elbow;

[0133] ρ is the fluid density;

[0134] g is the acceleration due to gravity;

[0135] z2 is the height of the point where p2 is tested, and z3 is the height of the point where p3 is tested. w =∑h f +∑h j (1)

[0136] Among them, ∑h f is the total along-the-line loss, which is a kind of energy loss occurring in the slowly varying flow area, ∑h j is the total local loss.

[0137] Among them, λ is the loss coefficient along the way, d is the diameter of the flow channel, and l is the flow channel length or pipe length; in laminar flow, Re is the Reynolds number.

[0138] Among them, h j is the local loss, which is an energy loss occurring in the rapid flow area, ζ is the local loss coefficient, and v is the flow velocity.

[0139] From the above formula, we can know that in this application, the energy loss h′ of the fluid passing through the pipe diameter enlargement section and multiple bends is w , the flow velocity v2 out of the pipe conforms to the following formula:

[0140] In order to match the driving speed of the driving electrode, the following corresponding relationship is established: v2=L*f (c)

[0141] Wherein, L is the length of the driving electrode;

[0142] f is the driving frequency of the driving electrode.

[0143] Formulas (a), (b), and (c) show the relationship between the length of the driving electrode and the driving frequency and the inflow velocity at the inlet of the digital microfluidic chip, as follows:

[0144] Wherein, a, b, ... z are used to distinguish different injection holes for adding oil phase;

[0145] v1 is the average flow velocity of all fluids in the flow channel at the entrance of the droplet deceleration area; v0 is the flow velocity of the aqueous phase at the injection hole, v a 、v b 、……v z are the flow rates of the oil phase at different injection holes. If there is only one injection hole for adding the oil phase, then v b 、……v z All are zero;

[0146] p2 is the pressure of all fluids in the flow channel at a certain point at the outlet of the droplet deceleration area, and p3 is the pressure of all fluids in the flow channel at a certain point at the junction of the pipe diameter enlargement section and the elbow;

[0147] h′ w is the total energy loss of all fluids in the flow channel in the droplet deceleration region;

[0148] ρ1 is the average density of all fluids in the flow channel at the outlet of the droplet deceleration zone; ρ0 is the density of the aqueous phase entering the flow channel through the injection hole, ρ a , ρ b 、……ρ z are the densities of the oil phase entering the flow channel through different injection holes, ρ a , ρ b 、……ρ z are the same or different. If there is only one injection hole for adding oil phase, then ρ b 、……ρ z All are zero;

[0149] L is the length of the driving electrode;

[0150] f is the driving frequency of the driving electrode;

[0151] D0 is the aperture of the injection hole for adding the aqueous phase, D a 、D b ,……D z are the apertures of different injection holes for adding the oil phase, and D1 is the diameter of the flow channel at the outlet of the droplet generation area.

[0152] For example, a microdroplet is generated at a speed of 5 mm / s. After passing through a hexagonal reservoir with an enlarged diameter, the speed drops to 1.25 mm / s. Simulations show that the average speed of the microdroplet decreases by 1.045 mm / s after each 180° elbow, a 16% speed loss. After passing through three 180° elbows, the speed of the microdroplet drops to 0.6 mm / s, as shown in Figure 4, matching the drive speed of the drive electrode.

[0153] In the digital microfluidic chip shown in Figure 3, the droplet deceleration region includes both the tube diameter enlargement section and the bend, and the tube diameter enlargement section is located between the droplet generation region and the bend. In other exemplary embodiments, the droplet deceleration region may include only the tube diameter enlargement section or the bend.

[0154] In an exemplary embodiment, the droplet deceleration region includes only the pipe diameter enlargement section but does not include the bend, which is equivalent to v2=v3;

[0155] According to the formula

[0156] The flow channel of the pipe diameter enlargement section is a horizontal flow channel, p1 = p2, h' w =0, the above formula is transformed into

[0157] Also because Therefore, v2=v3, which means the formula Applicable to the case where the droplet deceleration area only includes the tube diameter enlargement section.

[0158] In an exemplary embodiment, the droplet deceleration region includes only the bend but does not include the pipe diameter enlargement section, which is equivalent to v1=v3;

[0159] According to the formula

[0160] p1=p3,D1=D2,the above formula is transformed into the above formula (b) It complies with the Bernoulli equation when the liquid passes through a 180° elbow. Explain the formula Applicable to the case where the droplet deceleration area only includes the elbow.

[0161] In an exemplary embodiment of the present application, the droplet generation region 21 may be configured to have a flow focusing effect, so that the water phase and the oil phase entering the droplet generation region can generate water-in-oil micro droplets.

[0162] As shown in Figure 3, in an exemplary embodiment of the present application, the droplet generation area 21 can have a cross structure, and the cross structure is a "cross"-shaped cross or a "T"-shaped cross. For example, as shown in Figures 1 and 2, the droplet generation area can be composed of a cross-shaped flow channel. In addition, double T-shaped flow channels, Y-shaped flow channels, etc. can also be used. By utilizing the geometric characteristics of the intersection of the microchannels, the momentum change caused by the continuous phase shear force when the front of the dispersed phase liquid turns at the intersection is generated to generate microdroplets, which has a high-throughput two-phase separation capability. Among them, the horizontal main channel is the main channel for the cell suspension of the dispersed phase to flow into, and the continuous phase such as silicone oil material can flow into the side channel from the upper and lower inlets, and the outlet is on the right side of the main channel. By controlling the flow rate of the oil phase and the water phase in the chip, a plurality of microdroplets are formed, and the distribution of the microdroplets is relatively uniform.

[0163] In an exemplary embodiment of the present application, both the first transistor and the second transistor may be thin film transistors, and the photosensor may be a photodiode having a PIN junction.

[0164] Figure 5 is a schematic diagram of the structure of the second substrate of the digital microfluidic chip of the exemplary embodiment of the present application; Figure 6 is a top view of the pixel structure of the second substrate shown in Figure 5. The second substrate includes a driving device array and a photodetection device array located on a second base 01. The driving device array is formed by a plurality of driving devices, each including a driving electrode 31 and a first transistor 32 for controlling the driving electrode 31; the photodetection device array is formed by a plurality of photodetection devices, each including a photosensor 33 and a second transistor 34 for controlling the photosensor 33. The photosensor 33 is a photodiode with a PIN junction. The driving electrode 31 is electrically connected to the first transistor 32, and the photosensor 33 is electrically connected to the second transistor 34.

[0165] The first gate electrode G1 of the first transistor 32 and the second gate electrode G2 of the second transistor 34 are deposited on one side of the second substrate 01, and a first gate insulating layer 02 covering the first gate electrode G1 and the second gate electrode G2 is deposited. The first semiconductor layer and the first electrode layer 03 are deposited on the side of the first gate insulating layer 02 away from the second substrate 01. Different regions of the first semiconductor layer serve as the first channel region P1 of the first transistor 32 and the second channel region P2 of the second transistor 34, respectively. Different regions of the first electrode layer 03 serve as the source electrode and drain electrode of the first transistor 32 and the source electrode and drain electrode of the second transistor 34, respectively. The source electrode and drain electrode of the first gate electrode G1 are respectively connected to the first channel region P1 and expose the first channel region P1. The source electrode and drain electrode of the second transistor 34 are respectively connected to the second channel region P2 and expose the second channel region P2.

[0166] The bottom electrode 04 and the first insulating layer 05 of the photosensor 33 are deposited on the side of the first electrode layer 03 away from the second substrate 01; the photosensor 33 is electrically connected to the source electrode or drain electrode of the second transistor 34 through the bottom electrode 04; the photosensor 33 also includes a second semiconductor layer 041 located on the side of the bottom electrode 04 away from the second substrate 01 and a top electrode 042 located on the side of the second semiconductor layer 041 away from the second substrate 01.

[0167] The surfaces of the driving device array and the photoelectric detection device array are provided with a packaging layer 06 , a dielectric layer 07 and a second insulating layer 08 .

[0168] A top metal layer 09 used as a conductive line is deposited on the side of the top electrode 042 away from the second substrate 01. A retaining wall 15 and a second conductive layer 16 are deposited in sequence on the side of the top metal layer 09 away from the second substrate 01. The second conductive layer 16 includes a plurality of driving electrodes 31

[0169] A transparent dielectric layer 07 and a transparent hydrophobic layer 14 are sequentially deposited on the side of the second conductive layer 16 away from the second substrate 01. The hydrophobic layer 14 is located between the dielectric layer 07 and the intermediate layer. The dielectric layer 07 can be SiN x , SiO2, Resin and other materials, the hydrophobic layer 14 can be made of CYTOP, Teflon and other materials.

[0170] An embodiment of the present application also provides a use of the digital microfluidic chip as described above for cloning single cells.

[0171] FIG7 is a flow chart illustrating a digital microfluidic chip used to screen single cells according to an exemplary embodiment of the present application. As shown in FIG7 , the method for screening single cells using the digital microfluidic chip includes:

[0172] S10: injecting an oil phase and an aqueous solution containing the single cell into the digital microfluidic chip, wherein the aqueous solution generates microdroplets in the droplet generation area, and at least part of the microdroplets contain the single cell;

[0173] S20: The driving electrode drives the microdroplet containing the single cell to move to the digital microfluidic area, and drives the microdroplet not containing the single cell to move to the waste liquid area.

[0174] In an exemplary embodiment of the present application, step S10 may include:

[0175] injecting the oil phase and the aqueous solution containing the single cells into the droplet generation area through the injection hole;

[0176] The aqueous solution generates water-in-oil microdroplets in the droplet generation area, and at least part of the microdroplets contain the single cells.

[0177] In an exemplary embodiment of the present application, the method for screening single cells using the digital microfluidic chip may further include: after step S10 and before step S20,

[0178] The micro-droplets are decelerated when passing through the droplet deceleration area. The speed of the micro-droplets before entering the droplet deceleration area is v1, and the speed of the micro-droplets after passing through the droplet deceleration area is v2, where v1>v2;

[0179] The detection speed of the photoelectric detection device is v3, which is substantially the same as v2.

[0180] In an exemplary embodiment of the present application, the single cell may be a hybridoma single cell.

[0181] The present application also provides a method for screening positive single cells using the digital microfluidic chip described above. FIG8 is a flow chart of an exemplary embodiment of the present application using the digital microfluidic chip for screening positive single cells. As shown in FIG8 , the method for screening positive single cells using the digital microfluidic chip includes:

[0182] S100: injecting an oil phase and an aqueous solution containing the single cell into the digital microfluidic chip, wherein the aqueous solution generates microdroplets in the droplet generation area, and at least part of the microdroplets contain the single cell;

[0183] S200: The driving electrode drives the microdroplet containing the single cell to move to the digital microfluidic area, and drives the microdroplet not containing the single cell to move to the waste liquid area;

[0184] S300: injecting an antibody detection reagent into the digital microfluidic chip to detect whether the single cell secretes antibodies;

[0185] S400: Immunoluminescence detection is performed in the digital microfluidic area, and the driving electrode drives the microdroplets that do not contain the target antibody to move to the waste liquid area, and drives the microdroplets containing the target antibody to be screened out and remain in the digital microfluidic area. After screening, the single cell located in the microdroplet in the digital microfluidic area is the positive single cell.

[0186] In an exemplary embodiment of the present application, step S100 may include:

[0187] injecting the oil phase and the aqueous phase solution containing the single cells into the droplet generation area through the injection hole;

[0188] The aqueous solution generates water-in-oil microdroplets in the droplet generation area, and at least part of the microdroplets contain the single cells.

[0189] In an exemplary embodiment of the present application, the method for screening single cells using the digital microfluidic chip may further include: after step S100 and before step S200,

[0190] The micro-droplets are decelerated when passing through the droplet deceleration area. The speed of the micro-droplets before entering the droplet deceleration area is v1, and the speed of the micro-droplets after passing through the droplet deceleration area is v2, where v1>v2;

[0191] The detection speed of the photoelectric detection device is v3, which is substantially the same as v2.

[0192] In an exemplary embodiment of the present application, step S200 may include:

[0193] After being decelerated in the droplet deceleration area, the microdroplets arrive at the droplet deceleration area and the digital microfluidic area, and the photoelectric sensor detects light signals of different microdroplets and converts the detected light signals into electrical signals;

[0194] The first transistor controls the driving electrode to transport the microdroplet containing the single cell to the digital microfluidic area, and controls the driving electrode to transport the microdroplet not containing the single cell to a waste liquid area.

[0195] In an exemplary embodiment of the present application, step S200 may include:

[0196] After being decelerated in the droplet deceleration area, the microdroplets arrive at the droplet deceleration area and the digital microfluidic area, and the photoelectric sensor detects light signals of different microdroplets and converts the detected light signals into electrical signals;

[0197] The electrical signal data is transmitted to a host computer, which processes the electrical signal data and feeds it back to the first transistor;

[0198] The first transistor applies a driving signal to control the driving electrode to transport the microdroplets containing the single cell to the digital microfluidic area, and controls the driving electrode to transport the microdroplets not containing the single cell to the waste liquid area.

[0199] In an exemplary embodiment of the present application, step S300 may include:

[0200] injecting the target antigen and horseradish peroxidase / alkaline phosphatase labeled antibody into the droplet generation area through the injection hole;

[0201] The target antigen and horseradish peroxidase / alkaline phosphatase labeled antibody move to the digital microfluidic area and form a target antigen array and a horseradish peroxidase / alkaline phosphatase labeled antibody array that match the number of microdroplets located in the digital microfluidic area, respectively;

[0202] The target antigen and the horseradish peroxidase / alkaline phosphatase-labeled antibody enter the microdroplet located in the digital microfluidic area and mix with the single cell in the microdroplet to form a double antibody sandwich complex. The target antigen and the horseradish peroxidase / alkaline phosphatase-labeled antibody detect whether the single cell in the microdroplet secretes the target antibody.

[0203] In an exemplary embodiment of the present application, the method for screening positive single cells using the digital microfluidic chip may further include:

[0204] An enhancer and a luminescent agent are injected into the digital microfluidic chip. The enhancer and the luminescent agent enter the microdroplets located in the digital microfluidic area and produce a chemiluminescent effect with the double antibody sandwich complex in the microdroplets to enhance the intensity of the light signal of the microdroplets.

[0205] In an exemplary embodiment of the present application, step S400 may include:

[0206] The photoelectric sensor detects light signals of different micro-droplets and converts the detected light signals into electrical signals;

[0207] The first transistor controls the driving electrode to transport the microdroplets containing the target antibody to the digital microfluidic area, and controls the driving electrode to transport the microdroplets not containing the target antibody to a waste liquid area.

[0208] In an exemplary embodiment of the present application, step S400 may include:

[0209] The photoelectric sensor detects light signals of different micro-droplets and converts the detected light signals into electrical signals;

[0210] The electrical signal data is transmitted to a host computer, which processes the electrical signal data and feeds it back to the first transistor;

[0211] The first transistor applies a driving signal to control the driving electrode to transport the microdroplets containing the target antibody to the digital microfluidic area, and controls the driving electrode to transport the microdroplets not containing the target antibody to the waste liquid area.

[0212] In an exemplary embodiment of the present application, the method for screening positive single cells using the digital microfluidic chip may further include: after screening the positive single cells by the immunoluminescence detection,

[0213] The immunoluminescence detection is repeated to screen out positive single cells with desired secretion characteristics.

[0214] In an exemplary embodiment of the present application, repeatedly performing the immunoluminescence detection to screen out positive single cells with desired antibody secretion performance may include:

[0215] The first transistor controls the driving electrode to transport the positive single cells containing the desired antibody secretion performance to the digital microfluidic area, and transports the microdroplets containing the positive single cells that do not reach the desired antibody secretion performance to the waste liquid area.

[0216] In an exemplary embodiment of the present application, the single cell may be a hybridoma single cell.

[0217] In an exemplary embodiment, a method for screening positive single cells using the digital microfluidic chip may include:

[0218] (1) Preparation of fused cells: Myeloma cells and spleen lymphocytes were mixed in a culture dish outside the digital microfluidic chip, PEG was added to fuse the two cells, HAT medium (complete RPMI-1640 or DMEM medium, HT storage solution, A storage solution) was added, and then cultured in a 37°C incubator containing 6% CO2;

[0219] (2) High-throughput generation of hybridoma single cells: Hybridoma cells 40 can be added to the digital microfluidic chip about 2 weeks after fusion. Through the structure of the double T-shaped microchannel, the flow rate of the aqueous phase 50 and the oil phase 60 in the flow channel is controlled to generate a large number of microdroplets 70 containing hybridoma single cells;

[0220] (3) Microdroplet culture: The generated microdroplets have a high flow rate. In order to match the response time of the photoelectric detection device of the second substrate, a deceleration operation is required. The deceleration operation is performed in the droplet deceleration area. For example, the microdroplets containing hybridoma single cells are passed through the tube diameter enlargement section and multiple 180° bends, so that the speed of the microdroplets is reduced from v1 before entering the droplet deceleration area to v2 after passing through the droplet deceleration area; the decelerated microdroplets are driven by the driving electrode array to the detection area; the light signals generated by the microdroplets containing hybridoma single cells and the empty microdroplets 71 without hybridoma single cells are different. The difference in the response signal amount of the photoelectric detection device of the second substrate is used to distinguish the empty microdroplets 71 from the microdroplets 70 containing hybridoma single cells;

[0221] (4) Micro-droplet sorting: The first transistor controls the driving electrode array to transport the empty micro-droplets 71 to the waste liquid area, and transports the sorted micro-droplets 70 containing hybridoma single cells to the digital microfluidic area 23 to form a single cell array, as shown in FIG9 ;

[0222] (5) After the hybridoma single cell array is formed, the target antigen 80 and the horseradish peroxidase / alkaline phosphatase labeled antibody 90 are added to the system through the injection hole. The target antigen 80 array and the horseradish peroxidase / alkaline phosphatase labeled antibody 90 array matching the number of the hybridoma single cell array are formed by the manipulation of the driving electrode. The hybridoma single cell droplet is automatically mixed with the target antigen and the horseradish peroxidase / alkaline phosphatase labeled antibody on the platform to form a double antibody sandwich complex 100, as shown in FIG10 ;

[0223] (6) The enhancer 110 and the luminescent agent 120 are added to the system, and the double antibody sandwich complex undergoes a chemiluminescent reaction. As shown in FIG11 , the wavelength of the emitted light signal is in the 470 nm band, which is within the visible light range.

[0224] Figure 12 is a top view of the dual-antibody sandwich complex in a microdroplet undergoing chemiluminescence; Figure 13 is a schematic diagram of the detection principle of the photoelectric detection device. As shown in Figures 12 and 13, when a single hybridoma cell in the microdroplet secretes a specific, specific antibody, a highly specific chemiluminescent reaction occurs. The light signal triggers a response from the photoelectric detection device located on the second substrate, generating a photocurrent proportional to the incident light intensity. This light signal is then converted into an electrical signal for detection. The photoelectric detection device in this embodiment is a photodiode with a PIN junction, which converts light signals into electrical signals. The electrical signals vary in response to changes in light and generally operate in the visible light band, with a wavelength range of approximately 400nm to 760nm.

[0225] Figure 14 is a schematic diagram of the process of screening positive hybridoma cells before the separation of positive hybridoma cells from negative hybridoma cells; Figure 15 is a schematic diagram of the process of screening positive hybridoma cells after the separation of positive hybridoma cells from negative hybridoma cells; wherein WL represents the word line (WL) connected to the gate electrode of the first transistor, and BL represents the bit line (BL) connected to the source electrode of the first transistor. The optical signal generated by the positive hybridoma cell 72 is converted into an electrical signal through the photoelectric detection device on the second substrate. The first transistor controls the driving electrode to drive the negative hybridoma cell 73 that has not produced antibodies to automatically transfer to the waste liquid area, leaving the positive hybridoma cell 72 behind, and then undergoes recloning on the digital microfluidic chip and repeats the immunoluminescence detection process.

[0226] Figure 16 is a graph showing the relationship between the number of positive hybridoma cells and time. Table 1 shows the relationship between the amount of positive hybridoma cell signal and the number of positive hybridoma cells.

[0227] Table 1

[0228] As shown in Figure 16 and Table 1, if the cell activity is strong and the cell proliferation efficiency is high, the amount of secreted monoclonal antibodies will increase accordingly, which is reflected in the difference in the response time of the photoelectric detection device. Specific cell lines with high secretion are selected and moved outside the digital microfluidic chip for expanded culture or frozen storage.

[0229] Although the embodiments disclosed in this application are as described above, the contents described are merely embodiments adopted to facilitate understanding of this application and are not intended to limit this application. Any person skilled in the art may make any modifications and changes in the form and details of the implementation without departing from the spirit and scope disclosed in this application. However, the scope of protection of this application shall still be based on the scope defined by the appended claims.

Claims

1. A digital microfluidic chip, comprising: A first substrate, the first substrate comprising a first substrate, the first substrate having an injection hole; An intermediary layer, one side of which is connected to the first substrate, the intermediary layer comprising a flow channel connected to the injection hole, the flow channel sequentially comprising a droplet generation area, a droplet deceleration area and a digital microfluidic area; the droplet generation area is configured to generate micro droplets, and the droplet deceleration area is configured to decelerate the micro droplets; A second substrate, the second substrate being connected to a side of the interposer away from the first substrate; the second substrate comprising a driving electrode; The driving electrode is configured to drive the micro-droplets in the intermediary layer to move; The droplet deceleration region is configured to reduce the velocity of the micro-droplet to a value equal to L×f; Wherein, L is the length of the driving electrode; f is the driving frequency of the driving electrode.

2. The digital microfluidic chip according to claim 1, wherein: The droplet deceleration area is configured to reduce the speed of the micro-droplet from v1 to v2, where v2 = L×f; v1 and v2 satisfy: Wherein, v1 is the average flow velocity of all fluids in the flow channel at the inlet of the droplet deceleration region, v2 is the average flow velocity of all fluids in the flow channel at the outlet of the droplet deceleration region, and v2 is equal to the average flow velocity of all fluids in the flow channel in the digital microfluidic region; p1 is the pressure of all fluids in the flow channel at a certain point at the inlet of the droplet deceleration area, and p2 is the pressure of a certain point at the outlet of the droplet deceleration area; h′ w is the total energy loss per unit weight of the fluid in the flow channel in the droplet deceleration area; ρ1 is the average density of all fluids in the flow channel at the outlet of the droplet deceleration zone; If the flow channel in the droplet deceleration area is a horizontal flow channel, then p1 = p2, h′ w =0.

3. The digital microfluidic chip according to claim 2, comprising one injection hole for adding an aqueous phase and at least one injection hole for adding an oil phase; wherein, Wherein, a, b, ..., z are used to distinguish different injection holes for adding oil phase; v0 is the flow rate of the water phase at the injection hole, v a 、v b , ……v z are the flow rates of the oil phase at different injection holes. If there is only one injection hole for adding the oil phase, then v b , ……v z All are zero; ρ0 is the density of the water phase entering the flow channel through the injection hole, ρ a , b , ... ρ z are the densities of the oil phase entering the flow channel through different injection holes, ρ a , b , ... ρ z are the same or different. If there is only one injection hole for adding the oil phase, then ρ b , ... ρ z All are zero; D0 is the aperture of the injection hole used to add the aqueous phase, D a , D b , ...D z are the apertures of different injection holes for adding the oil phase, and D1 is the tube diameter of the flow channel at the outlet of the droplet generation area.

4. The digital microfluidic chip according to claim 2, wherein: The droplet deceleration area includes any one or both of a pipe diameter enlargement section and a curved pipe, and the flow channel of the pipe diameter enlargement section is a horizontal flow channel; The tube diameter enlargement section has a region with a tube diameter of D2, where D1<D2.

5. The digital microfluidic chip according to claim 2, wherein: The droplet generation region is configured to have a flow focusing effect, so that the water phase and the oil phase entering the droplet generation region can generate water-in-oil micro droplets.

6. The digital microfluidic chip according to claim 5, wherein: The flow channel in the droplet generation area has a cross structure, and the cross structure is a "X"-shaped cross, a "T"-shaped cross, or a "Y"-shaped cross.

7. The digital microfluidic chip according to any one of claims 1 to 6, wherein: The second substrate includes an array of driving devices and an array of photoelectric detection devices, wherein the driving devices include the driving electrodes and a first transistor for controlling the driving electrodes, and the photoelectric detection devices include a photoelectric sensor and a second transistor for controlling the photoelectric sensor; the photoelectric sensor is configured to detect an optical signal of a micro-droplet in the interposer and convert the optical signal into an electrical signal; The photodetection device is configured to have a detection speed that is the same as a driving speed of the driving electrode.

8. The digital microfluidic chip according to claim 7, wherein: The first transistor and the second transistor are both thin film transistors, and the photosensor is a photodiode having a PIN junction.

9. The digital microfluidic chip according to any one of claims 1 to 8, wherein: The first substrate further includes a transparent first conductive layer and a transparent hydrophobic layer, the first conductive layer is located on one side of the first base, the hydrophobic layer is located between the first conductive layer and the intermediate layer, and the injection hole penetrates the first conductive layer and the hydrophobic layer.

10. The digital microfluidic chip according to claim 8, wherein: The driving electrode is electrically connected to the first transistor; the photoelectric sensor is electrically connected to the second transistor, the first transistor, the photoelectric sensor and the second transistor are horizontally distributed on the second substrate, and the orthographic projection of the driving electrode on the second substrate covers the orthographic projections of the first transistor, the photoelectric sensor and the second transistor on the second substrate.

11. A use of the digital microfluidic chip according to any one of claims 1 to 10 for screening positive single cells, wherein the positive single cells are single cells that can secrete target antibodies, the use comprising: Injecting an oil phase and an aqueous solution containing the single cell into the digital microfluidic chip, wherein the aqueous solution generates microdroplets in the droplet generation area, and at least part of the microdroplets contain the single cell; The driving electrode drives the microdroplets containing the single cell to move to the digital microfluidic area, and drives the microdroplets not containing the single cell to move to the waste liquid area; Injecting an antibody detection reagent into the digital microfluidic chip to detect whether the single cell secretes antibodies; Immunoluminescence detection is performed in the digital microfluidic area, and the driving electrode drives the microdroplets that do not contain the target antibody to move to the waste liquid area, and drives the microdroplets containing the target antibody to be screened out and remain in the digital microfluidic area. After screening, the single cell located in the microdroplet in the digital microfluidic area is the positive single cell.

12. The use according to claim 11, wherein Injecting an oil phase and an aqueous solution containing the single cell into the digital microfluidic chip, wherein the aqueous solution generates microdroplets in the droplet generation area, comprises: Injecting the oil phase and the aqueous phase solution containing the single cells into the droplet generation area through the injection hole; The aqueous solution generates water-in-oil microdroplets in the droplet generation region, and at least a portion of the microdroplets contain the single cell.

13. The use according to claim 11, further comprising: After the micro-droplets are generated, the driving electrode drives the micro-droplets to move to the digital microfluidics area or the waste liquid area. The micro-droplets are decelerated when passing through the droplet deceleration area. The speed of the micro-droplets before entering the droplet deceleration area is v1, and the speed of the micro-droplets after passing through the droplet deceleration area is v2, where v1>v2.

14. The use according to claim 13, wherein The driving electrode drives the micro-droplet containing the single cell to move to the digital microfluidic area, and drives the micro-droplet not containing the single cell to move to the waste liquid area, including: After being decelerated in the droplet deceleration area, the micro-droplets arrive at the droplet deceleration area and the digital microfluidic area, and a photoelectric sensor is used to detect different optical signals of the micro-droplets and convert the detected optical signals into electrical signals; The first transistor is used to control the driving electrode to transport the micro-droplets containing the single cell to the digital microfluidic area, and the driving electrode is controlled to transport the micro-droplets not containing the single cell to the waste liquid area.

15. The use according to claim 11, wherein Injecting an antibody detection reagent into the digital microfluidic chip to detect whether the single cell secretes antibodies comprises: injecting the target antigen and the horseradish peroxidase / alkaline phosphatase labeled antibody into the droplet generation area through the injection hole; The target antigen and the horseradish peroxidase / alkaline phosphatase labeled antibody move to the digital microfluidic region, and form a target antigen array and a horseradish peroxidase / alkaline phosphatase labeled antibody array that match the number of microdroplets located in the digital microfluidic region, respectively; The target antigen and the antibody labeled with horseradish peroxidase / alkaline phosphatase enter the microdroplet located in the digital microfluidic area and mix with the single cell in the microdroplet to form a double antibody sandwich complex. The target antigen and the antibody labeled with horseradish peroxidase / alkaline phosphatase detect whether the single cell in the microdroplet secretes the target antibody.

16. The use according to claim 15, further comprising: An enhancer and a luminescent agent are injected into the digital microfluidic chip. The enhancer and the luminescent agent enter the microdroplets located in the digital microfluidic area and produce a chemiluminescent effect with the double antibody sandwich complex in the microdroplets to enhance the intensity of the light signal of the microdroplets.

17. The use according to any one of claims 11 to 16, wherein The driving electrode drives the microdroplets not containing the target antibody to move to the waste liquid area, and drives the microdroplets containing the target antibody to be screened out and remain in the digital microfluidic area, including: Using a photoelectric sensor to detect light signals of different micro-droplets and converting the detected light signals into electrical signals; The first transistor is used to control the driving electrode to transport the microdroplets containing the target antibody to the digital microfluidic area, and to control the driving electrode to transport the microdroplets not containing the target antibody to the waste liquid area.

18. The use according to any one of claims 11 to 16, further comprising: After the positive single cells are screened out by the immunoluminescence detection, The immunoluminescence detection is repeated to screen out positive single cells with desired secretion characteristics.

19. The use according to claim 18, wherein Repeat the immunoluminescence detection to screen out positive single cells with the desired secretion antibody performance, including: The first transistor is used to control the driving electrode to transport the positive single cells with the desired secretion antibody performance to the digital microfluidic area, and to transport the microdroplets containing the positive single cells that do not reach the desired secretion antibody performance to the waste liquid area.

20. The use according to any one of claims 11 to 19, wherein The single cell is a hybridoma single cell.

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