CN116408166B - Cell sorting active digital micro-fluidic device and implementation method thereof - Google Patents

Cell sorting active digital micro-fluidic device and implementation method thereof Download PDF

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CN116408166B
CN116408166B CN202310685560.4A CN202310685560A CN116408166B CN 116408166 B CN116408166 B CN 116408166B CN 202310685560 A CN202310685560 A CN 202310685560A CN 116408166 B CN116408166 B CN 116408166B
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row
column
pixel electrode
cell
power
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CN116408166A (en
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张帅龙
李凤刚
郭宗良
侯佳禄
符荣鑫
李航
覃珊
胡汉奇
毛亚楠
杜诗祺
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Beijing Institute of Technology BIT
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M47/00—Means for after-treatment of the produced biomass or of the fermentation or metabolic products, e.g. storage of biomass
    • C12M47/04—Cell isolation or sorting
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
    • B01L3/502769—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by multiphase flow arrangements
    • B01L3/502784—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by multiphase flow arrangements specially adapted for droplet or plug flow, e.g. digital microfluidics
    • B01L3/502792—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by multiphase flow arrangements specially adapted for droplet or plug flow, e.g. digital microfluidics for moving individual droplets on a plate, e.g. by locally altering surface tension
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M23/00—Constructional details, e.g. recesses, hinges
    • C12M23/02—Form or structure of the vessel
    • C12M23/16—Microfluidic devices; Capillary tubes
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
    • B01L2200/06—Fluid handling related problems
    • B01L2200/0626—Fluid handling related problems using levitated droplets
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
    • B01L2200/10—Integrating sample preparation and analysis in single entity, e.g. lab-on-a-chip concept
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/02—Identification, exchange or storage of information
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/16—Surface properties and coatings
    • B01L2300/161—Control and use of surface tension forces, e.g. hydrophobic, hydrophilic
    • B01L2300/165—Specific details about hydrophobic, oleophobic surfaces
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2400/00—Moving or stopping fluids
    • B01L2400/04—Moving fluids with specific forces or mechanical means
    • B01L2400/0403—Moving fluids with specific forces or mechanical means specific forces
    • B01L2400/0415—Moving fluids with specific forces or mechanical means specific forces electrical forces, e.g. electrokinetic
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2400/00—Moving or stopping fluids
    • B01L2400/04—Moving fluids with specific forces or mechanical means
    • B01L2400/0403—Moving fluids with specific forces or mechanical means specific forces
    • B01L2400/0415—Moving fluids with specific forces or mechanical means specific forces electrical forces, e.g. electrokinetic
    • B01L2400/0427—Electrowetting

Abstract

The invention discloses a cell sorting active digital micro-fluidic device and an implementation method thereof. According to the invention, the row-column address controller is used for carrying out row-column scanning control on the pixel electrodes, and the discrete micro-droplets are driven and controlled in real time based on the two-dimensional driving electrode array, so that the precision is high; according to the morphology and the motion characteristics of the cells, high-flux single-cell parallel selection is realized; the invention can carry out intelligent automatic traversal selection on all cells in the micro-droplets, simplifies complicated manual operation steps in the cell selection workflow on the basis of ensuring the activity of the selected cells, and provides a flexible and efficient realization way for single cell selection, in particular for precise selection of sperms in a standardized and procedural mode; not only is not limited by physical micro-channels and structures, but also the movement path and volume of the micro-droplets can be flexibly set.

Description

Cell sorting active digital micro-fluidic device and implementation method thereof
Technical Field
The invention relates to a medical health data technology, in particular to a cell sorting active digital micro-fluidic device and an implementation method thereof.
Background
This is particularly important for cell sorting, especially for sperm cell screening. Semen contains white blood cells, epithelial cells and some nutrient substances suitable for sperm life, the volume ratio of sperm is only 10%, and the situation that normal forms of sperm are less than 4% of all sperm is likely to be faced in actual operation due to various pathological reasons, so that higher requirements are put on sperm sorting. Therefore, the screening platform for researching the simple and quick operation and low cost, accurately separating sperms, minimizing the damage to sperms and guaranteeing the activity of sperms is particularly critical.
Currently, the methods for screening sperm commonly used in clinic mainly comprise a density gradient centrifugation method, a direct upstream method, a simple washing method and the like. The density gradient centrifugation method separates sperm by means of the motility of motile sperm and the density difference of various cells, so that the sperm forms soft sediment at the bottom of a centrifuge tube. The method has the advantages of higher sperm recovery rate and stable result, but excessive centrifugation operation is easy to cause peroxidation damage and breakage of sperm DNA, so that the quality of sperm is reduced, and the like. The direct upstream method utilizes the motion capability of the motile sperms from upstream to the surface level of the culture solution, thereby achieving the purpose of removing seminal plasma and impurities in the seminal plasma so as to obtain pure sperms. The method has the advantages of simple operation and low cost, but has relatively low recovery rate and long time, and is not suitable for treating the condition of fewer sperms. The simple washing method is to dilute the semen by the sperm culture solution and directly centrifugally collect the precipitated sperm mass, and the method can recover more sperms, but dead sperms and other cell fragments may exist due to poor separation effect. In addition to these three traditional methods, more novel sperm screening methods have been proposed in recent years, with magnetic activated sperm cell sorting (magnetic activated sperm cell sorting, MACS), hyaluronic acid binding (hyaluronic acid binding) and morphological examination (ultra morphological sperm assessment) being representative. However, according to researches, the novel methods still have the defects in terms of effectiveness and stability, and the whole process has the problems of complex and repeated operation, difficult control of operation quality, manual operation errors and the like.
Digital microfluidic (digital microfluidics, DMF) technology is a novel droplet manipulation technology based on microelectrode arrays to achieve precise control of discrete droplets (including movement, merging, dispensing, splitting, etc.), which utilizes the dielectric wetting phenomenon of droplets on a hydrophobized surface to achieve precise manipulation of microdroplets. The control mode has the characteristics of high parallelism and full automation, and can realize multichannel real-time controllable reaction. The technology can ensure that sperms are in stable fluid, can complete separation through the steps of movement, splitting and the like of liquid drops under the condition of no damage, and can screen sperms with parameters meeting requirements through observing and analyzing the forms and the movement states of sperms in each liquid drop. The technology can form stable fluid for enabling sperms to freely move, reduces manual operation, avoids mechanical damage caused by sperms, and ensures activity of sperms. The DMF technology is adopted in the scheme, so that time and cost are reduced, more accurate cell separation can be realized, and the sperm recovery rate is higher; meanwhile, the technology can finish automatic sorting work under machine vision, and can capture living sperm with single vitality and good morphology, and simultaneously save the vitality and the morphology information of the sperm for clinical use and tracing; the technology can be applied to screening and detection of sperm libraries and artificial insemination of animal husbandry to obtain more high-quality varieties, and can play a great role in the reproduction protection of endangered species. The operation of separating sperms on the integrated DMF platform is simple and convenient, compared with the centrifugal method, the method has less damage, can observe the screening result in real time, and has great advantages in improving the sperm separation efficiency and quantifying the sperm morphology and movement.
Disclosure of Invention
Based on the above technology and requirements, the invention provides a cell sorting active digital micro-fluidic device and an implementation method thereof.
An object of the present invention is to propose an active digital microfluidic device for cell sorting.
The cell sorting active digital micro-fluidic device of the invention comprises: the micro-droplet control chip, the row-column address controller, the camera, the displacement platform and the upper computer; the micro-droplet control chip is connected to the row-column address controller, and the row-column address controller is connected to the upper computer; the camera is positioned above the micro-droplet automatic control chip, and the micro-droplet automatic control chip is arranged on the displacement platform; the row-column address controller, the displacement platform and the camera are connected to the upper computer;
the micro-droplet control chip comprises an upper polar plate, a lower polar plate and a conductive gasket; the lower polar plate sequentially comprises a lower substrate, an electrode array, a dielectric layer and a lower hydrophobic layer from bottom to top; an electrode array is arranged on the lower substrate, and comprises a two-dimensional driving electrode array, a ground electrode and a contact electrode; the ground electrode is grounded; the two-dimensional driving electrode array comprises M rows and N columns of driving electrodes, M row conductors and N column conductors, M and N are natural numbers which are more than or equal to 2, each driving electrode comprises a three-wire bidirectional switch, a capacitor and a pixel electrode, and the three-wire bidirectional switch comprises a first main terminal, a grid electrode and a second main terminal; in the driving electrode of the ith row and the jth column, i is more than or equal to 1 and less than or equal to M, j is more than or equal to 1 and less than or equal to N, a second main terminal of the three-wire bidirectional switch is connected to one end of a capacitor and a pixel electrode, the other end of the capacitor is grounded, a grid electrode of the three-wire bidirectional switch is connected to the ith row wire, a first main terminal of the three-wire bidirectional switch is connected to the jth column wire, the M row wire and the N column wire are respectively connected to a row and column address controller through contact electrodes, when the jth column wire and the ith row wire are simultaneously electrified, the pixel electrode positioned in the ith row and the jth column is electrified, the jth column wire is electrified, and the pixel electrode positioned in the ith row and the jth column is electrified, so that the corresponding pixel electrode is controlled to be electrified and disconnected; in order to avoid signal crosstalk of multi-column parallel control and simultaneously improve scanning efficiency, the two-dimensional driving electrode array drives the pixel electrodes in a column-by-column scanning mode, so that power-on control is realized: the power of the row wire of the pixel electrode which is required to be switched from the power-on state to the power-off state in the j column is cut off, so that the power-off control of the pixel electrode which is required to be switched from the power-on state to the power-off state in the j column is realized, then the power-on of the row wire which is required to be switched from the power-off state to the power-on state in the j column is realized, the power-on control of the pixel electrode which is not required to be switched from the power-off state in the j column is always kept, the power-on state refresh of the pixel electrode in the j column is controlled, and the power-on control of the pixel electrode in the two-dimensional pixel electrode array is realized through the scanning column by column; covering a dielectric layer on the two-dimensional driving electrode array, covering a lower hydrophobic layer on the dielectric layer, and positioning the micro liquid drops on the lower hydrophobic layer; the upper polar plate sequentially comprises a transparent upper substrate, a transparent conductive layer and a transparent upper hydrophobic layer, wherein the transparent upper substrate, the transparent conductive layer and the transparent upper hydrophobic layer are made of transparent materials, one or more sample injection holes and one or more sample removal holes are respectively formed in the upper polar plate, and the sample injection holes penetrate through the lower surface of the transparent upper substrate to the upper surface of the transparent upper hydrophobic layer; the upper polar plate is reversely buckled on the lower polar plate, the transparent upper hydrophobic layer faces downwards to the lower hydrophobic layer of the lower polar plate, the transparent conductive layer is connected to the ground electrode through the conductive gasket, and the conductive gasket is positioned between the transparent upper hydrophobic layer of the upper polar plate and the lower hydrophobic layer of the lower polar plate; the cell suspension is injected onto the lower hydrophobic layer of the lower polar plate through the sample injection hole and can flow between the lower hydrophobic layer and the transparent upper hydrophobic layer;
Injecting cell suspension onto the lower hydrophobic layer of the lower polar plate through the sample injection hole, wherein the cell suspension covers a plurality of rows and columns of pixel electrodes, and the lower polar plate is not covered with the cell suspension and is positioned in the edge area of the lower polar plate to serve as a waste liquid area; the upper computer controls the displacement platform to drive the micro-droplet control chip to move, so that the cell suspension liquid positioned between the lower hydrophobic layer and the transparent upper hydrophobic layer is positioned in the imaging visual field of the camera, the camera collects the image of the cell suspension liquid, the image is input into the upper computer, a trained model is stored in the upper computer, the morphological characteristics and the movement characteristics of cells are obtained, and the cells meeting the requirements are selected according to a set threshold value to serve as target cells; the upper computer is positioned on the pixel electrode of the ith row and the jth column according to the position of the target cell acquired by the image of the camera, the upper computer controls the pixel electrode of the ith row where the target cell is positioned to be electrified through the row-column address controller, simultaneously, the pixel electrodes of the ith row and the lower k alternate rows are electrified, namely the pixel electrodes of the ith-2 row to the ith-k-1 row and the ith+2 row to the ith+k+1 row are electrified, k is a natural number more than or equal to 1, simultaneously, the pixel electrodes of the upper row and the lower adjacent row are powered off, the electrified pixel electrodes generate dielectric wetting force, and the cell suspension is respectively pulled to the upper side and the lower side so as to split the cell suspension into three parts along the upper direction and the lower direction, and the suspension containing the target cell forms a long strip shape distributed along the rows and is positioned on the pixel electrode of the ith row; the upper computer controls the pixel electrode of the j-th column of the ith row and the pixel electrodes of the left and right two columns which are alternately arranged to be electrified through the row and column address controller, namely, the pixel electrodes of the j-2 th column to the j-m-1 th column and the j+2 th column to the j+m+1 th column of the ith row are electrified, m is a natural number more than or equal to 1, meanwhile, the pixel electrodes of the left and right adjacent columns are powered off, dielectric wetting force is generated by the electrified pixel electrodes, and the cell suspension is pulled to the left and right sides respectively, so that the cell suspension on the pixel electrode of the ith row is split into three parts along the left and right directions, and the suspension containing target cells forms micro drops and is positioned on the pixel electrode of the j-th column of the ith row; the upper computer controls the row-column address controller to sequentially turn on and off the pixel electrodes on the paths leading to the waste liquid area, and the cell suspension without target cells is collected and led into the waste liquid area; the upper computer sets a moving path of the cell suspension containing the target cells, then controls the row-column address controller to cut off the power of a pixel electrode where the cell suspension containing the target cells is located, the next pixel electrode located on the moving path is electrified, the electrified pixel electrode generates dielectric wetting force, the cell suspension containing the target cells moves to the next pixel electrode under the action of the dielectric wetting force, and the upper computer controls the row-column address controller to sequentially cut off the power of the pixel electrodes located on the moving path respectively, so as to drive the cell suspension containing the target cells to move according to the set moving path until a sample containing the cell suspension containing the target cells moves to a designated position is moved out of the hole; the sample removing hole at the designated position is connected to a negative pressure pump through a liquid transferring gun or through a connecting pipe, and the selected target cells are removed from the chip through the sample removing hole, so that a cell suspension liquid only containing the target cells is finally obtained for subsequent application.
The lower substrate is one of glass, silicon, paper, polyester film and PCB (printed circuit board); the upper substrate is made of transparent material glass or an acrylic plate; the dielectric layer is used for accumulatingThe charge prevents the electrode from being broken down in the operation process of liquid drops, and the dielectric layer is made of parylene, SU-8 photoresist and SiO 2 、Si 3 N 4 、Al 2 O 3 Polydimethyl siloxane or parylene; the lower and upper hydrophobic layers are made of transparent materials, and are consolidated by spin coating and baking process with Teflon, CYTOP dispersion or FluoPel.
The conductive gaskets are arranged between the upper hydrophobic layer and the lower hydrophobic layer and used for supporting the upper polar plate, the conductive gaskets are arranged on the upper surface of the ground electrode of the lower polar plate and are symmetrically arranged in a strip shape, a plurality of centrally symmetrical or annular shapes, and the upper hydrophobic layer of the upper polar plate is positioned on the conductive gaskets; a through hole is formed in the upper hydrophobic layer corresponding to the conductive gasket, and the transparent conductive layer is connected to the conductive gasket through a wire through the through hole; the transparent conductive layer adopts indium tin oxide; the conductive gasket is made of conductive adhesive tape or colloid mixed with conductive microspheres, the thickness of the conductive adhesive tape is 0.1-500 mu m, and the diameter of the conductive microspheres is 0.1-500 mu m.
The pixel electrode, the ground electrode and the contact electrode are made of one of polysilicon, metal and metal oxide; the size of the pixel electrode is 0.1 μm by 0.1 μm to 500 μm by 500 μm. The size of the micro-droplets is 1nL to 125 mu L. i-k-1 is more than or equal to 1, and i+k+1 is less than or equal to M; j-m-1 is more than or equal to 1, and j+m+1 is less than or equal to N.
The cells to which the invention is directed are animal or human cells, such as sperm cells, circulating tumor cells, hybridoma B cells, T cells, NK cells, or the like.
Another object of the present invention is to provide a method for implementing a cell sorting active digital microfluidic device.
The invention discloses a realization method of an active digital micro-fluidic device for cell sorting, which comprises the following steps:
1) Injecting cell suspension onto the lower hydrophobic layer of the lower polar plate through the sample injection hole, wherein the cell suspension covers a plurality of rows and columns of pixel electrodes, and the lower polar plate is not covered with the cell suspension and is positioned in the edge area of the lower polar plate to serve as a waste liquid area;
2) The displacement platform is controlled by the upper computer to drive the micro-droplet control chip to move, so that the cell suspension liquid positioned between the lower hydrophobic layer and the transparent upper hydrophobic layer is positioned in the imaging view field of the camera, and the camera acquires the image of the sample through the transparent upper polar plate;
3) The camera inputs the collected image of the cell suspension into the upper computer;
4) The upper computer stores a trained model to obtain morphological characteristics and movement characteristics of cells, and selects cells meeting the requirements according to a set threshold value to serve as target cells;
5) The upper computer is positioned on the pixel electrode of the ith row and the jth column according to the position of the target cell acquired by the image of the camera, the upper computer controls the pixel electrode of the ith row where the target cell is positioned to be electrified through the row-column address controller, simultaneously, the pixel electrodes of the ith row and the lower k alternate rows are electrified, namely the pixel electrodes of the ith-2 row to the ith-k-1 row and the ith+2 row to the ith+k+1 row are electrified, k is a natural number more than or equal to 1, simultaneously, the pixel electrodes of the upper row and the lower adjacent row are powered off, the electrified pixel electrodes generate dielectric wetting force, and the cell suspension is respectively pulled to the upper side and the lower side so as to split the cell suspension into three parts along the upper direction and the lower direction, and the suspension containing the target cell forms a long strip shape distributed along the rows and is positioned on the pixel electrode of the ith row; the upper computer controls the pixel electrode of the j-th column of the ith row and the pixel electrodes of the left and right two columns which are alternately arranged to be electrified through the row and column address controller, namely, the pixel electrodes of the j-2 th column to the j-m-1 th column and the j+2 th column to the j+m+1 th column of the ith row are electrified, m is a natural number more than or equal to 1, meanwhile, the pixel electrodes of the left and right adjacent columns are powered off, dielectric wetting force is generated by the electrified pixel electrodes, and the cell suspension is pulled to the left and right sides respectively, so that the cell suspension on the pixel electrode of the ith row is split into three parts along the left and right directions, and the suspension containing target cells forms micro drops and is positioned on the pixel electrode of the j-th column of the ith row;
6) The upper computer controls the row-column address controller to sequentially turn on and off the pixel electrodes on the paths leading to the waste liquid area, and the cell suspension without target cells is collected and led into the waste liquid area;
7) The upper computer sets a moving path of the cell suspension containing the target cells, then controls the row-column address controller to cut off the power of a pixel electrode where the cell suspension containing the target cells is located, the next pixel electrode located on the moving path is electrified, the electrified pixel electrode generates dielectric wetting force, the cell suspension containing the target cells moves to the next pixel electrode under the action of the dielectric wetting force, and the upper computer controls the row-column address controller to sequentially cut off the power of the pixel electrodes located on the moving path respectively, so as to drive the cell suspension containing the target cells to move according to the set moving path until a sample containing the cell suspension containing the target cells moves to a designated position is moved out of the hole;
8) The sample removing hole at the designated position is connected to a negative pressure pump through a liquid transferring gun or through a connecting pipe, and the selected target cells are removed from the chip through the sample removing hole, so that a cell suspension liquid only containing the target cells is finally obtained for subsequent application.
Cell selection of a plurality of cell suspensions is performed in parallel in different areas of the digital microfluidic chip according to the above procedure.
In the step 4), the upper computer selects cells meeting the requirements as target cells according to a set threshold, and the method comprises the following steps:
i. collecting images of a plurality of (more than five hundred) cells to form a cell data set;
step ii, based on the neural network, establishing a cell target detection model and an instance segmentation model;
establishing a cell assessment model based on the kinematic and morphological characteristics of the cell;
training a cell target detection model, an example segmentation model and a cell evaluation model through a cell data set respectively, inputting the cell target detection model, the example segmentation model and the cell evaluation model into the cell data set, and outputting the cell target detection model, the example segmentation model and the cell evaluation model into morphological characteristics and movement characteristics of cells;
v, a camera collects cell images, and inputs the cell images into a trained model to obtain morphological characteristics and movement characteristics of cells;
setting the threshold values of the morphological characteristics and the movement characteristics of the cells, and selecting the cells meeting the requirements as target cells according to the set threshold values.
In step 5), the voltage applied to the pixel electrode is 5V to 150V.
In order to avoid signal crosstalk of multi-column parallel control and simultaneously improve scanning efficiency, a two-dimensional driving electrode array drives pixel electrodes in a column-by-column scanning mode to realize power-on and power-off control of the pixel electrodes on a moving path, and the method comprises the following steps:
i. The power-off of the jth column conductor is realized, and the power-off control of the pixel electrode in the jth column, which is required to be switched from the power-on state to the power-off state, is realized by powering on the row conductor where the pixel electrode in the jth column, which is required to be switched from the power-on state to the power-off state, is located;
the j-th column conductor is electrified, and meanwhile, the row conductor where the pixel electrode in the j-th column needs to be switched from the power-off state to the power-on state is electrified, so that the power-on control of the pixel electrode in the j-th column needs to be switched from the power-off state to the power-on state is realized;
and thirdly, the row conductor of the pixel electrode which does not need to be switched in the on-off state in the jth column is always kept powered off, so that the on-off state refreshing of the pixel electrode in the jth column is controlled, and the on-off control of the pixel electrode in the two-dimensional pixel electrode array is realized through scanning column by column.
The invention has the advantages that:
according to the invention, the row-column address controller is used for carrying out row-column scanning control on the pixel electrodes, so that the number of driving electrodes is reduced, and the complex wiring problem between the pixel electrodes is solved; the real-time driving control of discrete micro-droplets is realized based on the two-dimensional driving electrode array, so that the precision is high; according to the morphology and the motion characteristics of the cells, high-flux single-cell parallel selection is realized; under the condition that cell samples are relatively rare and precious, the intelligent automatic traversing and selecting device can carry out intelligent automatic traversing and selecting on all cells in micro-droplets, simplifies complicated manual operation steps in a cell selecting workflow on the basis of guaranteeing the activity of the selected cells, and provides a flexible and efficient realization way for single cell sorting in a standardized and procedural mode, in particular for precise selecting of sperms; compared with the traditional single-cell droplet generation method in the micro-channel, the single-cell droplet generation method is not limited by the physical micro-channel and the structure, and the movement path and the volume of the micro-droplet can be flexibly set; according to the invention, the micro-droplets containing single cells are independently controlled through the programmable pixel electrode, and an electronic code can be distributed to each selected single cell based on a vision system, so that the target cell calibration and tracking are not required by the traditional bar code.
Drawings
FIG. 1 is a block diagram of a sperm sorting active digital microfluidic device of the present invention;
FIG. 2 is a cross-sectional view of a micro-droplet manipulation chip of one embodiment of a sperm sorting active digital microfluidic device of the present invention;
FIG. 3 is a schematic diagram of one drive electrode of a micro-droplet manipulation chip of one embodiment of a sperm-sorting active digital microfluidic device of the present invention;
fig. 4 is a schematic diagram of an embodiment of the active digital microfluidic device for sperm sorting according to the present invention for driving movement of a sperm suspension containing target cells, where (a) - (f) are schematic diagrams of recognition, pixel electrode power-on control for row separation, pixel electrode power-on control for column separation, droplet movement path after column separation and separation, and results of continuous sorting, respectively.
Detailed Description
The invention will be further elucidated by means of specific embodiments in conjunction with the accompanying drawings.
As shown in fig. 1, the cell sorting active digital microfluidic device of the present embodiment includes: the micro-droplet control chip, the row-column address controller, the camera, the displacement platform and the upper computer; the micro-droplet control chip is connected to the row-column address controller, and the row-column address controller is connected to the upper computer; the camera is positioned above the micro-droplet automatic control chip, and the micro-droplet automatic control chip is arranged on the displacement platform; the row-column address controller, the displacement platform and the camera are connected to the upper computer;
As shown in fig. 2, the micro-droplet manipulation chip comprises an upper plate, a lower plate and a conductive pad; the lower polar plate sequentially comprises a lower substrate 6, an electrode array, a dielectric layer 5 and a lower hydrophobic layer 4 from bottom to top; an electrode array is arranged on the lower substrate, and comprises a two-dimensional driving electrode 7 array, a ground electrode 8 and a contact electrode; the ground electrode is grounded; the two-dimensional driving electrode array comprises M rows, N columns, M row conductors and N column conductors, M and N are natural numbers which are more than or equal to 2, each driving electrode comprises a three-wire bidirectional switch, a capacitor and a pixel electrode 7.1, and the three-wire bidirectional switch comprises a first main terminal, a grid electrode and a second main terminal; as shown in fig. 3, in the driving electrode of the ith row and the jth column, 1.ltoreq.i.ltoreq.m, 1.ltoreq.j.ltoreq.n, a second main terminal of the three-wire bidirectional switch is connected to one end of the capacitor and the pixel electrode, the other end of the capacitor is grounded, a gate of the three-wire bidirectional switch is connected to the ith row wire, a first main terminal of the three-wire bidirectional switch is connected to the jth column wire, the M row wire and the N column wire are respectively connected to the row and column address controller through contact electrodes, when the jth column wire and the ith row wire are simultaneously electrified, the pixel electrode positioned in the ith row and the jth column is electrified, the jth column wire is electrified, and the pixel electrode positioned in the jth row and the jth column is powered off, so that the corresponding pixel electrode is controlled to be electrified and powered off; in order to avoid signal crosstalk of multi-column parallel control and simultaneously improve scanning efficiency, the two-dimensional driving electrode array drives the pixel electrodes in a column-by-column scanning mode, so that power-on control is realized: the power of the row wire of the pixel electrode which is required to be switched from the power-on state to the power-off state in the j column is cut off, so that the power-off control of the pixel electrode which is required to be switched from the power-on state to the power-off state in the j column is realized, then the power-on of the row wire which is required to be switched from the power-off state to the power-on state in the j column is realized, the power-on control of the pixel electrode which is not required to be switched from the power-off state in the j column is always kept, the power-on state refresh of the pixel electrode in the j column is controlled, and the power-on control of the pixel electrode in the two-dimensional pixel electrode array is realized through the scanning column by column; covering a dielectric layer on the two-dimensional driving electrode array, covering a lower hydrophobic layer on the dielectric layer, and positioning the micro liquid drops on the lower hydrophobic layer; the upper polar plate sequentially comprises a transparent upper substrate 1, a transparent conductive layer and a transparent upper hydrophobic layer 2, wherein the transparent upper substrate, the transparent conductive layer and the transparent upper hydrophobic layer are made of transparent materials, a plurality of sample injection holes 10 and a plurality of sample removal holes 11 are formed in the upper polar plate, and the sample injection holes and the sample removal holes penetrate through the lower surface of the transparent upper substrate to the upper surface of the transparent upper hydrophobic layer; the upper polar plate is reversely buckled on the lower polar plate, the transparent upper hydrophobic layer faces downwards to the lower hydrophobic layer of the lower polar plate, the transparent conductive layer is connected to the ground electrode through the conductive gasket 3, and the conductive gasket is positioned between the transparent upper hydrophobic layer of the upper polar plate and the lower hydrophobic layer of the lower polar plate; the sperm suspension 9 is injected onto the lower hydrophobic layer of the lower polar plate through the sample injection hole and can flow between the lower hydrophobic layer and the transparent upper hydrophobic layer; a liquid 12 for preventing evaporation is filled between the transparent upper hydrophobic layer of the upper polar plate and the lower hydrophobic layer of the lower polar plate;
In the embodiment, sperm cells are screened, sperm suspension is injected into a lower hydrophobic layer of a lower polar plate through a sample injection hole, the sperm suspension covers a plurality of rows and columns of pixel electrodes, the lower polar plate is not covered with the sperm suspension, and the edge area of the lower polar plate is used as a waste liquid area; the upper computer controls the displacement platform to drive the micro-droplet control chip to move, so that the sperm suspension liquid positioned between the lower hydrophobic layer and the transparent upper hydrophobic layer is positioned in the imaging visual field of the camera, the camera collects images of the sperm suspension liquid, the images are input into the upper computer, a trained model is stored in the upper computer, the morphological characteristics and the movement characteristics of sperms are obtained, and sperms meeting the requirements are selected according to a set threshold value to serve as target cells; the upper computer is used for configuring a unique electronic code for target cells and is used for target cell calibration and tracking; as shown in fig. 4, the upper computer is located on the pixel electrodes of the fourth row and the fourth column according to the position of the target cells acquired by the image of the camera, the upper computer controls the pixel electrodes of the third row where the target cells are located to be electrified through the row-column address controller, meanwhile, the pixel electrodes of the first row and the fifth row and the sixth row are electrified, and meanwhile, the pixel electrodes of the upper row and the lower row are powered off, the electrified pixel electrodes generate dielectric wetting force, and the sperm suspension is pulled to the upper side and the lower side respectively, so that the sperm suspension is split into three parts along the upper direction, the suspension containing the target cells forms a long strip shape distributed along the row and is located on the pixel electrodes of the third row, in fig. 3, "on" indicates that the pixel electrodes are electrified, and "off" indicates that the pixel electrodes are powered off; the upper computer controls the pixel electrodes of the fourth column of the third row where the target sperm suspension is located and the pixel electrodes of the left two columns and the right one column which are alternately electrified through the row-column address controller, namely, the pixel electrodes of the first column, the second column and the sixth column are electrified, meanwhile, the pixel electrodes of the left and right adjacent columns are powered off, dielectric wetting force is generated by the electrified pixel electrodes, and the sperm suspension is respectively pulled to the left and right sides, so that the sperm suspension on the pixel electrodes of the third row is split into three parts along the left and right directions, and the suspension containing target cells forms micro drops and is positioned on the pixel electrodes of the fourth column of the third row; the upper computer controls the row-column address controller to turn on and off the pixel electrode, and the sperm suspension without target cells is collected and introduced into the waste liquid area; the upper computer sets a moving path of the sperm suspension containing the target cells, and then controls the row-column address controller to respectively electrify pixel electrodes positioned on the moving path in sequence, so as to drive the sperm suspension containing the target cells to move according to the set moving path until the sperm suspension containing the target cells is moved to a sample moving hole at a specified position; selecting sperms of a plurality of sperm suspensions, and operating in parallel in different areas of the digital microfluidic chip according to the process; the sperm suspension containing the target cells is removed from the micro-droplet control chip at the sample removal hole at the designated position through a pipette or a connecting pipe, and finally the sperm suspension containing only the target cells is obtained and used for subsequent pathological analysis, assisted reproduction, cell injection and other applications.
In this embodiment, the lower substrate and the upper substrate are glass; the material of the dielectric layer adopts parylene; the lower hydrophobic layer and the upper hydrophobic layer are fixedly connected by adopting Teflon through spin coating and baking processes; the pixel electrode, the ground electrode and the contact electrode adopt metal chromium; the size of the pixel electrode is 60 μm×60 μm; the transparent conductive layer adopts indium tin oxide; the conductive pad is made of conductive adhesive tape with thickness of 10 μm.
The implementation method of the cell sorting active digital microfluidic device of the embodiment screens sperm cells, and comprises the following steps:
1) Injecting the sperm suspension onto the lower hydrophobic layer of the lower polar plate through the sample injection hole, wherein the sperm suspension covers a plurality of rows and columns of pixel electrodes, and the lower polar plate is not covered with the sperm suspension and is positioned in the edge area of the lower polar plate to serve as a waste liquid area;
2) The displacement platform is controlled by the upper computer to drive the micro-droplet control chip to move, so that the sperm suspension liquid positioned between the lower hydrophobic layer and the transparent upper hydrophobic layer is positioned in the imaging view field of the camera, and the camera acquires the image of the sample through the transparent upper polar plate;
3) The camera inputs the acquired image of the sperm suspension into the upper computer;
4) The upper computer stores a trained model to obtain morphological characteristics and movement characteristics of sperms, and selects sperms meeting the requirements as target cells according to a set threshold value; the upper computer is used for configuring a unique electronic code for target cells and is used for target cell calibration and tracking;
5) The upper computer is positioned on the pixel electrodes of the fourth row of the third row according to the position of the target cells acquired by the image of the camera, the upper computer controls the pixel electrodes of the third row of the target cells to be electrified through the row-column address controller, simultaneously the pixel electrodes of the first row and the fifth row and the sixth row are electrified, simultaneously the pixel electrodes of the second row and the fourth row which are the upper adjacent row and the lower adjacent row are powered off, dielectric wetting force is generated by the electrified pixel electrodes, and sperm suspension is pulled to the upper side and the lower side respectively, so that the sperm suspension is split into three parts along the upper direction and the lower direction, and the suspension containing the target cells forms long strips distributed along the rows and is positioned on the pixel electrodes of the third row; the upper computer controls the pixel electrodes of a fourth column of a third row where the target sperm suspension is located and the pixel electrodes of left and right two rows and an alternate column respectively to be electrified through a row-column address controller, namely the pixel electrodes of the fourth column and the pixel electrodes of the left two columns and the right one row and the alternate column are electrified, namely the pixel electrodes of the first column and the second column of the third row and the pixel electrodes of the sixth column are simultaneously electrified, and simultaneously the pixel electrodes of the left and right adjacent columns, namely the pixel electrodes of the third column and the fifth column are powered off, dielectric wetting force is generated by the electrified pixel electrodes, and the sperm suspension is pulled to the left and right sides respectively, so that the sperm suspension on the pixel electrodes of the third row is split into three parts along the left and right directions, and the suspension containing target cells forms micro droplets and is positioned on the pixel electrodes of the fourth column of the third row;
6) The upper computer controls the row-column address controller to sequentially turn on and off the pixel electrodes on the paths leading to the waste liquid area, and the sperm suspension without target cells is collected and introduced into the waste liquid area;
7) The upper computer sets a moving path of the sperm suspension containing the target cells, then controls the row-column address controller to cut off the power of a pixel electrode where the sperm suspension containing the target cells is located, the next pixel electrode on the moving path is electrified, the electrified pixel electrode generates dielectric wetting force, the sperm suspension containing the target cells moves to the next pixel electrode under the action of the dielectric wetting force, and the upper computer controls the row-column address controller to sequentially cut off the power of the pixel electrodes on the moving path respectively, so as to drive the sperm suspension containing the target cells to move according to the set moving path until a sample moving hole for moving the sperm suspension containing the target cells to a designated position; selecting sperms of a plurality of sperm suspensions, and operating in parallel in different areas of the digital microfluidic chip according to the process;
8) The sample removing hole at the designated position is connected to a negative pressure pump through a liquid transferring gun or through a connecting pipe, and the selected target cells are removed from the chip through the sample removing hole, so that the sperm suspension only containing the target cells is finally obtained and used for rear-end pathology analysis, auxiliary reproduction, cell injection and other applications.
In the step 4), the upper computer selects the sperms meeting the requirements as target cells according to the set threshold, and the method comprises the following steps:
i. collecting images of more than five hundred sperms to form a sperm data set;
establishing a sperm target detection model and an example segmentation model based on the neural network;
establishing a sperm evaluation model based on the kinematic and morphological characteristics of the sperm;
training a sperm target detection model, an example segmentation model and a sperm evaluation model through a sperm data set respectively, inputting the sperm data set into the sperm target detection model, the example segmentation model and the sperm evaluation model, and outputting the sperm data set into morphological characteristics and movement characteristics of sperms;
v, a camera collects sperm images, and inputs the sperm images into a trained model to obtain morphological characteristics and movement characteristics of sperms;
selecting the sperms meeting the requirements according to the set threshold value to be used as target cells.
In step 6), in order to avoid signal crosstalk of multi-column parallel control and simultaneously improve scanning efficiency, the two-dimensional driving electrode array drives the pixel electrode in a column-by-column scanning manner, so as to realize power-on and power-off control of the pixel electrode on a moving path, and the method comprises the following steps:
i. the power-off of the jth column conductor is realized, and the power-off control of the pixel electrode in the jth column, which is required to be switched from the power-on state to the power-off state, is realized by powering on the row conductor where the pixel electrode in the jth column, which is required to be switched from the power-on state to the power-off state, is located;
The j-th column conductor is electrified, and meanwhile, the row conductor where the pixel electrode in the j-th column needs to be switched from the power-off state to the power-on state is electrified, so that the power-on control of the pixel electrode in the j-th column needs to be switched from the power-off state to the power-on state is realized;
and thirdly, the row conductor of the pixel electrode which does not need to be switched in the on-off state in the jth column is always kept powered off, so that the on-off state refreshing of the pixel electrode in the jth column is controlled, and the on-off control of the pixel electrode in the two-dimensional pixel electrode array is realized through scanning column by column.
Finally, it should be noted that the examples are disclosed for the purpose of aiding in the further understanding of the present invention, but those skilled in the art will appreciate that: various alternatives and modifications are possible without departing from the spirit and scope of the invention and the appended claims. Therefore, the invention should not be limited to the disclosed embodiments, but rather the scope of the invention is defined by the appended claims.

Claims (10)

1. A cell sorting active digital microfluidic device, the cell sorting active digital microfluidic device comprising: the micro-droplet control chip, the row-column address controller, the camera, the displacement platform and the upper computer; the micro-droplet control chip is connected to the row-column address controller, and the row-column address controller is connected to the upper computer; the camera is positioned above the micro-droplet automatic control chip, and the micro-droplet automatic control chip is arranged on the displacement platform; the row-column address controller, the displacement platform and the camera are connected to the upper computer;
The micro-droplet control chip comprises an upper polar plate, a lower polar plate and a conductive gasket; the lower polar plate sequentially comprises a lower substrate, an electrode array, a dielectric layer and a lower hydrophobic layer from bottom to top; an electrode array is arranged on the lower substrate, and comprises a two-dimensional driving electrode array, a ground electrode and a contact electrode; the ground electrode is grounded; the two-dimensional driving electrode array comprises M rows and N columns of driving electrodes, M row conductors and N column conductors, M and N are natural numbers which are more than or equal to 2, each driving electrode comprises a three-wire bidirectional switch, a capacitor and a pixel electrode, and the three-wire bidirectional switch comprises a first main terminal, a grid electrode and a second main terminal; in the driving electrode of the ith row and the jth column, i is more than or equal to 1 and less than or equal to M, j is more than or equal to 1 and less than or equal to N, a second main terminal of the three-wire bidirectional switch is connected to one end of a capacitor and a pixel electrode, the other end of the capacitor is grounded, a grid electrode of the three-wire bidirectional switch is connected to the ith row wire, a first main terminal of the three-wire bidirectional switch is connected to the jth column wire, the M row wire and the N column wire are respectively connected to a row and column address controller through contact electrodes, when the jth column wire and the ith row wire are simultaneously electrified, the pixel electrode positioned in the ith row and the jth column is electrified, the jth column wire is electrified, and the pixel electrode positioned in the ith row and the jth column is electrified, so that the corresponding pixel electrode is controlled to be electrified and disconnected; in order to avoid signal crosstalk of multi-column parallel control and simultaneously improve scanning efficiency, the two-dimensional driving electrode array drives the pixel electrodes in a column-by-column scanning mode, so that power-on control is realized: the power of the row wire of the pixel electrode which is required to be switched from the power-on state to the power-off state in the j column is cut off, so that the power-off control of the pixel electrode which is required to be switched from the power-on state to the power-off state in the j column is realized, then the power-on of the row wire which is required to be switched from the power-off state to the power-on state in the j column is realized, the power-on control of the pixel electrode which is not required to be switched from the power-off state in the j column is always kept, the power-on state refresh of the pixel electrode in the j column is controlled, and the power-on control of the pixel electrode in the two-dimensional pixel electrode array is realized through the scanning column by column; covering a dielectric layer on the two-dimensional driving electrode array, covering a lower hydrophobic layer on the dielectric layer, and positioning the micro liquid drops on the lower hydrophobic layer; the upper polar plate sequentially comprises a transparent upper substrate, a transparent conductive layer and a transparent upper hydrophobic layer, wherein the transparent upper substrate, the transparent conductive layer and the transparent upper hydrophobic layer are made of transparent materials, one or more sample injection holes and one or more sample removal holes are respectively formed in the upper polar plate, and the sample injection holes penetrate through the lower surface of the transparent upper substrate to the upper surface of the transparent upper hydrophobic layer; the upper polar plate is reversely buckled on the lower polar plate, the transparent upper hydrophobic layer faces downwards to the lower hydrophobic layer of the lower polar plate, the transparent conductive layer is connected to the ground electrode through the conductive gasket, and the conductive gasket is positioned between the transparent upper hydrophobic layer of the upper polar plate and the lower hydrophobic layer of the lower polar plate; the cell suspension is injected onto the lower hydrophobic layer of the lower polar plate through the sample injection hole and can flow between the lower hydrophobic layer and the transparent upper hydrophobic layer;
Injecting cell suspension onto the lower hydrophobic layer of the lower polar plate through the sample injection hole, wherein the cell suspension covers a plurality of rows and columns of pixel electrodes, and the lower polar plate is not covered with the cell suspension and is positioned in the edge area of the lower polar plate to serve as a waste liquid area; the displacement platform is controlled by the upper computer to drive the micro-droplet control chip to move, so that the cell suspension liquid positioned between the lower hydrophobic layer and the transparent upper hydrophobic layer is positioned in the imaging view field of the camera; the camera collects images of the cell suspension and inputs the images into the upper computer; the upper computer stores a trained model to obtain morphological characteristics and movement characteristics of cells, and selects cells meeting the requirements according to a set threshold value to serve as target cells; the upper computer is positioned on the pixel electrode of the ith row and the jth column according to the position of the target cell acquired by the image of the camera, the upper computer controls the pixel electrode of the ith row where the target cell is positioned to be electrified through the row-column address controller, simultaneously, the pixel electrodes of the ith row and the lower k alternate rows are electrified, namely the pixel electrodes of the ith-2 row to the ith-k-1 row and the ith+2 row to the ith+k+1 row are electrified, k is a natural number more than or equal to 1, simultaneously, the pixel electrodes of the upper row and the lower adjacent row are powered off, the electrified pixel electrodes generate dielectric wetting force, and the cell suspension is respectively pulled to the upper side and the lower side so as to split the cell suspension into three parts along the upper direction and the lower direction, and the suspension containing the target cell forms a long strip shape distributed along the rows and is positioned on the pixel electrode of the ith row; the upper computer controls the pixel electrode of the j-th column of the ith row and the pixel electrodes of the left and right two columns which are alternately arranged to be electrified through the row and column address controller, namely, the pixel electrodes of the j-2 th column to the j-m-1 th column and the j+2 th column to the j+m+1 th column of the ith row are electrified, m is a natural number more than or equal to 1, meanwhile, the pixel electrodes of the left and right adjacent columns are powered off, dielectric wetting force is generated by the electrified pixel electrodes, and the cell suspension is pulled to the left and right sides respectively, so that the cell suspension on the pixel electrode of the ith row is split into three parts along the left and right directions, and the suspension containing target cells forms micro drops and is positioned on the pixel electrode of the j-th column of the ith row; the upper computer controls the row-column address controller to sequentially turn on and off the pixel electrodes on the paths leading to the waste liquid area, and respectively receives and introduces the cell suspension which does not contain target cells into the waste liquid area; the upper computer sets a moving path of the cell suspension containing the target cells, then controls the row-column address controller to cut off the power of a pixel electrode where the cell suspension containing the target cells is located, the next pixel electrode located on the moving path is electrified, the electrified pixel electrode generates dielectric wetting force, the cell suspension containing the target cells moves to the next pixel electrode under the action of the dielectric wetting force, and the upper computer controls the row-column address controller to sequentially cut off the power of the pixel electrodes located on the moving path respectively, so as to drive the cell suspension containing the target cells to move according to the set moving path until a sample containing the cell suspension containing the target cells moves to a designated position is moved out of the hole; the sample removing hole at the designated position is connected to a negative pressure pump through a liquid transferring gun or through a connecting pipe, and the selected target cells are removed through the sample removing hole, so that a cell suspension liquid only containing the target cells is finally obtained for subsequent application.
2. The cell sorting active digital microfluidic device of claim 1, wherein the pixel electrode, ground electrode and contact electrode are one of polysilicon, metal and metal oxide.
3. The cell sorting active digital microfluidic device of claim 1, wherein the pixel electrode has dimensions of 1 μm x 1 μm to 500 μm x 500 μm.
4. The cell sorting active digital microfluidic device according to claim 1, wherein the conductive pad is disposed between the upper hydrophobic layer and the lower hydrophobic layer for supporting the upper electrode plate, and the conductive pad is disposed on the upper surface of the ground electrode of the lower electrode plate.
5. The cell sorting active digital microfluidic device according to claim 1, wherein a through hole is formed at a position of the upper hydrophobic layer corresponding to the conductive pad, and the transparent conductive layer is connected to the conductive pad through a wire through the through hole.
6. The cell sorting active digital microfluidic device according to claim 1, wherein said lower substrate is one of glass, silicon, paper, mylar, and printed circuit board; the upper substrate is a transparent material.
7. The cell sorting active digital microfluidic device of claim 1, wherein the lower and upper hydrophobic layers are made of transparent hydrophobic materials.
8. A method of implementing the cell sorting active digital microfluidic device of claim 1, comprising the steps of:
1) Injecting cell suspension onto the lower hydrophobic layer of the lower polar plate through the sample injection hole, wherein the cell suspension covers a plurality of rows and columns of pixel electrodes, and the lower polar plate is not covered with the cell suspension and is positioned in the edge area of the lower polar plate to serve as a waste liquid area;
2) The displacement platform is controlled by the upper computer to drive the micro-droplet control chip to move, so that the cell suspension liquid positioned between the lower hydrophobic layer and the transparent upper hydrophobic layer is positioned in the imaging view field of the camera, and the camera acquires the image of the sample through the transparent upper polar plate;
3) The camera inputs the collected image of the cell suspension into the upper computer;
4) The upper computer stores a trained model to obtain morphological characteristics and movement characteristics of cells, and selects cells meeting the requirements according to a set threshold value to serve as target cells;
5) The upper computer is positioned on the pixel electrode of the ith row and the jth column according to the position of the target cell acquired by the image of the camera, the upper computer controls the pixel electrode of the ith row where the target cell is positioned to be electrified through the row-column address controller, simultaneously, the pixel electrodes of the ith row and the lower k alternate rows are electrified, namely the pixel electrodes of the ith-2 row to the ith-k-1 row and the ith+2 row to the ith+k+1 row are electrified, k is a natural number more than or equal to 1, simultaneously, the pixel electrodes of the upper row and the lower adjacent row are powered off, the electrified pixel electrodes generate dielectric wetting force, and the cell suspension is respectively pulled to the upper side and the lower side so as to split the cell suspension into three parts along the upper direction and the lower direction, and the suspension containing the target cell forms a long strip shape distributed along the rows and is positioned on the pixel electrode of the ith row; the upper computer controls the pixel electrode of the j-th column of the ith row and the pixel electrodes of the left and right two columns which are alternately arranged to be electrified through the row and column address controller, namely, the pixel electrodes of the j-2 th column to the j-m-1 th column and the j+2 th column to the j+m+1 th column of the ith row are electrified, m is a natural number more than or equal to 1, meanwhile, the pixel electrodes of the left and right adjacent columns are powered off, dielectric wetting force is generated by the electrified pixel electrodes, and the cell suspension is pulled to the left and right sides respectively, so that the cell suspension on the pixel electrode of the ith row is split into three parts along the left and right directions, and the suspension containing target cells forms micro drops and is positioned on the pixel electrode of the j-th column of the ith row;
6) The upper computer controls the row-column address controller to sequentially turn on and off the pixel electrodes on the paths leading to the waste liquid area, and introduces the cell suspension which does not contain target cells into the waste liquid area;
7) The upper computer sets a moving path of the cell suspension containing the target cells, then controls the row-column address controller to cut off the power of a pixel electrode where the cell suspension containing the target cells is located, the next pixel electrode located on the moving path is electrified, the electrified pixel electrode generates dielectric wetting force, the cell suspension containing the target cells moves to the next pixel electrode under the action of the dielectric wetting force, and the upper computer controls the row-column address controller to sequentially cut off the power of the pixel electrodes located on the moving path respectively, so as to drive the cell suspension containing the target cells to move according to the set moving path until a sample containing the cell suspension containing the target cells moves to a designated position is moved out of the hole;
8) The sample removing hole at the designated position is connected to a negative pressure pump through a liquid transferring gun or through a connecting pipe, and the selected target cells are removed from the chip through the sample removing hole, so that a cell suspension liquid only containing the target cells is finally obtained for subsequent application.
9. The method according to claim 8, wherein in step 4), the host computer selects cells meeting the requirements as target cells according to a set threshold, comprising the steps of:
i. Collecting images of a plurality of cells to form a cell data set;
step ii, based on the neural network, establishing a cell target detection model and an instance segmentation model;
establishing a cell assessment model based on the kinematic and morphological characteristics of the cell;
training a cell target detection model, an example segmentation model and a cell evaluation model through a cell data set respectively, inputting the cell target detection model, the example segmentation model and the cell evaluation model into the cell data set, and outputting the cell target detection model, the example segmentation model and the cell evaluation model into morphological characteristics and movement characteristics of cells;
v, a camera collects cell images, and inputs the cell images into a trained model to obtain morphological characteristics and movement characteristics of cells;
setting the threshold values of the morphological characteristics and the movement characteristics of the cells, and selecting the cells meeting the requirements as target cells according to the set threshold values.
10. The implementation method of claim 8, wherein in step 5), in order to avoid crosstalk of signals controlled by multiple columns in parallel and improve scanning efficiency at the same time, the two-dimensional driving electrode array drives the pixel electrodes in a column-by-column scanning manner, so as to realize power-on and power-off control of the pixel electrodes on a moving path, and the implementation method comprises the following steps:
i. the power-off of the jth column conductor is realized, and the power-off control of the pixel electrode in the jth column, which is required to be switched from the power-on state to the power-off state, is realized by powering on the row conductor where the pixel electrode in the jth column, which is required to be switched from the power-on state to the power-off state, is located;
The j-th column conductor is electrified, and meanwhile, the row conductor where the pixel electrode in the j-th column needs to be switched from the power-off state to the power-on state is electrified, so that the power-on control of the pixel electrode in the j-th column needs to be switched from the power-off state to the power-on state is realized;
and thirdly, the row conductor of the pixel electrode which does not need to be switched in the on-off state in the jth column is always kept powered off, so that the on-off state refreshing of the pixel electrode in the jth column is controlled, and the on-off control of the pixel electrode in the two-dimensional pixel electrode array is realized through scanning column by column.
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CN114778422A (en) * 2022-04-19 2022-07-22 北京理工大学 Automatic cell sorting system and method based on micro-droplet control technology

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