Digital micro-fluidic chip and system for drug screening

Abstract

The invention discloses a digital micro-fluidic chip and system for drug screening, and relates to the technical field of biology. The digital micro-fluidic chip adopts a double-layer composite electrode structure of an upper polar plate and a lower polar plate which are arranged in parallel, and a space for liquid drops to run is formed between the upper polar plate and the lower polar plate; and the lower polar plate adopts a stacking process and is composed of a printed circuit substrate, an electrode array, a dielectric layer and a hydrophobic coating from bottom to top. The digital microfluidic system takes the chip as a core and integrates a driving module, a high-precision temperature control module, a real-time optical detection module, a visual interaction interface, automatic operation software, image analysis software and a standardized kit. Through design of the digital micro-fluidic chip, cooperation of all modules of the digital micro-fluidic system and dynamic regulation and control of distribution of liquid drops, high-throughput screening of drug-target interaction is achieved, the drug screening efficiency and the automation level are remarkably improved, and the digital micro-fluidic chip is suitable for cell, tissue, organ, cell cluster or organoid level drug screening.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biotechnology, in particular to a digital microfluidic chip and system for drug screening, which is especially suitable for high-throughput drug activity detection and dose-effect relationship analysis at the level of cells, tissues, organs and organoids. BACKGROUND

[0002] Traditional drug screening methods mainly rely on microplate or microfluidic channel chips, which have problems such as large reagent consumption, limited throughput, complex operation, etc. Microplate screening usually requires a large number of drug compounds and cell samples, resulting in high cost, and it is difficult to achieve accurate regulation of dynamic concentration gradient. Although microfluidic channel chips can reduce reagent consumption, their fixed channel structure limits the flexibility and scalability of the reaction unit, which cannot meet the needs of high-throughput drug screening. In addition, the detection process of traditional methods relies on manual operation or complex external equipment, which is prone to errors and difficult to achieve full-process automation control.

[0003] Digital microfluidic technology is a technology that precisely controls the movement of discrete droplets through electrical signals. Its core is based on the principle of electrowetting, which adjusts the hydrophilic and hydrophobic properties of the electrode surface by outputting voltage, thereby driving the droplets to move on the surface of the electrode array and realizing operations such as droplet formation, movement, splitting, and merging. Compared with traditional microfluidic channel chips, digital microfluidic chips have the advantages of no channel design, high flexibility, and programmable control. This technology has been successfully applied in nucleic acid detection, protein analysis, cell culture, and other fields, showing the potential of low reagent consumption, high integration, and automation.

[0004] Currently, a few studies have attempted to use digital microfluidic technology for drug screening, but the functions are relatively single, usually only supporting static drug reactions, and cannot generate dynamic concentration gradients. In addition, the number of reaction units of existing chips is limited, making it difficult to meet the needs of automated high-throughput screening. The integration of detection modules and driving modules is insufficient, which also limits the real-time and accuracy of data. Therefore, it is urgent to develop a new type of digital microfluidic system to realize high-throughput, low-cost, and full-process automation of drug screening by optimizing chip design and improving multi-module collaboration capabilities. SUMMARY

[0005] The present application aims to address the lack of high-throughput drug screening based on digital microfluidic chips in the prior art, and provides a digital microfluidic chip and system for high-throughput drug screening. It takes advantage of the programmable nature of digital microfluidic technology to meet the urgent needs of drug research and development for efficient and low-cost screening, while opening up new technical paths for personalized medicine research.

[0006] To achieve the above object, the present application provides the following technical solutions.

[0007] The present application provides a digital microfluidic chip, comprising an upper plate and a lower plate.

[0008] The upper plate and the lower plate are arranged in parallel to form a space for liquid droplet operation, and the distance between the upper plate and the lower plate is 50-1000 microns.

[0009] The lower plate comprises the following layers arranged from bottom to top: a printed circuit substrate, a driving electrode, a dielectric layer and a hydrophobic layer.

[0010] The upper plate comprises the following layers arranged from top to bottom: an ITO glass layer and a hydrophobic layer, and the upper plate is provided with a liquid injection port for adding solution.

[0011] According to the arrangement of the driving electrode, the space between the upper plate and the lower plate for liquid droplet operation is divided into a liquid storage area and a reaction area.

[0012] The liquid storage area is arranged on the left side, the right side and the bottom of the chip, wherein the left and right side liquid storage areas each comprise a plurality of independent liquid storage pools, and the bottom liquid storage area is configured with a plurality of liquid storage pools, and the liquid storage pools on each side are arranged in parallel at equal distances. The liquid storage pools are used to store cell suspension, drug solution and required reagent solution, etc.; the reaction area comprises a plurality of reaction pools, and one reaction pool is composed of 6 electrodes, and forms a reaction unit in the form of a 3x2 electrode array; the cell suspension, the drug solution and the reagent solution, etc. are respectively distributed from the liquid storage pools through cross matrix arrangement to realize high-efficiency distribution, and then react in the reaction pools.

[0013] Further, the upper plate is further provided with an oil injection port and an oil outlet port; the oil injection port and the oil outlet port can be arranged diagonally.

[0014] Further, the diameter of the liquid injection port can be set according to sample requirements, and the diameters of the oil injection port and the oil outlet port can be 1mm.

[0015] Further, the upper plate and the lower plate can be sealed by double-sided adhesive tape and UV glue.

[0016] Further, the dielectric layer is one or more of SiO2, Al2O3, Parylene C and PDMS.

[0017] Further, the hydrophobic layer is one or more of Teflon AF and CYTOP.

[0018] Further, the driving electrode uses metal Cu or Au as the electrode layer material; the dielectric layer is prepared by chemical vapor deposition on the electrode layer using Parylene C; and the hydrophobic layer is prepared after spin coating and drying of the hydrophobic layer material on the dielectric layer.

[0019] Further, the specific shape of the driving electrode is a puzzle shape; the size of the driving electrode is 3 mm*3 mm; the driving electrode has 36 rows and 49 columns in total.

[0020] Further, the left and right liquid storage areas each contain 8 independent liquid storage pools, and the lower liquid storage area is configured with 12 liquid storage pools. The number of reaction pools in the reaction area is 80-120.

[0021] Preferably, the number of reaction pools in the reaction area is 96.

[0022] In another aspect, the present application provides a digital microfluidic system, which takes the above-mentioned digital microfluidic chip as the core, and further comprises a driving module, a high-precision temperature control module, a real-time optical detection module, a visual interactive interface, an automatic operation software, an image analysis software and a standardized reagent kit; the driving module is mainly composed of a development board, a flexible flat cable and an adapter plate, and is used for accurately controlling the movement of droplets, and the digital microfluidic chip is connected by inserting the adapter plate; the high-precision temperature control module is mainly composed of a temperature controller and a semiconductor refrigerating sheet, and is arranged below the lower electrode plate and used for accurately controlling the temperature of the lower electrode plate; the real-time optical detection module is arranged above the chip and used for fluorescence imaging and dynamic monitoring; the automatic operation software is used for process editing and data analysis, and the droplets move according to the requirements by inputting commands in advance; the image analysis software is used for analyzing the cell images in the droplets; and the standardized reagent kit is used for maintaining a suitable environment for sample culture and realizing cell staining detection.

[0023] In still another aspect, the present application provides a group of drugs for application to the above-mentioned digital microfluidic chip or the above-mentioned digital microfluidic system, which includes but is not limited to cisplatin, carboplatin, oxaliplatin and lobaplatin.

[0024] In still another aspect, the present application provides a drug screening method based on the above-mentioned digital microfluidic system, which comprises the following steps:

[0025] S1. The biological sample suspension is introduced into the liquid storage area through the liquid injection port of the chip, cell droplets are formed from the liquid storage area, and are distributed to the reaction pools, and are cultured at 37 DEG C and 5% CO2 for 12-72 h;

[0026] S2. The drug to be screened and the culture solution are loaded into the independent liquid storage pools of the chip respectively, drug droplets and culture solution droplets are generated respectively, the volume ratio of the drug droplets to the culture solution droplets is controlled to control the movement and mixing of the droplets on the electrode array surface, and drug droplets containing different drug components or a series of concentration gradients are obtained;

[0027] S3. Delivering the drug droplet to the cells in the reaction pool, maintaining the 37 DEG C culture condition to continue culturing for 6-72h, and collecting images in real time by the real-time optical detection module;

[0028] S4. Calculating the sample survival rate after staining, and generating a dose-effect curve.

[0029] In step S1, the biological sample comprises cells, tissues, organs, cell clusters or organoids, etc.

[0030] In step S2, the series of concentration gradients are 0 μg / ml, 10 μg / ml, 20 μg / ml, 50 μg / ml and 100 μg / ml.

[0031] In step S4, the method for calculating the sample survival rate is as follows: introducing 0.4% trypan blue dye, acting for 3 minutes, automatically capturing the images of each reaction unit by a microscopic imaging system, and quantitatively calculating the cell survival rate by image analysis software.

[0032] The present application fully gives the programmable advantage of the digital microfluidic technology, and develops a high-throughput drug screening system based on the digital microfluidic chip technology, which can realize automatic drug screening under laboratory conditions, and provides a powerful technical tool for innovative drug research and development and drug efficacy evaluation. The method meets the urgent needs of high-throughput, high-efficiency, automation and low-cost screening for current drug research and development, and opens up a new technical path for personalized medicine research. On the basis of the microfluidic cell culture technology, the drug screening chip system supporting multi-parameter detection is constructed, so as to provide a new solution for accelerating the new drug discovery process and optimizing the clinical medication scheme.

[0033] Compared with the prior art, the present application has the beneficial effects that:

[0034] 1. High-throughput screening capability: through the optimized electrode array design, the parallel test of dozens of drugs can be completed in dozens of reaction pools at a time in a single experiment, the screening efficiency is significantly improved, and the large-scale compound library screening demand is met.

[0035] 2. Dynamic concentration gradient control: the programmable droplet control technology can be used to accurately generate and adjust the drug concentration gradient, and the fine study of the dose-effect relationship is realized.

[0036] 3. Multi-component drug combination analysis capability: droplets containing multiple different drug components can be formed, and the synergistic effect and antagonistic effect between multiple drug components can be studied.

[0037] 4. Real-time dynamic monitoring: the multi-modal detection module is integrated, the change of the cell state during the drug action can be continuously monitored, and the kinetic data of the drug action are obtained.

[0038] 5. Micro-reagent consumption: nanoliter-scale reaction system greatly reduces the use of precious drugs, compounds and cell samples, and reduces screening costs.

[0039] 6. Full-process automation: from cell culture, drug loading to result analysis, the whole process is automatically controlled, reducing human operation errors and improving data reliability.

[0040] 7. Closed operation environment: the fully enclosed chip design avoids external pollution and prevents the spread of potentially harmful drugs, ensuring the safety of experimenters.

[0041] 8. Flexible scalability: modular design facilitates system configuration adjustment according to different screening needs, and is suitable for screening of various drug types such as small molecules, antibodies and cell therapy drugs.

[0042] 9. Multi-level biological structure screening capability: in addition to drug screening on cells, tissues, organs, cell clusters and organoids can also be formed in the reaction area, thereby realizing high-throughput drug screening on tissues, organs, cell clusters and organoids. BRIEF DESCRIPTION OF DRAWINGS

[0043] Fig. 1 It is a cross-sectional view of the microfluidic chip involved in the present application.

[0044] Fig. 2 It is a structural schematic diagram of the printed circuit board involved in the present application.

[0045] Fig. 3 It is a structural schematic diagram of the upper electrode plate of the microfluidic chip involved in the present application.

[0046] The various marks in the figure are: 10, upper electrode plate, 11, liquid injection port, 12, oil injection port, 13, ITO glass layer, 14, hydrophobic layer; 20, lower electrode plate, 21, printed circuit board, 22, driving electrode, 23, dielectric layer, 24, hydrophobic layer, 30, liquid storage area, 31, liquid storage pool, 40, reaction area, 41, reaction pool. DETAILED DESCRIPTION

[0047] In order to better understand the present application, the present application will be further described in detail below in conjunction with the embodiments and drawings, but those skilled in the art will appreciate that the following embodiments are not a limitation on the scope of protection of the present application, and any changes and variations made on the basis of the present application are within the scope of protection of the present application.

[0048] The embodiments described herein are illustrative and exemplary only and are not to be construed as limiting the scope of the present application. Any further modifications of the embodiments described herein, which are apparent to those skilled in the art to which the present application pertains, are deemed to be within the scope of the present application. The drawings of the present specification are schematic representations for purposes of the specified embodiments and are exemplary only, and are not to be construed as limiting the scope of the present application. Note that the drawings are not necessarily to scale. Identical or similar reference numerals are used to represent identical or similar parts throughout the specification.

[0049] The experimental methods used in the following examples are conventional unless otherwise specified. The materials, reagents, etc. used in the following examples are commercially available unless otherwise specified.

[0050] Example 1 Construction of a digital microfluidic chip

[0051] Reference Figs. 1-3 The digital microfluidic chip described in the present embodiment includes an upper plate 10 and a lower plate 20. The upper plate 10 and the lower plate 20 are arranged in parallel to form a space for the movement of liquid droplets. In the present embodiment, the distance between the upper plate 10 and the lower plate 20 is set to 300 μm. The upper plate 10 and the lower plate 20 are sealed to enable closed operation throughout. The entire drug screening process is performed in the digital microfluidic chip, and there is no need to open the cover for operation, thereby completely eliminating the risk of aerosol contamination and improving the safety of the experiment and the reliability of the results. In the present embodiment, the upper plate 10 and the lower plate 20 are sealed by polyimide double-sided tape (thickness 300 μm) and acrylic ester UV glue, and the shear strength after curing is ≥ 15 MPa, and the liquid leakage rate is 0. The movement of liquid droplets 1 is controlled between the upper plate 10 and the lower plate 20. The distance between the upper plate 10 and the lower plate 20 is 50-1000 microns to allow the movement of liquid droplets.

[0052] The lower plate 20 includes, from bottom to top, a printed circuit board 21, a driving electrode 22, a dielectric layer 23, and a hydrophobic layer 24.

[0053] The printed circuit board 21 is made of FR-4 epoxy resin material with a thickness of 1.6 mm.

[0054] The driving electrode 22 uses metal Au as the electrode layer material. The thickness of the driving electrode 22 is 3 μm, the line width is 80 μm, the adjacent spacing is 30 μm, and the driving electrode 22 is arranged in 36 rows x 49 columns, with a total of 1074 independent electrodes.

[0055] The medium layer 23 is made of Parylene C by chemical vapor deposition method on the electrode layer, and is an insulating material with high dielectric constant, uniform thickness and strong anti-breakdown capability. In this embodiment, Parylene C is used as the medium layer 23, with a thickness of 2.5 μm, a dielectric constant of 3.1, and a breakdown field strength of 250 V / μm.

[0056] The hydrophobic layer 24 is made of Teflon AF. The hydrophobic layer 24 is made on the medium layer 23 by spin coating and drying the hydrophobic layer material, with a spin coating speed of 3000 rpm, drying at 80°C for 45 min, a thickness of 100 nm, and a contact angle of 120°.

[0057] Meanwhile, an Au material grounding electrode is arranged on the lower electrode plate 20.

[0058] The upper electrode plate 10 is sequentially provided with an ITO glass layer 13 and a hydrophobic layer 14 from top to bottom.

[0059] The upper electrode plate 10 is provided with a liquid injection port 11 above the upper electrode plate 10 for adding solution, and the diameter of the liquid injection port 11 is 1.5 mm, and the inner wall contact angle is 115°.

[0060] The upper electrode plate 10 is further provided with an oil injection port 12 above the upper electrode plate 10, which can be used to add silicone oil. The silicone oil is slowly added in the oil injection port 12 to fill the interlayer gap inside the chip, provide a stable medium environment for droplet movement, and prevent chemical pollution. The diameter of the oil injection port 12 can be 1 mm.

[0061] The upper electrode plate 10 is further provided with an oil outlet (not shown in the figure), and the diameter of the oil outlet can be 1 mm, which can be diagonally arranged with the oil injection port 12.

[0062] The ITO glass layer 13 of the upper electrode plate 10 is in contact with the grounding electrode of the lower electrode plate 20 through conductive silver paste, and finally connected to the grounding of the driving circuit board through a flexible flat cable.

[0063] The space between the upper electrode plate 10 and the lower electrode plate 20 is divided into a liquid storage area 30 and a reaction area 40 by the arrangement of the driving electrode 22.

[0064] The liquid storage area 30 includes a plurality of liquid storage pools 31. In this embodiment, 8 liquid storage pools are arranged on the left side of the chip, 8 liquid storage pools are arranged on the right side of the chip, and 12 liquid storage pools are arranged below the chip. The capacity of a single liquid storage pool is 8 μL, and the distance between the centers of adjacent liquid storage pools is 9 mm. The liquid storage pools can be used to store cell suspension, drug solution and required reagent solution respectively. The storage of cell suspension, drug solution and staining solution is not limited to a specific position. For example, cell suspension can be stored in the left liquid storage area, the right liquid storage area or the lower liquid storage area of the digital microfluidic chip.

[0065] The reaction zone 40 contains a plurality of reaction pools 41, 96 of which are provided in this embodiment, each of which is formed by 3*2 electrodes, with an effective reaction area of 54mm2, and each electrode can accommodate a 1ul droplet. The reaction zone is the area where the cell suspension, drug solution and staining solution react. The efficient mixing of the cell suspension, drug solution and staining solution is achieved through the cross array.

[0066] The ITO glass layer 13 of the upper electrode plate 10 is connected with the ground electrode of the lower electrode plate 20 through conductive silver glue, and then connected with the ground of the driving circuit board through the flexible flat cable; the insulating silicon oil (viscosity 80cSt, 25 DEG C) is injected from the oil injection port 12 to fill the gap between the layers, and the droplet movement medium environment is formed.

[0067] Experiments show that: the driving electrode can realize precise control of the droplet, the movement speed is 3mm / s, and the path deviation is less than or equal to 0.05mm; there is no cross contamination between the liquid storage pools, the droplet mixing uniformity is greater than or equal to 95%, and the basic requirements of drug screening are met.

[0068] Embodiment 2 Integration of digital microfluidic system

[0069] The digital microfluidic chip of the application is equipped with a high-precision driving module, combined with automatic operation software, to realize precise movement and processing of droplets, and meet the needs of complex chemical and biological experiments.

[0070] The embodiment provides an integrated digital microfluidic system, which aims to realize the whole-process automatic screening from cell culture, drug loading to drug efficacy analysis. The system comprises the digital microfluidic chip, a driving module, a high-precision temperature control module, a real-time optical detection module, a visual interactive interface, automatic operation software, image analysis software and a standardized reagent kit.

[0071] The digital microfluidic chip is used as a core screening platform, and the electrode array is used to generate a drug gradient and co-culture with cells.

[0072] The driving module is responsible for regulating and controlling the precise distribution and mixing of droplets. In this embodiment, the driving module is composed of an STM32H743ZI development board, a flexible flat cable and an adapter plate, with an output voltage of 0-300V, an adjustment accuracy of 1V, a response time of less than or equal to 10ms, and 1074 driving electrodes that can be independently controlled. The droplet delivery repeatability is less than or equal to 0.05mm.

[0073] The high-precision temperature control module maintains the optimum environmental conditions for cell culture. In this embodiment, the high-precision temperature control module comprises a TC-720 temperature controller and a 50W semiconductor refrigerating sheet. The high-precision temperature control module is arranged below the lower electrode plate, and the temperature control range is 4-50 DEG C, the temperature control accuracy is ± 0.1 DEG C, and the fluctuation is less than or equal to 0.05 DEG C under the condition of 37 DEG C constant temperature.

[0074] The real-time optical detection module integrates optical imaging and fluorescence microscopy technology to monitor the state change of cells in real time.

[0075] The automatic operation software realizes programmed control of the whole process, and can automatically complete the establishment of drug concentration gradient, timed drug administration, continuous observation and the like according to a preset scheme.

[0076] The image analysis software is used to quantitatively calculate the cell survival rate, and the image analysis software can be self-programmed software or existing general software, and in this embodiment, ImageJ with version number 1.8.0_345 is used.

[0077] The standardization kit contains pH 7.3 cell buffer, 10 μg / mL AO / EB staining solution and 5 μm standard microspheres (CV≤3%).

[0078] The whole digital microfluidic system realizes high-throughput, automatic and standardized operation of the drug screening process by cooperating with each functional module. Experiments show that the whole process response time is less than or equal to 500 ms from parameter setting to data export, which can meet the demand of automatic screening of the whole process.

[0079] The working principle of the present application is as follows:

[0080] The drug solution to be tested and the cell suspension are loaded into the liquid storage pools 31 on both sides of the chip through the liquid injection port 11; the drug droplets and cell culture droplets are formed by controlling the driving electrode 22, and are moved to the reaction area 40 according to the preset path; the drug and the cell are precisely co-cultured in the reaction area 40, and the temperature control module maintains a constant temperature environment of 37 DEG C to ensure the activity of the cell; during the culture process, the integrated real-time optical detection module monitors the morphological change of the cell and the fluorescence labeling signal in real time; after the predetermined action time is completed, the high-resolution microscopic imaging system captures the images of each reaction pool 41; finally, the data analysis software automatically calculates the cell survival rate and other pharmacodynamic parameters, generates a dose-effect curve, and intuitively presents the screening results on the display module. The whole operation process realizes the whole process automation from sample loading to result analysis, and ensures the efficiency and repeatability of drug screening.

[0081] The integrated digital microfluidic system of the present application covers sample pretreatment, biochemical reaction, real-time detection and feedback, data processing and result display. The system uses functional modules with small volume, light weight and high integration; integration is realized as much as possible, unnecessary functions and connections between functions are reduced; and reasonable overall layout is realized.

[0082] Example 3: HeLa cell drug screening based on the system

[0083] This embodiment uses the system of Example 2 to screen four platinum drugs, cisplatin, carboplatin, oxaliplatin and lobaplatin, using HeLa cells as a model. The drug screening method steps are as follows:

[0084] 1. Cell culture: HeLa cell suspension (concentration 2 x 10 5 cells / mL) is introduced into the left reservoir by the chip, and the driving electrode controls the formation of 400 nL cell droplets to be distributed to 96 reaction pools; after conventional culture for 24 h, the cell adhesion rate is 85%.

[0085] 2. Drug gradient preparation: four drugs are loaded into the right reservoir (initial concentration 10 μmol / L), and the culture solution is loaded into the lower reservoir; control to generate 100 nL drug droplets and 300 nL culture solution droplets, and after movement and combination, stand for 1.5 min to form 5 concentration gradients of 10, 5, 2.5, 1.25 and 0.625 μmol / L.

[0086] 3. Drug action and monitoring: deliver each concentration of drug droplets to the culture unit and maintain 37°C ± 0.1°C culture for 24 h; during the period, collect bright field and fluorescence images every 2 h by real-time optical detection module to monitor cell morphological changes.

[0087] 4. Result analysis: add AO / EB double staining solution (10 μg / mL, 100 nL) and act for 3 min, and collect fluorescence images; calculate cell survival rate (surviving cell number / total cell number x 100%) by image analysis software, and the results are shown in Table 1.

[0088] Table 1

[0089]

[0090] The system detection error is ≤4%, the repeatability RSD is ≤3%, and the experiment proves that the system is stable and reliable.

[0091] Example 4 MCF7 breast cancer cell drug screening based on the system

[0092] This embodiment uses the system of Example 2 to screen the efficacy of paclitaxel using MCF7 breast cancer cells as a model. The steps are as follows:

[0093] 1. MCF7 cell suspension (concentration 3 x 10 5 cells / mL) is introduced into the left reservoir, and 400 nL droplets are distributed to the reaction area, and cultured in the reaction pool 41 of the chip for 24 h;

[0094] 2. Load 0.02 μmol / mL paclitaxel solution into the right reservoir, control the delivery of paclitaxel solution to the reaction pool 41 through the driving electrode 22 for treatment for 24 h, maintain the culture condition at 37℃, and continuously act for 6 h, during which the cell morphological changes are monitored in real time through the integrated optical module;

[0095] 3. Introduce AO / EB double staining solution through the injection port 11, act for 3 min, automatically capture the images of each reaction unit by using the microscopic imaging system, and quantitatively calculate the cell survival rate through the image analysis software.

[0096] The results are in good consistency with the results of the traditional microwell plate method.

[0097] Example 5 Drug screening of liver cancer organoids based on the system

[0098] In this example, the system of Example 2 is used to screen the drug efficacy of sorafenib by taking liver cancer organoids as a model, and the specific steps are as follows:

[0099] 1. Take fresh liver cancer tissue, prepare organoid suspension (50 / μL) after 0.2 mg / mL collagenase digestion and filtration; introduce the left reservoir to form 500 nL droplets (containing 25 organoids) and distribute them to the reaction area 40; add organoid culture medium for conventional culture for 72 h, and the organoid formation rate is ≥90% (diameter 100-200 μm).

[0100] 2. Load sorafenib (initial concentration 20 μmol / L) into the right reservoir, prepare 1.25, 2.5, 5, 10, and 20 μmol / L five concentration gradients by mixing 100 nL drug droplets with different volumes of culture solution droplets; deliver to the reaction area 40, and culture for 48 h, collect bright field images every 6 h to monitor the morphology of organoids.

[0101] 3. Add Calcein-AM / PI double staining solution (5 μmol / L each) and incubate for 15 min, collect fluorescence images; calculate the organoid survival rate (green fluorescence area / total organoid area x 100%), and the IC50 is 6.2 μmol / L, which is consistent with the screening results of the conventional method, proving that the system can be used for organoid screening.

[0102] Example 6 Drug screening of tumor tissue blocks based on the system

[0103] In this example, the system of Example 2 is used to screen the drug efficacy of gefitinib by taking lung cancer tissue blocks as a model, and the specific steps are as follows:

[0104] 1. Fresh lung cancer tissue was taken and cut into tissue blocks with a diameter of 300-500 μm; a custom-made enlarged injection port (2 mm in diameter) was used to introduce the lower reservoir, and the electrode was driven to form a 800 nL droplet containing a single tissue block, which was then dispensed into the reaction zone 40. A 3x2 electrode array was used to wrap the tissue block to prevent movement deviation.

[0105] 2. Drug gradient and effect: Gefitinib (initial concentration 5 μmol / L) was loaded into the right reservoir, and 100 nL of the drug droplet was mixed with different volumes of culture solution droplets to prepare 5 concentration gradients of 0.3125, 0.625, 1.25, 2.5, and 5 μmol / L. The mixture was delivered to the reaction zone 40, and the culture was performed for 72 h. Bright-field images were collected every 12 h to monitor the morphological integrity of the tissue block.

[0106] 3. PI staining solution (10 μg / mL) was added and incubated for 30 min, and fluorescence images were collected. The fluorescence positive rate of the tissue block (red fluorescence area / tissue block area x 100%) was calculated, and the IC50 was 1.8 μmol / L. The survival status of the tissue block was consistent with the pathological section results with a consistency of ≥85%, and the applicability of the system for high-throughput screening of the tissue block was verified.

[0107] Example 7 In vitro liver lobule drug screening based on the system

[0108] In this example, the system of Example 2 was used to screen the hepatotoxicity of acetaminophen (APAP) and the intervention effect of the antidote N-acetylcysteine (NAC) on the rat in vitro liver lobule. The system highlights the high-throughput manipulation and multi-index analysis capabilities for functional organ units. The specific steps are as follows:

[0109] 1. Fresh rat liver was taken and perfused with 0.05% collagenase solution, and then dissected to obtain intact liver lobules (volume about 1 mm³, containing functional units such as hepatic sinusoids, hepatocytes, and Kupffer cells). After dispensing into the reaction zone 40, the droplet generator was used to generate a 1 μL culture droplet containing a single liver lobule, which was then dispensed into a 3x2 array reaction zone. The liver lobules were cultured dynamically at 37°C for 24 h, and the survival rate was ≥95%.

[0110] 2. Paracetamol (initial concentration 20 mmol / L) and N-acetylcysteine (initial concentration 40 mmol / L) were loaded into the two reservoirs, respectively, and two groups of screening systems were prepared by mixing 100 nL of drug droplets with different volumes of culture solution droplets: ① APAP group alone: the concentration gradient was 0.5, 1, 2.5, 5, 10 mmol / L; ② APAP+NAC combination group: the concentration of APAP was fixed at 5 mmol / L, and the concentration gradient of NAC was 2.5, 5, 10, 20, 40 mmol / L. The drug droplets were delivered to the corresponding reaction pool, and the perfusion culture was continued for 48 h, and bright field images were collected every 12 h to monitor the morphological integrity of liver lobules.

[0111] 3. After the culture ended, the culture solution was collected to detect the activities of alanine transaminase (ALT) and aspartate transaminase (AST), and the enzyme release rate was calculated; Calcein-AM / PI double staining solution (5 μmol / L each) was added and incubated for 20 min, and fluorescence images were collected by a high-content imaging system to calculate the proportion of living cells. Compared with the traditional isolated liver perfusion model, the correlation coefficient of the chip screening results reached 0.92, and a single experiment could complete parallel analysis of 32 samples, verifying the applicability of the system to high-throughput screening of organ units.

[0112] Example 7 Drug screening of liver cancer cell clusters based on the system

[0113] In this embodiment, the system of Example 2 was used to screen the efficacy of doxorubicin on liver cancer cell clusters, which embodied the screening advantages of three-dimensional cell aggregates, and the specific steps were as follows:

[0114] 1. HepG2 cell suspension (concentration 5×10 5 individuals / mL) was introduced into the left reservoir, and the electrode was controlled to form 600 nL droplets which were dispensed into the reaction area 40; by adjusting the electrode voltage (150-200 V), the cells were induced to aggregate into spheres, and the cell clusters with a diameter of 150-200 μm were formed after conventional culture for 48 h, and the sphere formation rate was ≥85%.

[0115] 2. Doxorubicin (initial concentration 2 μmol / L) was loaded into the right reservoir to prepare 5 concentration gradients of 0.125, 0.25, 0.5, 1, and 2 μmol / L; it was delivered to the reaction area 40, and conventional culture was carried out for 48 h, and FITC channel fluorescence images were collected every 6 h to monitor the apoptosis signal.

[0116] 3. After AO / EB staining, the images were collected, the survival rate of the cell clusters was calculated, and the IC50 was 0.65 μmol / L. The difference from the two-dimensional cell screening results (IC50 0.32 μmol / L) was consistent with the characteristics of three-dimensional culture drug sensitivity, and the experiment proved that the system of the application could be used for drug screening at the level of cell clusters.

[0117] While the foregoing describes specific embodiments of the application, one of ordinary skill in the art will further appreciate that the specific exemplary embodiments described are meant to be illustrative only and are not intended to limit the scope of the application. Changes, modifications, and equivalents which would occur to one skilled in the art upon a reading of the foregoing description are meant to be encompassed within the scope of the application.

Claims

1. A digital microfluidic chip for drug screening, characterized in that, The upper plate and the lower plate are arranged in parallel to form a space for liquid drop running; the upper plate and the lower plate are sealed by double-sided adhesive tape and UV glue, and the distance between the upper plate and the lower plate is 50-1000 microns; The lower plate comprises, from bottom to top, a printed circuit substrate, an arrayed driving electrode, a dielectric layer and a hydrophobic layer; The upper plate comprises, from top to bottom, an ITO glass layer and a hydrophobic layer, and is provided with a liquid injection port, an oil injection port and an oil outlet port; The space between the upper plate and the lower plate for liquid drop running is divided into a liquid storage area and a reaction area according to the arrangement of the driving electrode; the liquid storage area comprises a plurality of groups of independent liquid storage pools arranged on the left side, the right side and the bottom, for respectively storing cell suspension, drug solution and required reagent solution; The reaction area comprises a plurality of reaction pools, each reaction pool being composed of a 3×2 electrode array to form a reaction unit; The cell suspension, the drug solution and the reagent solution are respectively mixed in a high efficiency through the cross matrix arrangement from the liquid storage pool, and then react in the reaction pool.

2. The digital microfluidics chip for drug screening according to claim 1, wherein, The left side and the right side of the liquid storage area are each provided with 8 independent liquid storage pools, and the bottom of the liquid storage area is provided with 12 independent liquid storage pools; the number of reaction pools in the reaction area is 80-120.

3. The digital microfluidics chip for drug screening according to claim 1, wherein, The dielectric layer is one or more of SiO2, Al2O3, Parylene C, SU-8 and PDMS; the hydrophobic layer is one or more of Teflon-AF and CYTOP.

4. The digital microfluidics chip for drug screening according to claim 1, wherein, The arrayed driving electrode is arranged in a 36-row×49-column array, and the size of a single driving electrode is 3mm×3mm, and the shape is a puzzle shape; the electrodes connecting the liquid storage area and the reaction area in the arrayed driving electrode are arranged in a cross matrix, the output electrodes of the left side, the right side and the bottom liquid storage pools form cross paths with the input electrodes of each reaction pool in the reaction area, and the cell suspension, the drug solution and the reagent solution are respectively distributed to any reaction pool in a high efficiency through the cross matrix arrangement from the liquid storage pool, and then react in the reaction pool.

5. A digital microfluidics system for drug screening, characterized in that, The digital microfluidic chip for drug screening comprises the chip according to any one of claims 1-4, and further comprises a driving module, a high-precision temperature control module, a real-time optical detection module, an automatic operation software, an image analysis software, a visual interactive interface and a standardized reagent kit; The driving module is composed of a development board, a flexible flat cable and an adapter plate; the digital microfluidic chip is connected by inserting the adapter plate; the driving module is electrically connected with the digital microfluidic chip to control the movement of liquid drops; The high-precision temperature control module is composed of a temperature controller and a semiconductor refrigeration sheet, and is arranged below the lower plate; the high-precision temperature control module is thermally coupled with the chip to control the culture temperature; the real-time optical detection module is arranged above the chip, and is used for dynamically collecting sample images in the reaction pool; the automatic operation software is used for process editing and data analysis, and controls the movement of liquid drops through a preset command; The image analysis software is used for quantitative analysis of sample images; the visual interactive interface is used for operation control and result display; and the standardized reagent kit is used for sample culture and staining detection.

6. A drug screening method using the digital microfluidic chip according to claims 1 to 4 or the digital microfluidic system according to claim 5, characterized in that, The method comprises the following steps: S1. The biological sample suspension is introduced into the storage area through the injection port of the digital microfluidic chip. Under the control of the driving module, cell droplets are formed and distributed to the reaction pool, which is cultured under suitable culture conditions maintained by the high-precision temperature control module; S2. The drug to be screened and the culture solution are loaded into separate storage pools of the digital microfluidic chip. Under the control of the driving electrode, drug droplets and culture solution droplets are generated, respectively. The driving module controls the movement and mixing of the drug droplets and the culture solution droplets on the surface of the electrode array to obtain drug droplets containing different drug components or a series of concentration gradients; S3. The drug droplets are delivered to the reaction pool of the digital microfluidic chip, and the culture is continued under the culture conditions controlled by the high-precision temperature control module. During this period, the real-time optical detection module dynamically collects images of the samples in the reaction pool; S4. After staining with the staining solution in the standard kit, the sample survival rate is calculated by image analysis software to generate a dose-effect curve.

7. The drug screening method according to claim 6, characterized in that, The biological sample in step S1 includes cells, tissues, organs, cell clusters, or organoids. The suitable culture conditions are 37℃, 5% CO2, and the culture time is 12-72h.

8. The drug screening method according to claim 6, characterized in that, The drug to be screened in step S2 includes but is not limited to cisplatin, carboplatin, oxaliplatin, and lobaplatin.

9. The drug screening method according to claim 6, characterized in that, The culture time in step S3 is 6-72h, and the real-time optical detection module collects images at intervals of 2-12h.

10. The drug screening method according to claim 6, characterized in that, In step S4, the method for calculating the sample survival rate by image analysis software is as follows: introduce 0.4% trypan blue staining solution and act for 3 minutes. The microscopic imaging system automatically captures images of each reaction unit, and the image analysis software quantitatively calculates the cell survival rate.

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