Microfluidic electronic chip, organoid model preparation method, and protein analysis method

Abstract

A microfluidic electronic chip, an organoid model preparation method, and a protein analysis method. The microfluidic electronic chip comprises a lab-on-chip and an electrode array, wherein the lab-on-chip comprises chambers for accommodating substances to be cultured; and the electrode array is connected to the chambers, and the electrode array is used for performing stimulation and electrophysiological monitoring on said substances in the chambers. The organoid model preparation method and the protein analysis method are both executed on the basis of the microfluidic electronic chip.

Description

Microfluidic electronic chips, organoid model preparation methods, and protein analysis methods Technical Field

[0001] The present application relates to the field of biomedical engineering technology, and in particular to a microfluidic electronic chip, an organoid model preparation method, and a protein analysis method. Background Art

[0002] Microfluidics (Lab on a chip) uses micro- and nano-fabrication technologies to miniaturize complex biological or chemical reactions onto tiny chips. By manipulating the movement of microfluidics within channels and reaction chambers, automated and rapid experimental operations are achieved. By integrating genomics, immunology, clinical chemistry diagnostics, and organoids, microfluidics can enable DNA sequencing, protein / amino acid / cell detection, early disease diagnosis, precise drug screening, and in vitro culture of organoids. This technology comprehensively advances the automated, personalized, and integrated diagnosis and treatment of clinical medicine, making significant contributions to biology, chemistry, materials science, and clinical medicine. For example, organ chips are microfluidic chips designed using micro- and nanofabrication techniques. They combine precise microfluidic manipulation with the integration of cellular and biological tissues to create in vitro organ physiological microsystems that mimic real-world physiological environments or functions. By simulating the biochemical and mechanical interactions between cells, these microchips can simulate human tissues or organs, allowing the feedback and response of organ microsystems to external stimuli to be tested. This provides more accurate drug evaluation results than those achieved with two-dimensional static cell culture and animal experiments. These applications have been widely applied in toxicity assessment, disease modeling, and drug screening. At present, many human organ-like chips such as brain, heart, liver, kidney, lung, intestine and multi-organ mixture have been widely developed based on microfluidic chips.

[0003] For brain-on-a-chip and heart-on-a-chip, electrophysiological information is a key indicator for evaluating the functional performance of in vitro brain-on-a-chip and heart-on-a-chip reconstruction. Real-time monitoring and feedback of electrophysiological parameters can accurately provide feedback on drug toxicity and performance assessment. However, existing organ-on-a-chips are mostly fabricated using microfluidics, which cannot be integrated with electrophysiological acquisition systems, making it impossible to monitor the dynamic changes of biological tissues in the organ-on-a-chip over long periods of time.

[0004] Summary of the Invention

[0005] This application aims to solve at least one of the technical problems existing in the prior art.

[0006] To this end, this application proposes a microfluidic electronic chip that can quickly and conveniently monitor and process the potential of cultured materials, and has broad application potential in many fields such as analytical detection, labeling sequencing, organ chips and drug screening.

[0007] This application also proposes a method for preparing an organoid model.

[0008] This application also proposes a DNA-protein binding analysis method.

[0009] This application also proposes a method for analyzing RNA-protein binding.

[0010] This application also proposes a protein binding analysis method.

[0011] This application also proposes a liposome transfection method.

[0012] This application also proposes an electroporation transfection method.

[0013] The microfluidic electronic chip according to the first embodiment of the present application includes:

[0014] A microfluidic chip comprising one or more chambers for accommodating substances to be cultured;

[0015] An electrode array is connected to the chamber and is used to stimulate and electrophysiologically monitor the substance to be cultured in the chamber.

[0016] According to some embodiments of the present application, the microfluidic chip includes a control input component, there are multiple chambers, and multiple chambers are connected to the control input component, and the control input component is used to control the material to be cultured to enter the corresponding chamber.

[0017] According to some embodiments of the present application, the control input component includes a micro-channel, a micro-valve body arranged on the micro-channel, and a stepped multi-concentration liquid separation device, and the stepped multi-concentration liquid separation device is connected to the chamber through the micro-channel.

[0018] According to some embodiments of the present application, there are multiple microchannels and multiple microvalve bodies, wherein the multiple microvalve bodies are controlled and processed so that different substances to be cultured enter different chambers through different microchannels, and each chamber corresponds to a multi-channel electrode in the electrode array.

[0019] According to some embodiments of the present application, an information acquisition and processing system is further included, and the electrode array is connected to the information acquisition and processing system.

[0020] According to some embodiments of the present application, the information acquisition and processing system includes a processing circuit unit, a controller and a control circuit unit, and the electrode array, the processing circuit unit, the controller, the control circuit unit and the control input component are connected in sequence.

[0021] According to some embodiments of the present application, the information acquisition and processing system further includes a communication module, which is connected to the controller.

[0022] According to some embodiments of the present application, the processing circuit unit includes a preprocessing circuit subunit and an analog-to-digital conversion subunit, and the electrode array, the preprocessing circuit subunit, the analog-to-digital conversion subunit and the controller are connected in sequence.

[0023] According to some embodiments of the present application, the substance to be cultured includes at least one of the following:

[0024] Cells, tissues, DNA, RNA, proteins and metabolic molecules.

[0025] According to some embodiments of the present application, the cells include at least any one of the following types: vascular cells, brain cells, liver cells, lung cells, kidney cells, heart cells, spleen cells and intestinal cells.

[0026] According to the second embodiment of the present application, the method for preparing an organoid model is implemented based on the microfluidic electronic chip of the first embodiment, and the method includes:

[0027] injecting stem cells into the chambers of the microfluidic electronic chip in sequence and culturing them to generate organoid spheroids;

[0028] performing enzymatic hydrolysis on the organoid spheroids to form self-assembled cells in each chamber of the microfluidic electronic chip, and adding the self-assembled cells to the lower chamber;

[0029] Cultivating the self-assembled cells so that the self-assembled cells generate an initial organoid model;

[0030] culturing the initial organoid model to form organoids;

[0031] The organoid is intermittently electrically stimulated based on the electrode array, and the electrophysiological information of the organoid is collected to construct a functional organoid model.

[0032] According to some embodiments of the present application, the organoids include at least any one of the following types: vascular organoids, brain organoids, heart organoids, liver organoids, lung organoids, kidney organoids, spleen organoids, and intestinal organoids.

[0033] According to some embodiments of the present application, the vascular organoids include endothelial cells, fibroblasts and smooth muscle cells.

[0034] According to some embodiments of the present application, the brain-like organ includes brain-like stem cells, neuronal cells and glial cells.

[0035] According to some embodiments of the present application, the heart-like organ comprises cardiomyocytes, endocardial cells, epicardial cells and cardiovascular cells.

[0036] According to the DNA-protein binding analysis method of the third embodiment of the present application, which is implemented based on the microfluidic electronic chip of the first aspect, the method includes:

[0037] preparing a DNA template of a protein transcription factor, wherein the DNA template carries an antibody tag;

[0038] Building a library based on the DNA template, adding a fluorescent marker, and obtaining a sample DNA dilution solution;

[0039] Spotting the sample DNA dilution solution into the chamber of the microfluidic electronic chip;

[0040] Surface patterning of the chamber is performed based on biotinylated bovine serum albumin, phosphate buffered saline, and biotinylated antibodies;

[0041] Loading an in vitro transcription / translation protein transcription factor mixture into the chamber, so that the in vitro transcription / translation protein transcription factor binds to the DNA template in the sample DNA dilution solution to obtain a binding molecule, and the binding molecule is captured and deposited by the biotinylated antibody covered in the chamber;

[0042] washing the chamber with phosphate buffer to remove unbound molecules and capture the bound molecules;

[0043] The microfluidic chip is scanned using a fluorescence microscope to quantify the binding molecules.

[0044] According to the RNA protein binding analysis method of the fourth embodiment of the present application, which is implemented based on the microfluidic electronic chip of the first aspect, the method includes:

[0045] Prepare protein and store it in a preset low-temperature environment;

[0046] Prepare RNA library transcription template dilutions based on DNA oligonucleotides;

[0047] Spotting the RNA library transcription template dilution into the chamber of the microfluidic electronic chip;

[0048] The chamber surface is patterned based on biotinylated bovine serum albumin, phosphate buffer, and capture probes containing fluorescent markers;

[0049] loading an in vitro transcription mixture into the chamber, so that the RNA library transcription template in the RNA library transcription template dilution solution is transcribed to obtain transcribed RNA, and the transcribed RNA is hybridized with the capture probe;

[0050] Thawing the protein, mixing the thawed protein with an antibody to obtain a protein mixture; and loading the protein mixture into the chamber so that the transcribed RNA binds to the protein to obtain a binding molecule;

[0051] washing the chamber with phosphate buffer to remove unbound molecules and capture the bound molecules;

[0052] The microfluidic chip is scanned using a fluorescence microscope to quantify the binding molecules.

[0053] According to a protein binding analysis method according to a fifth embodiment of the present application, which is implemented based on the microfluidic electronic chip of the first aspect, the method includes:

[0054] A DNA template for preparing a protein, wherein the protein comprises a target protein and a binding protein; the target protein comprises a first amino acid chain or molecule at its N-terminus and a second amino acid chain or molecule at its C-terminus; and the binding protein comprises a third amino acid chain or molecule at its C-terminus;

[0055] spotting the DNA template into the chamber of the microfluidic electronic chip;

[0056] Surface patterning of the chamber is performed based on biotinylated bovine serum albumin, phosphate buffer, and anti-amino acid chain or molecule antibodies;

[0057] Loading an in vitro transcription / translation protein transcription factor mixture into the chamber, so that the in vitro transcription / translation protein transcription factor binds to the DNA template to obtain a binding molecule, and the binding molecule is captured and deposited by the anti-amino acid chain or molecule antibody covered in the chamber;

[0058] loading an antibody solution into the chamber, so that the protein binds in the antibody solution to obtain a binding molecule;

[0059] washing the chamber with phosphate buffer to remove unbound molecules and capture the bound molecules;

[0060] The microfluidic chip is scanned using a fluorescence microscope to quantify the binding molecules.

[0061] According to the liposome transfection method of the sixth embodiment of the present application, which is implemented based on the microfluidic electronic chip of the first aspect, the method includes:

[0062] coating the fixed adhesion on a glass slide in the chamber of the microfluidic electronic chip;

[0063] preparing a liposome sample and transferring the liposome sample onto the glass slide coated with the fixed adhesive;

[0064] Plasma-treating the microfluidic electronic chip loaded with the liposome sample;

[0065] preparing a cell culture fluid, and injecting the cell culture fluid into the microfluidic electronic chip to culture and transfect cells in the cell culture fluid;

[0066] injecting culture medium into the microfluidic electronic chip to promote cell surface attachment;

[0067] The microfluidic chip was scanned using a fluorescence microscope to measure the transfection efficiency.

[0068] According to the electroporation transfection method of the seventh embodiment of the present application, which is implemented based on the microfluidic electronic chip of the first aspect, the method includes:

[0069] printing a plasmid containing a fluorescent protein onto the microfluidic electronic chip;

[0070] preparing a cell culture fluid, and injecting the cell culture fluid into the microfluidic electronic chip to culture and transfect cells in the cell culture fluid;

[0071] Stimulating cells in the cell culture medium based on the electrode array of the microfluidic electronic chip, so that holes are temporarily formed in the cell membranes of the cells, and the plasmid enters the cells through the holes;

[0072] injecting culture medium into the microfluidic electronic chip to promote cell surface attachment;

[0073] The microfluidic chip was scanned using a fluorescence microscope to measure the transfection efficiency.

[0074] The microfluidic electronic chip according to the embodiment of the present application has at least the following beneficial effects: the microfluidic electronic chip includes a microfluidic chip, wherein the microfluidic chip includes a chamber for accommodating a substance to be cultured; the microfluidic electronic chip also includes an electrode array, and the electrode array is connected to the chamber, so that the electrode array can stimulate or monitor the substance to be cultured in the chamber; through the above settings, the potential of the substance to be cultured can be quickly and conveniently monitored, and it has broad application potential in many fields such as analysis and detection, marker sequencing, organ chips, and drug screening.

[0075] Other features and advantages of the present application will be described in the following description, and in part will become apparent from the description, or will be understood by practicing the present application. The purposes and other advantages of the present application can be achieved and obtained through the structures particularly pointed out in the description, claims and drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0076] The accompanying drawings are used to provide a further understanding of the technical solution of the present application and constitute a part of the specification. Together with the embodiments of the present application, they are used to explain the technical solution of the present application and do not constitute a limitation on the technical solution of the present application.

[0077] FIG1 is a schematic structural diagram of a microfluidic electronic chip provided in one embodiment of the present application;

[0078] FIG2 is a schematic structural diagram of a microfluidic chip provided in one embodiment of the present application;

[0079] FIG3 is a schematic diagram of signal transmission of a microfluidic electronic chip provided in one embodiment of the present application;

[0080] FIG4 is a schematic diagram of the electrode array distribution provided by one embodiment of the present application;

[0081] FIG5 is an enlarged schematic diagram of the internal structure of a chamber of a single microfluidic chip provided in one embodiment of the present application;

[0082] FIG6 is a flow chart of a method for preparing an organoid model provided in one embodiment of the present application;

[0083] FIG7 is a flow chart of a DNA protein binding analysis method provided in one embodiment of the present application;

[0084] FIG8 is a flow chart of an RNA protein binding analysis method provided in one embodiment of the present application;

[0085] FIG9 is a flow chart of a protein binding analysis method provided in one embodiment of the present application;

[0086] FIG10 is a flow chart of a liposome transfection method provided in one embodiment of the present application;

[0087] FIG11 is a flow chart of an electroporation transfection method provided in one embodiment of the present application.

[0088] Reference numerals:

[0089] Microfluidic chip 10, chamber 100, stepped multi-concentration liquid separation device 200, microvalve body 300, microchannel 400, culture medium and reagent injection port 500, cell screening inlet 600, waste liquid outlet 700, electrode array 800, information acquisition and processing system 900, controller 910, control circuit unit 920, analog-to-digital conversion subunit 930, preprocessing circuit subunit 940, communication module 950. DETAILED DESCRIPTION

[0090] In order to make the purpose, technical solutions and advantages of this application more clear, the following further describes this application in detail with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain this application and are not intended to limit this application.

[0091] In the description of this application, "several" means one or more, "many" means more than two, "greater than," "less than," and "exceed" are understood to exclude the number itself, while "above," "below," and "within" are understood to include the number itself. The terms "first" and "second" are used solely to distinguish technical features and are not to be construed as indicating or implying relative importance, or as implicitly specifying the number or order of the technical features indicated.

[0092] In the description of this application, unless otherwise clearly defined, terms such as setting, installing, and connecting should be understood in a broad sense, and technicians in the relevant technical field can reasonably determine the specific meanings of the above terms in this application based on the specific content of the technical solution.

[0093] The present application provides a microfluidic electronic chip, an organoid model preparation method, a DNA-protein binding analysis method, an RNA-protein binding analysis method, a protein binding analysis method, a liposome transfection method, and an electroporation transfection method, wherein the microfluidic electronic chip includes a microfluidic chip, wherein the microfluidic chip includes a chamber for accommodating a substance to be cultured; the microfluidic electronic chip also includes an electrode array, and the electrode array is connected to the chamber so that the electrode can stimulate or monitor the substance to be cultured in the chamber; through the above settings, the potential monitoring and processing of the substance to be cultured can be performed quickly and conveniently, and it has broad application potential in many fields such as analysis and detection, labeling sequencing, organ chips, and drug screening.

[0094] The embodiments of the present application are further described below with reference to the accompanying drawings.

[0095] As shown in Figure 1, a microfluidic electronic chip provided by one embodiment of the present application includes a microfluidic chip 10 and an electrode array 800, wherein the microfluidic chip 10 includes a chamber 100 for accommodating substances to be cultured; the microfluidic electronic chip also includes an electrode array 800, and the electrode array 800 is connected to the chamber 100, so that the electrode array 800 can stimulate or monitor the substances to be cultured in the chamber 100.

[0096] It should be noted that the microfluidic chip 10 can be injected with a substance to be cultured, and the substance to be cultured can be cultured in the chamber 100 of the microfluidic chip 10; since the electrode array 800 is connected to the chamber 100 of the microfluidic chip 10, the electrode array 800 can be used to electrically stimulate the substance to be cultured in the chamber 100 of the microfluidic chip 10, and the electrode array 800 can be used to receive the electrical signal fed back by the substance to be cultured, so that the potential of the substance to be cultured can be monitored and processed conveniently and quickly, and it has broad application potential in many fields such as analysis and detection, labeling sequencing, organ chips and drug screening.

[0097] In some specific embodiments of the present application, the microfluidic chip 10 includes a control input component, and there are multiple chambers 100. The multiple chambers 100 are connected to the control input component, and the control input component is used to control the cultured material to enter the corresponding chamber 100.

[0098] It should be noted that the control input component is connected to the chamber 100, and the control input component can be used to control the entry of the substance to be cultured into the corresponding chamber 100; wherein, there are multiple chambers 100, and each chamber 100 can be used to accommodate different substances to be cultured. Based on the control input component, different substances to be cultured can be guided to different chambers 100, and then different substances to be cultured can be cultured in the microfluidic chip 10.

[0099] As shown in Figure 2, in some specific embodiments of the present application, the control input component includes a micro-channel 400, a micro-valve body 300 arranged on the micro-channel 400, and a stepped multi-concentration liquid separation device 200, and the stepped multi-concentration liquid separation device 200 is connected to the chamber 100 through the micro-channel 400.

[0100] It should be noted that the control input component may include a micro-channel 400 and a step-by-step multi-concentration liquid separation device 200, and a plurality of culture fluid and reagent injection ports 500 may be provided at the inlet position of the step-by-step multi-concentration liquid separation device 200; through the plurality of culture fluid and reagent injection ports 500, the step-by-step multi-concentration liquid separation device 200 can mix the input culture fluid and reagent in a step-by-step manner to obtain culture fluid and reagents of different concentrations, and by controlling the microvalve body 300 on the micro-channel 400, the culture fluid and reagents of different concentrations are drained into different chambers 100, so that different culture operations can be performed in different chambers 100. For example, five culture fluid and reagent injection ports 500 are provided at the inlet of the stepped multi-concentration liquid separation device 200. Different culture fluids are injected into the five culture fluid and reagent injection ports 500, respectively. After the stepped mixing process of the stepped multi-concentration liquid separation device 200, culture fluids of different concentrations can be obtained. Then, the working state of the microvalve bodies 300 on different microchannels 400 is controlled, so that culture fluids of different concentrations can enter different chambers 100 of the microfluidic chip 10. It is worth noting that different chambers 100 can also be connected to the waste liquid outlet 700 through the microchannel 400. After the material culture operation is completed, the relevant material to be cultured can be discharged from the waste liquid outlet 700 through the waste liquid outlet 700, and the relevant chamber 100 is emptied to prepare for subsequent culture work. Each chamber 100 can be arranged with genes and proteins by a multi-array spotter, and cells can be loaded into the corresponding chamber 100 by the multi-array spotter or through the microfluidic cell screening inlet 600. The culture medium and cells enter the chamber 100, and the cultured material of the above-mentioned embodiment can be formed in the chamber 100.

[0101] Specifically, different culture fluids can enter the stepped multi-concentration liquid separation device 200 under the control of high-precision liquid pumps and valves; and cell screening can inject the corresponding cells into the corresponding chambers 100 under the action of the cell screening liquid pumps and valves; the microvalves can drain different culture fluids and cells into the corresponding chambers 100; because the microfluidic chip 10 is provided with a multi-channel electrode array 800, the multi-channel electrode array 800 can stimulate the substances to be cultured in different chambers 100 in the microfluidic chip 10 and collect and process physiological electrical signals.

[0102] Specifically, the chambers 100 in the microfluidic chip 10 can be arranged in an array. For example, they can be arranged in a 16*96 array, i.e., there are a total of 1536 chambers 100, each of which is connected to a microchannel 400. By controlling the microvalve bodies 300 on the microchannels 400, different substances to be cultured can be directed into different chambers 100. The microfluidic chip 10 can utilize a combination array of binary valve patterns. By controlling different microvalve bodies 300, the flow of liquid within the microchannels 400 can be manipulated. For example, 1024 microchannels 400, 1000 chambers 100, and 3574 microvalve bodies 300 can be provided. The microfluidic chip 10 can be used to implement functions such as sample preparation, chemical reactions, separation and mixing, detection and analysis, cell culture, and sorting and sequencing, and has broad application potential in a wide range of fields, including analytical testing, marker sequencing, organ chips, and drug screening. The microfluidic chip 10 adopts a multi-layer binary valve combination array mode, which can achieve precise control of up to thousands of channel chambers, thereby realizing high-throughput analysis of different samples. Compared with the consumption of traditional chemical detection reagents, it can reduce several orders of magnitude, and the cost of sample detection and screening is significantly reduced.

[0103] In some specific embodiments of the present application, there are multiple microchannels 400 and multiple microvalve bodies 300, wherein the multiple microvalve bodies 300 are controlled and processed so that different substances to be cultured enter different chambers 100 through different microchannels 400, and each chamber 100 corresponds to an electrode in the electrode array 800.

[0104] As shown in Figures 2 and 4, there can be multiple chambers 100, and different chambers 100 can be distributed in an array, with each chamber 100 corresponding to an electrode. Therefore, the electrode array 800 of the microfluidic electronic chip can be distributed in a multi-channel array to form a multi-channel electrode array; the electrode array 800 includes a multi-channel electrode array 800 based on gold, platinum, or graphene prepared by micro-nano processing; the multi-channel electrode array 800 ensures stable electrical signal monitoring during the culture process; micro-nano processing technology includes but is not limited to micro-nano processing methods such as photolithography, laser printing, or magnetron sputtering to ensure the processing accuracy and stability of the electrode array. The multi-channel electrode array 800 prepared in combination with micro-nano processing technology can be well integrated with cells or biological samples to achieve accurate acquisition of electrical signal information and stimulation feedback.

[0105] In some specific embodiments of the present application, the electrode array 800 can be used to stimulate the cultured material in the chamber 100 and to collect electrical signals fed back by the cultured material in the chamber 100. Specifically, the multi-channel electrode array 800 is integrated at the bottom of the microfluidic chip 10 and structurally includes recording or stimulation sites, connecting wires, and a packaging insulation layer. The cells or tissues cultured in the microfluidic chip 10 are in contact with the exposed electrode sites, so that external electrical stimulation can be applied through the electrode sites or electrophysiological signals can be collected in real time.

[0106] As shown in Figure 5, the electrode array 800 is arranged at the bottom of the microfluidic chip 10. The electrode array 800 includes electrode sites, connecting wires and a packaging insulation layer, wherein the electrode sites are in contact with the substance to be cultured in the chamber 100, and the electrode sites can electrically stimulate the substance to be cultured in the chamber 100 or collect and record physiological electrical signals fed back by the substance to be cultured in the chamber 100; specifically, a silicon dioxide substrate is provided below the electrode array 800 made of platinum and gold, and the electrode array 800 can be in contact with DNA or protein above. Cell tissue is provided above the DNA or protein, and the cell tissue is surrounded by polydimethylsiloxane. Silicon nitride ceramic is also provided between the polydimethylsiloxane and the electrode array 800. The electrode array 800 is in contact with the substance to be cultured, and then the electrode array 800 can stimulate the substance to be cultured and collect and process physiological electrical signals.

[0107] As shown in FIG. 1 and FIG. 3 , in some specific embodiments of the present application, an information acquisition and processing system 900 is further included, and the electrode array 800 is connected to the information acquisition and processing system 900 .

[0108] It should be noted that the electrode array 800 is connected to the information acquisition and processing system 900 , so that the information acquisition and processing system 900 can perform potential stimulation control processing and physiological electrical signal acquisition processing on the electrode array 800 .

[0109] In some specific embodiments of the present application, the information acquisition and processing system 900 includes a processing circuit unit, a controller 910 and a control circuit unit 920, and the electrode array 800, the processing circuit unit, the controller 910, the control circuit unit 920 and the control input component are connected in sequence.

[0110] It should be noted that the processing circuit unit can process the electrical signals collected by the electrode array 800, and then transmit the processed collection signals to the controller 910. Then, the controller 910 can control and process the control circuit unit 920 according to the received collection signals, so as to control and process the working state of the control input component, so as to electrically stimulate the material to be cultured in the chamber 100 and collect and process physiological electrical signals.

[0111] As shown in FIG. 3 , in some specific embodiments of the present application, the information collection and processing system 900 further includes a communication module 950 , which is connected to the controller 910 .

[0112] It should be noted that the communication module 950 is connected to the controller 910, and the controller 910 can send the received detection information related to the electrode array 800 to the monitoring terminal through the communication module 950, and the detection personnel can quickly and easily understand the detection status of the electrode array 800 through the monitoring terminal. Among them, the communication module 950 may include an antenna or a data cable. The monitoring terminal can be a mobile phone, a tablet or a computer. Through the information acquisition and processing system 900, the physiological environment and growth status of the sample or cell tissue in the chamber 100 of the microfluidic chip 10 can be displayed in real time, providing a personalized monitoring platform.

[0113] In some specific embodiments of the present application, the processing circuit unit includes a preprocessing circuit subunit 940 and an analog-to-digital conversion subunit 930, and the electrode array 800, the preprocessing circuit subunit 940, the analog-to-digital conversion subunit 930 and the controller 910 are connected in sequence.

[0114] It should be noted that the processing circuit unit includes a pre-processing circuit sub-unit 940 and an analog-to-digital conversion sub-unit 930, wherein the pre-processing circuit sub-unit 940 may include a filtering unit and an amplifying unit, so that the amplifying unit can amplify the physiological electrical signals collected by the electrode array 800, and then the amplified electrical signals need to be processed by the filtering unit for interference removal; then the electrical signals after interference removal can be transmitted to the controller 910.

[0115] Specifically, the control circuit unit 920 may include a boost circuit unit and a drive circuit unit. The controller 910 may control the microvalve body 300 or the high-precision injection pump of the microfluidic chip 10 through the boost circuit unit and the drive circuit unit, so that different culture fluids enter different chambers 100; since the substances to be cultured in the chamber 100 of the microfluidic chip 10 need to be cultured in a suitable environment, such as suitable temperature, humidity and carbon dioxide concentration, relevant sensors may be provided around the microfluidic electronic chip, and the environment may be detected by the relevant sensors, and the detected environmental signals may be transmitted to the controller 910, and then the controller 910 may control and process the environment of the microfluidic electronic chip, such as adjusting the temperature, humidity and carbon dioxide concentration.

[0116] As shown in Figure 3, specifically, the preprocessing circuit subunit 940 is used to filter and amplify the voltage or current signals collected by the electrode array 800 or the temperature sensor, humidity sensor, and carbon dioxide sensor; the analog-to-digital conversion subunit 930 is used to convert the preprocessed signal into a digital signal that can be recognized by subsequent equipment; the control circuit unit 920 is used to generate the voltage output required for voltage stimulation, or the high current output required for the current stimulation constant current source, or drive a high-precision injection pump, or control the heating source, humidifier and carbon dioxide valve, etc.; the controller 910 can be programmed to process the data collected by the temperature sensor, humidity sensor, and carbon dioxide sensor, receive instructions issued by the monitoring terminal, transmit the received data to the monitoring terminal, and output digital signals to control the various parts in the control circuit unit 920 for control processing.

[0117] It is worth noting that the microfluidic chip 10 involved in the embodiments of the present application can include organ chips, droplet chips, and high-throughput sequencing chips. The microfluidic chip 10 adopts a modular design, which can be standardized and highly freely matched with the information acquisition and processing system 900, and has strong scalability. The substances to be cultured in the chamber 100 of the embodiments of the present application can include cells, microorganisms, proteins, DNA, RNA, and organoids.

[0118] Through the above technical solution, the microfluidic electronic chip provided in the embodiment of the present application has the characteristics of high precision, high throughput and high intelligence, and can realize the fusion of the microfluidic chip 10 and the flexible electrode, thereby preparing an integrated electronic chip for sample separation and screening, cell screening and culture, and real-time monitoring and feedback, which has broad use value and clinical application prospects.

[0119] In some specific embodiments of the present application, the substance to be cultured may include at least one of the following: cells, tissues, DNA, RNA, proteins, and metabolic molecules. All of the above substances can be injected into the chamber 100 to form the substance to be cultured.

[0120] In some specific embodiments of the present application, the cells may include at least any one of the following types: vascular cells, brain cells, liver cells, lung cells, kidney cells, heart cells, spleen cells and intestinal cells.

[0121] As shown in FIG6 , in some embodiments of the present application, one embodiment of the present application further provides a method for preparing an organoid model, which is implemented based on the microfluidic electronic chip of the above embodiment, and the method includes but is not limited to steps S110, S120, S130, S140, and S150:

[0122] Step S110, injecting stem cells into the chambers of the microfluidic electronic chip in sequence to culture and generate organoid spheroids;

[0123] Step S120, performing enzymatic hydrolysis on the organoid spheroids to form self-assembled cells in each chamber of the microfluidic electronic chip, and adding the self-assembled cells to the lower chamber;

[0124] Step S130, culturing the self-assembled cells so that the self-assembled cells generate an initial organoid model;

[0125] Step S140, culturing the initial organoid model to form organoids;

[0126] Step S150 , intermittently electrically stimulating the organoid based on the electrode array and collecting electrophysiological information of the organoid to construct a functional organoid model.

[0127] It should be noted that in the process of preparing the organoid model, stem cells are first injected into the chambers of the microfluidic electronic chip in sequence to culture and generate organoid spheroids; then the organoid spheroids are enzymatically hydrolyzed to form self-assembled cells in each chamber of the microfluidic electronic chip, and the self-assembled cells are added to the lower chamber; then the self-assembled cells are cultured so that the self-assembled cells generate an initial organoid model; then the initial organoid model is cultured to form an organoid; finally, the organoid is intermittently electrically stimulated based on the electrode array and the electrophysiological information of the organoid is collected to construct a functional organoid model.

[0128] It is worth noting that the preparation method of the above-mentioned organoid models is general, and vascular organoid models, brain organoid models, heart organoid models, liver organoid models, lung organoid models, kidney organoid models, spleen organoid models and intestinal organoid models can all be obtained through the above-mentioned preparation method.

[0129] In some embodiments of the present application, the organoids include at least one of the following types: vascular organoids, brain organoids, heart organoids, liver organoids, lung organoids, kidney organoids, spleen organoids, and intestinal organoids. Vascular organoids include endothelial cells, fibroblasts, and smooth muscle cells; brain organoids include brain stem cells, neurons, and glial cells; and heart organoids include cardiomyocytes, endocardial cells, epicardial cells, and cardiovascular cells.

[0130] For example, for vascular organoids, microfluidic electronic chips can be used for organ chip culture. The main modules of the microfluidic electronic chip include vascular network chip, microfluidic organ chip and high-throughput electrode array. The vascular network chip is connected to the external liquid supply pump, which circulates liquid to provide cell nutrients; the microfluidic organ chip consists of biological tissue chambers and microfluidic connecting pipes, which simulate the cell microbial environment and provide a cell growth scenario; the high-throughput electrode array is connected to the upper cell chamber. The high-throughput integrated microfluidic electronic chip mainly includes a chamber, a vascular network chip, a high-throughput flexible electrode array and a circulating fluid supply system; the chamber is connected to an external high-precision injection pump to control the fluid supply; the vascular network chip is composed of endothelial cells, fibroblasts and smooth muscle cells, which are co-cultured in microchannels to provide the nutrients required for organ culture; the flexible neural electrode is prepared using micro-nano processing technology combined with flexible conductive materials, and the organ tissue is cultured above the flexible electrode array, and has monitoring and stimulation functions; in addition, the external system of the electronic chip contains temperature control, humidity control and carbon dioxide supply systems to ensure the good growth of the organ chip.

[0131] Specifically, the process of preparing vascular networks and brain organ chips can include the following steps: uniformly mixing polydimethylsiloxane and its curing agent in a mass ratio of 10:1, vacuum degassing and pouring on a silicon wafer template prepared by photolithography, peeling off the polydimethylsiloxane template from the silicon wafer after curing, and punching holes at relevant entrance and exit positions; then using microfluidic printing technology to fill the liquid metal particles after ultrasound into the microfluidic electrode channel, pouring stretchable polymer material polyurethane on top, and peeling off from the polydimethylsiloxane channel after the polymer film is formed to obtain the liquid metal electrode transferred to the polyurethane film, and then electrostatically bonding the liquid metal particles to the electrode. An insulating layer is encapsulated above the electrode array to produce a liquid metal electrode array; then the polydimethylsiloxane chip and the flexible electrode array are bonded by a plasma cleaning machine to obtain a microfluidic electronic chip model; the microfluidic chip is connected to the external liquid supply system through microchannels to achieve circulating liquid supply; then endothelial cells, fibroblasts and smooth muscle cells are mixed and injected into the vascular chip pipeline to construct a vascular network chip; neural stem cells are injected into the brain chip pipeline, and the neural stem cells grow above the electrode array monitoring site and are co-cultured with the vascular network to construct a vascularized organ chip; the obtained vascularized brain chip is then cultured in a 37°C, 5% CO2 environment, and the chip pipeline is connected to the external circulating liquid supply system through a peristaltic pump to provide the nutrients required for cell culture; finally, the high-throughput electrode array is connected to the external electrophysiological acquisition system equipment to observe the physiological state of the cultured neural stem cells in real time.

[0132] As shown in FIG7 , in some embodiments of the present application, one embodiment of the present application further provides a DNA-protein binding analysis method, which is implemented based on the microfluidic electronic chip of the above embodiment, and the method includes but is not limited to steps S210, S220, S230, S240, S250, S260, and S270:

[0133] Step S210, preparing a DNA template of a protein transcription factor, wherein the DNA template has an antibody tag;

[0134] Step S220, constructing a library based on the DNA template, adding a fluorescent marker, and obtaining a sample DNA dilution solution;

[0135] Step S230 , spotting the sample DNA dilution solution into the chamber of the microfluidic electronic chip;

[0136] Step S240 , patterning the surface of the chamber based on biotinylated bovine serum albumin, phosphate buffer, and biotinylated antibody;

[0137] Step S250, loading the in vitro transcription / translation protein transcription factor mixture into the chamber, so that the in vitro transcription / translation protein transcription factor binds to the DNA template in the sample DNA dilution solution to form a binding molecule, and the binding molecule is captured and deposited by the biotinylated antibody covered in the chamber;

[0138] Step S260, washing the chamber with phosphate buffer to remove unbound molecules and capture bound molecules;

[0139] Step S270 , scanning the microfluidic chip using a fluorescence microscope to quantify the bound molecules.

[0140] It should be noted that in the process of DNA protein binding analysis, the DNA template of the protein transcription factor must first be prepared, and the DNA template has an antibody label; then a library is constructed based on the DNA template, and a fluorescent label is added to obtain a sample DNA dilution solution; then the sample DNA dilution solution is spotted into the chamber of the microfluidic electronic chip; then the surface of the chamber is patterned based on biotinylated bovine serum albumin, phosphate buffer and biotinylated antibody; then the in vitro transcription / translation protein transcription factor mixture is loaded into the chamber, so that the in vitro transcription / translation protein transcription factor binds to the DNA template in the sample DNA dilution solution to obtain a bound molecule, and the bound molecule is captured and deposited by the biotinylated antibody covered in the chamber; then the chamber is cleaned with phosphate buffer to remove unbound molecules and capture bound molecules; finally, the microfluidic chip is scanned using a fluorescence microscope to quantify the bound molecules.

[0141] In order to more clearly illustrate the specific process of the DNA protein binding analysis method provided in the embodiments of the present application, a specific example is given below.

[0142] Synthesis of DNA templates for protein transcription factors: DNA templates for protein expression are generated via a two-step polymerase chain reaction (PCR). In the first step, gene-specific primers are used to amplify the DNA sequence of the target transcription factor. During amplification, an upstream Kozak sequence, a C-terminal 6-histidine tag (His6), and a terminator are added. In the second step, universal primers are used to add the upstream T7 promoter 5′UTR, 3′ poly-A tail, and downstream T7 terminator 3′UTR to the amplified product.

[0143] DNA library construction: The sequence to be tested is hybridized with a Cy5-labeled oligonucleotide, and then amplified using the Klenow fragment (exo-) digested by DNA polymerase. During the amplification reaction, the Klenow fragment adds complementary bases to the DNA sequence of the hybrid product and simultaneously trims off unpaired bases at the ends of the single-stranded DNA, making the synthesized DNA chain more accurate and complete. Ultimately, double-stranded DNA labeled with Cy5 is formed. After synthesis, the Cy5-labeled double-stranded DNA is added to 70 μL of a solution containing 0.5% bovine serum albumin (BSA), 0.125% polyethylene glycol (PEG), and 1.25 mg / mL D-(+)-trehalose dihydrate to prevent irreversible binding of the DNA to the printed slide. The DNA is diluted to a final concentration of 2 μmol / L, and serial dilutions are prepared and loaded into a well plate.

[0144] DNA printing: Sample DNA from a well plate is spotted onto an epoxy-coated glass slide in microarray format using a QArray2 DNA microarray chip. The device is assembled by manually aligning the array using a stereomicroscope and bonding overnight in a 40°C incubator. The neck valve is closed to protect the DNA chamber.

[0145] To pattern the surface of a single reaction chamber, 30 μL of 2 mg / mL biotinylated bovine serum albumin (BSA-biotin) was introduced into the device, where the biotin covalently bound to the epoxy resin. After 2-3 minutes, the outlet valve was closed and pressure was continued for 5-10 minutes to ensure that all parts of the device were filled with liquid and to expel any air bubbles. The outlet valve was opened to allow reagent flow and the device was rinsed with phosphate-buffered saline (PBS). A 0.5 mg / mL solution of Neutravidin in PBS was perfused for 20-30 minutes to completely cover the device surface, followed by a 10-minute rinse with PBS. The push-button valve was closed, and biotinylated bovine serum albumin (BSA-biotin) was added to partially cover the device surface. The push-button valve was opened, and 30 μL of a 1:1 biotinylated anti-His6 antibody in 2% bovine serum albumin (BSA) was added for 20 minutes. The device was then rinsed with PBS for another 10 minutes to complete the surface patterning.

[0146] In vitro transcription / translation of protein transcription factors, binding of proteins to DNA: Prepare 25 μL of TNT T7 in vitro transcription / translation reagent and add 1 μL of tRNA Lys-boron-dipyrromethene (BODIPY)-Fl and 2 μL of transcription factor template. Open the sandwich valve, load the mixture into the device, and rinse for 5-7 minutes until the mixture is completely filled. Close the sandwich valve, while preventing the reaction mixture and DNA from adjacent unit reaction chambers from mixing. Open the neck valve, allowing the solution to enter the DNA reaction chamber and dissolve the Cy5-labeled sample DNA. The unit reaction chamber temperature is maintained at 37°C. BODIPY- and His-tagged transcription factors are synthesized by in vitro transcription / translation according to the template. The synthesized transcription factors can bind to the sample DNA and be captured by the anti-His6 antibody coated on the device surface before deposition. The reaction lasts for 90 minutes.

[0147] The button valve captures surface-bound molecules: Once the protein transcription factor and DNA binding reaches a stable state and the reaction is complete, the button valve and neck valve are closed to capture the surface-bound molecules. Wash with phosphate-buffered saline (PBS) for 5 minutes to remove unbound molecules.

[0148] Fluorescence microscope scanning chip:

[0149] Following the above steps, the chip is scanned using a fluorescence microscope, measuring the Cy5 and BODIPY channels to quantify protein transcription factor binding to DNA. The Cy5 intensity within the DNA chamber reflects the concentration of free DNA available for binding [D]. The BODIPY intensity after capture by the push-button valve reflects the number of surface-bound protein molecules; the Cy5 intensity reflects the number of protein-bound DNA molecules. The ratio of Cy5 to BODIPY fluorescence (r) reflects the concentration of protein bound to DNA. The binding affinity, Kd, ​​is calculated using the following formula: Kd is the DNA concentration at which half of the binding sites are occupied when the reaction reaches equilibrium.

[0150] For in vitro transcription / translation of protein transcription factors, the protein is bound to DNA. Alternatively, in vitro transcription / translation of the protein transcription factor can be performed on the chip first. Open the sandwich valve, close the neck valve, and introduce 50 μL of the synthesized protein transcription factor into the device. Allow the protein to flow for 40 minutes. Open the neck valve and close the sandwich valve to allow the protein solution to enter the DNA chamber and dissolve the DNA.

[0151] As shown in FIG8 , in some embodiments of the present application, an embodiment of the present application further provides an RNA protein binding analysis method, which is implemented based on the microfluidic electronic chip of the above embodiment, and the method includes but is not limited to steps S310, S320, S330, S340, S350, S360, S370, and S380:

[0152] Step S310, preparing protein and storing it in a preset low-temperature environment;

[0153] Step S320, preparing a dilution of RNA library transcription template based on the DNA oligonucleotide;

[0154] Step S330, spotting the RNA library transcription template dilution solution into the chamber of the microfluidic electronic chip;

[0155] Step S340 , patterning the surface of the chamber based on biotinylated bovine serum albumin, phosphate buffer, and a capture probe containing a fluorescent marker;

[0156] Step S350, loading the in vitro transcription mixture into the chamber, so that the RNA library transcription template in the RNA library transcription template dilution solution is transcribed to obtain transcribed RNA, and the transcribed RNA is hybridized with the capture probe;

[0157] Step S360, thawing the protein, mixing the thawed protein with the antibody to obtain a protein mixture; and loading the protein mixture into the chamber to allow the transcribed RNA to bind to the protein to obtain a binding molecule;

[0158] Step S370, washing the chamber with phosphate buffer to remove unbound molecules and capture bound molecules;

[0159] Step S380 , scanning the microfluidic chip using a fluorescence microscope to quantify the bound molecules.

[0160] It should be noted that in the process of RNA protein binding analysis, the protein is first prepared and stored in a preset low-temperature environment; then, an RNA library transcription template dilution is prepared based on DNA oligonucleotides; then the RNA library transcription template dilution is spotted into the chamber of the microfluidic electronic chip; then the surface of the chamber is patterned based on biotinylated bovine serum albumin, phosphate buffer and a capture probe containing a fluorescent marker; then the in vitro transcription mixture is loaded into the chamber so that the RNA library transcription template in the RNA library transcription template dilution is transcribed to obtain transcribed RNA, and the transcribed RNA is hybridized with the capture probe; then the protein is thawed, and the thawed protein is mixed with the antibody to obtain a protein mixture; and the protein mixture is loaded into the chamber so that the transcribed RNA is bound to the protein to obtain a bound molecule; then the chamber is cleaned with phosphate buffer to remove unbound molecules and capture bound molecules; finally, the microfluidic chip can be scanned using a fluorescence microscope to quantify the bound molecules.

[0161] In order to more clearly illustrate the specific process of the RNA protein binding analysis method provided in the embodiments of the present application, a specific example is given below.

[0162] Stem-Loop Binding Protein Expression: Cells were grown in suspension culture in SF900-II medium supplemented with 10% fetal bovine serum. A stem-loop binding protein target gene containing an N'-glutathione-S transferase (GST) tag was inserted into the transcriptome of an expression vector using recombinant technology. Cells were infected and cultured to produce the expressed stem-loop binding protein on a large scale. After 3 days, cells were pelleted by centrifugation and lysed in a buffer solution. The clarified lysate was then bound to Ni-NTA resin and the target stem-loop binding protein was eluted. Protein samples were aliquoted and stored at -80°C.

[0163] DNA oligonucleotides as transcription templates for the RNA library: Equal amounts (9 μmol / L) of the mutation-containing antisense oligonucleotide DNA and the sense T7 promoter oligonucleotide are annealed at 95°C for 1 minute, cooled to room temperature, and diluted 1000-fold in phosphate-buffered saline (PBS) to a final concentration of 10 mg / mL. The dilution is then loaded into the well plate. The antisense oligonucleotide will generate a poly-A tail in step 800.

[0164] RNA library transcription template printing: The steps are the same as the above DNA printing steps, except that the RNA library transcription template is printed on the microarray.

[0165] Unit reaction chamber surface patterning: The procedure for unit reaction chamber surface patterning is the same as that for DNA-protein binding analysis described above, except that a 5' biotin, 3' fluorescein (FAM) labeled poly-T single-stranded DNA capture probe is used instead of the biotinylated anti-His6 antibody.

[0166] RNA is transcribed in vitro and bound to the capture probe. Prepare the in vitro transcription mixture, open the sandwich valve, load the mixture into the device, and rinse for 5-7 minutes until the mixture is completely filled. Open the neck valve and allow the in vitro transcription mixture to flow into the DNA chamber. Close the neck valve and transcribe at 37°C for 30 minutes. Then rinse the chip with HBSEP buffer (HEPES Buffered Saline with EDTA and Surfactant Polysorbate). After rinsing, open the button valve and neck valve, close the sandwich valve, and hybridize the transcribed RNA with the capture probe in the detection chamber for 1 hour. The Iowa Black Fluorescent Quencher probe can be used for binding and the FAM channel can be fluorescently scanned to detect RNA binding.

[0167] Stem-loop binding protein antibody labeling and RNA-protein binding: Thaw the stem-loop binding protein. Mix 1 μL of stem-loop binding protein with 5 μL of 1.0 μg / μL Texas Red-labeled GST antibody and incubate the mixture at room temperature for 1 hour. Open the sandwich valve, close the neck valve, and introduce 50 μL of antibody-conjugated stem-loop binding protein into the device. Allow the flow to continue for 40 minutes. Open the neck valve, close the sandwich valve, and allow the protein solution to enter the DNA chamber and react with the RNA. The reaction continues for 90 minutes.

[0168] The push-button valve captures surface-bound molecules. The corresponding steps are the same as those described above for DNA-protein binding analysis.

[0169] Fluorescence microscopy: Scan the chip using a fluorescence microscope to measure the fluorescein (FAM) and Texas Red channels to quantify RNA-protein binding.

[0170] As shown in FIG9 , in some embodiments of the present application, one embodiment of the present application further provides a protein binding analysis method, which is implemented based on the microfluidic electronic chip of the above embodiment, and the method includes but is not limited to steps S410, S420, S430, S440, S450, S460, and S470:

[0171] Step S410, preparing a DNA template for a protein, wherein the protein includes a target protein and a binding protein; the target protein has a first amino acid chain or molecule at its N-terminus and a second amino acid chain or molecule at its C-terminus; and the binding protein has a third amino acid chain or molecule at its C-terminus;

[0172] Step S420, spotting the DNA template into the chamber of the microfluidic electronic chip;

[0173] Step S430 , patterning the surface of the chamber based on biotinylated bovine serum albumin, phosphate buffer, and anti-amino acid chain or molecule antibodies;

[0174] Step S440, loading the in vitro transcription / translation protein transcription factor mixture into the chamber, so that the in vitro transcription / translation protein transcription factor binds to the DNA template to obtain a binding molecule, and the binding molecule is captured and deposited by the anti-amino acid chain or molecule antibody covered in the chamber;

[0175] Step S450, loading the antibody solution into the chamber, so that the protein binds in the antibody solution to obtain a binding molecule;

[0176] Step S460, washing the chamber with phosphate buffer to remove unbound molecules and capture bound molecules;

[0177] In step S470 , the microfluidic chip is scanned using a fluorescence microscope to quantify the bound molecules.

[0178] It should be noted that in the process of protein binding analysis, a DNA template of the protein is first prepared, wherein the protein includes a target protein and a binding protein; the N-terminus of the target protein contains a first amino acid chain or molecule, and the C-terminus contains a second amino acid chain or molecule; the C-terminus of the binding protein contains a third amino acid chain or molecule; the DNA template is then spotted into the chamber of the microfluidic electronic chip; the surface of the chamber is then patterned based on biotinylated bovine serum albumin, phosphate buffer and anti-amino acid chain or molecule antibodies; the in vitro transcription / translation protein transcription factor mixture is then loaded into the chamber, so that the in vitro transcription / translation protein transcription factor binds to the DNA template to obtain a binding molecule, and the binding molecule is captured and deposited by the anti-amino acid chain or molecule antibody covered in the chamber; the antibody solution is then loaded into the chamber, so that the protein binds in the antibody solution to obtain a binding molecule; the chamber is then cleaned with phosphate buffer to remove unbound molecules and capture bound molecules; finally, the microfluidic chip is scanned using a fluorescence microscope to quantify the bound molecules. Among them, the first amino acid chain or molecule, the second amino acid chain or molecule and the third amino acid chain or molecule all act as labels; the first amino acid chain or molecule is used to detect protein expression content; the second amino acid chain or molecule is used for capture; and the third amino acid chain or molecule is used to detect the interaction force between proteins.

[0179] In order to more clearly illustrate the specific process of the protein binding analysis method provided in the embodiments of the present application, a specific example is given below.

[0180] Protein DNA template synthesis: The steps for synthesizing the DNA template for protein transcription factors in the DNA-protein binding analysis method are the same. Synthesize the protein of interest and potential binding proteins. The N-terminus of the protein of interest contains a c-myc tag for detecting protein expression levels and a T7 tag for capture at the C-terminus. The C-terminus of the potential binding protein contains a 6-histidine tag (His6) for detecting protein-protein interactions.

[0181] DNA printing: The steps for DNA printing of protein transcription factors are the same as those in the DNA-protein binding analysis method described above; the difference is that the protein of interest and the DNA template of the potential binding protein are printed on the microarray at the same time.

[0182] Unit reaction chamber surface patterning: The procedure for unit reaction chamber surface patterning is the same as that for protein transcription factors in the above-mentioned DNA-protein binding analysis method, except that anti-T7 antibody is used instead of biotinylated anti-His6 antibody.

[0183] In vitro transcription / translation of protein-protein interactions; the in vitro transcription / translation steps are identical to those described above for DNA-protein binding analysis. Proteins synthesized within the DNA chamber interact with each other and are captured by anti-T7 antibodies coated on the device surface before deposition. The reaction proceeds at 30°C for 2.5 hours.

[0184] Antibody-labeled proteins: Cy3-labeled anti-c-myc antibody and Alexa647-labeled anti-His6 antibody were mixed in a 2% bovine serum albumin (BS) solution. Cy3 and Alexa647 serve as the acceptor and donor, respectively, for fluorescence resonance energy transfer (FRET). Open the sandwich valve, close the neck and button valves, and introduce the antibody solution for 5 minutes. Close the isolation and chamber valves, and allow the protein to bind to the antibody for 20 minutes.

[0185] A push-button valve captures surface-bound molecules, similar to the DNA-protein binding analysis method described above.

[0186] Fluorescence microscope scanning chip: Use a fluorescence microscope to scan the chip, measure the Cy3 and Alexa647 channels, and quantify the binding between proteins.

[0187] As shown in Figure 10, in some embodiments of the present application, one embodiment of the present application also provides a liposome transfection method, which is implemented based on the microfluidic electronic chip of the above embodiment, and the method includes but is not limited to step S510, step S520, step S530, step S540, step S550 and step S560.

[0188] Step S510 , coating the fixed adhesion substance on a glass slide in a chamber of a microfluidic electronic chip;

[0189] Step S520, preparing a liposome sample and transferring the liposome sample to a glass slide coated with a fixed adhesion substance;

[0190] Step S530, subjecting the microfluidic electronic chip loaded with the liposome sample to plasma treatment;

[0191] Step S540 , preparing a cell culture solution and injecting the cell culture solution into the microfluidic electronic chip to culture and transfect the cells in the cell culture solution;

[0192] Step S550, injecting culture medium into the microfluidic electronic chip to promote cell surface attachment;

[0193] Step S560: Scan the microfluidic chip using a fluorescence microscope to measure the transfection efficiency.

[0194] It should be noted that during the liposome transfection process, the fixed adhesion is first coated on the glass slide in the chamber of the microfluidic electronic chip, then the liposome sample is prepared and the liposome sample is transferred to the glass slide coated with the fixed adhesion; then the microfluidic electronic chip loaded with the liposome sample is plasma treated; then the microfluidic electronic chip loaded with the liposome sample is plasma treated; then the cell culture fluid is prepared and the cell culture fluid is injected into the microfluidic electronic chip to culture and transfect the cells in the cell culture fluid; then the culture medium is injected into the microfluidic electronic chip to promote cell surface attachment; finally, the microfluidic chip is scanned using a fluorescence microscope to measure the transfection efficiency.

[0195] It is worth noting that transfection is usually performed on high-throughput microarrays, but this device requires manual cell culture and cannot precisely control the cell environment. Transfection through high-throughput integrated microfluidic electronic chips can simplify the process of cell loading and culture, and implement multiple transfections. Microfluidic electronic chips can strictly regulate the culture environment and use the physical isolation characteristics of the chamber to avoid the problem of cross-contamination between transfection reactions. Microfluidic electronic chips are combined with high-content imaging and analysis technologies to achieve real-time observation and analysis of cells during the transfection process, providing more cellular-level information and helping to gain a deeper understanding of the dynamic changes and cellular responses during the transfection process.

[0196] It is worth noting that the fixed adhesion substance can be connected to the liposome sample by physical adhesion, intermolecular force or chemical bond, so that the liposome sample is fixed on the microfluidic chip; wherein the fixed adhesion substance can be poly-L-lysine.

[0197] In order to more clearly illustrate the specific process of the liposome transfection method provided in the examples of the present application, a specific example is given below.

[0198] The microarray surface was patterned using poly-L-lysine (PLL). The slide was immersed in a culture dish containing 57% ethanol and 10% NaOH and incubated for 2 hours to remove surface contamination. The slide was then thoroughly rinsed with ultrapure water and air-dried. 25 μL of 0.1% poly-L-lysine (PLL) solution was added to 50 μL of 0.225 mol / L boric acid solution (pH 8.4) to prepare a poly-L-lysine (PLL) solution, which was then loaded into the well plate for later use. The poly-L-lysine (PLL) was adsorbed onto the slide surface using an array needle through electrostatic interaction. Four prints were performed on each spot, with an interval of approximately 8 minutes between each print to ensure that the arrangement of the spots matched the chambers on the microfluidic chip. Two hours after patterning, the microarray surface was rinsed with ultrapure water, blown dry with nitrogen, and stored in a desiccator.

[0199] 1.5 μg of plasmid containing fluorescent protein was added to 5 μL of 0.2 mol / L sucrose-EC buffer, the mixture was vortexed for 10 seconds and incubated for 15 minutes. 12.7 μL of 0.5% gelatin solution and 12.7 μL of 0.1% fibronectin solution were added and pipetted up and down to mix to form a liposome sample, and the sample was loaded into a well plate for later use. The liposome sample was transferred to a slide coated with poly-L-lysine (PLL) using an array needle, and printed once at each point. Poly-L-lysine (PLL) acts as a binder to fix the liposome molecules on the surface of the slide, providing stability and spatial organization for the liposome sample, facilitating precise control of the position of the liposome sample on the slide and facilitating subsequent analysis.

[0200] The liposome-loaded microarray was plasma-treated with a PDMS chip, and the device was manually aligned and assembled using a stereomicroscope. This chip assembly was then connected to a microfluidic device, with each liposome molecule enclosed within a unit reaction chamber.

[0201] A pressure source connected to a manifold allows manipulation of specific controls and flow lines on the chip. The manifold's ports can be individually controlled and connected to the chip components via flexible tubing. A mixture containing filtered, CO2-independent culture medium, 10% fetal bovine serum (FBS), an antibiotic-antimycotic solution, and the glutamine-containing culture medium supplement GlutaMax is connected to the microfluidic device, providing the necessary culture environment for cells. Cell culture and experiments can be performed under microfluidic conditions.

[0202] 800,000 cells at 70–80% confluence were centrifuged and resuspended in 20 μL of PBS. The cells were loaded into a flexible tube connected to the manifold and chip. Valves controlling the flow rate were used to purge the cells onto the chip at a rate of 27 μL / min. All valves were opened to facilitate filling the entire chip. When a sufficient number of cells (~70% confluence) was achieved in the chamber, the chamber divider valve was opened to capture the cells. The chip temperature was maintained at 37°C for cell culture and transfection.

[0203] One hour after cell loading, the cells are pulsed with culture medium to promote cell surface attachment. The culture medium flows through the channel and the cell capture chamber is closed. This process lasts for 5 minutes. The flow of culture medium is stopped, and the valve connecting the cell capture chamber and the medium channel is opened to allow medium diffusion. This process lasts for 1 hour. Subsequently, the valves of the top and bottom chambers are opened, and the culture medium continues to flow at a rate of 1 mL / h. Wastewater can be removed from the chip assembly through the hose through the outlet valve. Bubbles generated during perfusion can be reduced by pressurizing and closing the outlet valve.

[0204] 48 hours after the cells were introduced into the chip, cellular protein expression was monitored using a fluorescence microscope to count the number of fluorescent cells and measure the transfection efficiency.

[0205] As shown in Figure 11, in some embodiments of the present application, one embodiment of the present application also provides an electroporation transfection method, which is implemented based on the microfluidic electronic chip of the above embodiment, and the method includes but is not limited to step S610, step S620, step S630, step S640 and step S650.

[0206] Step S610, printing a plasmid containing a fluorescent protein onto a microfluidic electronic chip;

[0207] Step S620 , preparing a cell culture solution and injecting the cell culture solution into the microfluidic electronic chip to culture and transfect the cells in the cell culture solution;

[0208] Step S630 , stimulating cells in the cell culture medium based on the electrode array of the microfluidic electronic chip, so that pores are temporarily formed in the cell membranes, and the plasmids enter the cells through the pores;

[0209] Step S640, injecting culture medium into the microfluidic electronic chip to promote cell surface attachment;

[0210] Step S650: Scan the microfluidic chip using a fluorescence microscope to measure the transfection efficiency.

[0211] It should be noted that during the electroporation transfection process, the plasmid containing the fluorescent protein is first printed onto the microfluidic electronic chip; then the cell culture fluid is prepared and injected into the microfluidic electronic chip to culture and transfect the cells in the cell culture fluid; then the cells in the cell culture fluid are stimulated based on the electrode array of the microfluidic electronic chip, so that holes are temporarily formed in the cell membrane, and the plasmid enters the cell through the holes; then the culture medium is injected into the microfluidic electronic chip to promote cell surface attachment; finally, the microfluidic chip is scanned using a fluorescence microscope to measure the transfection efficiency.

[0212] In order to more clearly illustrate the specific process of the electroporation transfection method provided in the embodiments of the present application, a specific example is given below.

[0213] A plasmid containing a fluorescent protein is printed onto a microfluidic electronic chip. Cells are loaded and cultured using the liposome transfection method described above. Electrodes are then applied to the microarray chip to create transient pores in the cell membrane, allowing the plasmid to enter the cells. Post-transfection is performed using the liposome transfection method described above. Transfection efficiency is measured using fluorescence using the liposome transfection method described above.

[0214] Those skilled in the art will appreciate that all or some of the steps and systems in the method disclosed above can be implemented as software, firmware, hardware, and appropriate combinations thereof. Some physical components or all physical components can be implemented as software executed by a processor, such as a central processing unit, a digital signal processor, or a microprocessor, or implemented as hardware, or implemented as an integrated circuit, such as an application-specific integrated circuit. Such software can be distributed on a computer-readable medium, and the computer-readable medium can include computer storage media (or non-transitory media) and communication media (or temporary media). As known to those skilled in the art, the term computer storage media is included in any method or technology for storing information (such as computer-readable instructions, data structures, program modules, or other data) and is volatile and non-volatile, removable, and non-removable. Computer storage media includes, but is not limited to, RAM, ROM, EEPROM, flash memory, or other memory technology, CD-ROM, digital versatile disks (DVD), or other optical disk storage, magnetic cassettes, magnetic tapes, disk storage, or other magnetic storage devices, or any other medium that can be used to store desired information and can be accessed by a computer. Furthermore, as is well known to those skilled in the art, communication media typically embodies computer-readable instructions, data structures, program modules, or other data in a modulated data signal such as a carrier wave or other transport mechanism, and may include any information delivery media.

[0215] The above is a specific description of the preferred implementation of the present application, but the present application is not limited to the above implementation mode. Technical personnel familiar with the field can also make various equivalent modifications or substitutions without violating the protection of the present application. These equivalent modifications or substitutions are all included in the scope defined by the claims of the present application.

Claims

1. A microfluidic electronic chip, characterized in that: include: A microfluidic chip, the microfluidic chip comprising one or more chambers for containing a substance to be cultured; An electrode array is connected to the chamber and is used for stimulating and electrophysiologically monitoring the substance to be cultured in the chamber.

2. The microfluidic electronic chip according to claim 1, characterized in that: The microfluidic chip comprises a control input component, the microfluidic chip comprises a plurality of chambers, the plurality of chambers are connected to the control input component, and the control input component is used to control the substances to be cultured to enter the corresponding chambers; Preferably, the control input component comprises a micro-channel, a micro-valve body arranged on the micro-channel and a stepped multi-concentration liquid separation device, and the stepped multi-concentration liquid separation device is connected to the chamber through the micro-channel; Preferably, the control input component includes a plurality of the microchannels and a plurality of the microvalve bodies, wherein the plurality of the microvalve bodies are controlled so that different substances to be cultured enter into different chambers through different microchannels, and each chamber corresponds to an electrode in the electrode array.

3. The microfluidic electronic chip according to claim 2, characterized in that: It also includes an information acquisition and processing system, and the electrode array is connected to the information acquisition and processing system; Preferably, the information acquisition and processing system comprises a processing circuit unit, a controller and a control circuit unit, and the electrode array, the processing circuit unit, the controller, the control circuit unit and the control input component are connected in sequence; Preferably, the information collection and processing system further comprises a communication module, and the communication module is connected to the controller; Preferably, the processing circuit unit comprises a pre-processing circuit sub-unit and an analog-to-digital conversion sub-unit, and the electrode array, the pre-processing circuit sub-unit, the analog-to-digital conversion sub-unit and the controller are connected in sequence.

4. The microfluidic electronic chip according to claim 1, characterized in that: The substance to be cultured includes at least one of the following: cells, tissues, DNA, RNA, proteins, and metabolic molecules; Preferably, the cells include at least any one of the following types: vascular cells, brain cells, liver cells, lung cells, kidney cells, heart cells, spleen cells and intestinal cells.

5. A method for preparing an organoid model, characterized in that: The method is implemented based on the microfluidic electronic chip according to any one of claims 1 to 4, and comprises: injecting stem cells into the chambers of the microfluidic electronic chip in sequence and culturing them to generate organoid spheroids; Performing enzymatic hydrolysis on the organoid spheroids to form self-assembled cells in each chamber of the microfluidic electronic chip, and adding the self-assembled cells to the lower chamber; Cultivating the self-assembled cells so that the self-assembled cells generate an initial organoid model; culturing the initial organoid model to form organoids; The organoid is intermittently electrically stimulated based on the electrode array, and the electrophysiological information of the organoid is collected to construct a functional organoid model.

6. The method for preparing an organoid model according to claim 5, characterized in that: The organoids include at least any one of the following types: vascular organoids, brain organoids, heart organoids, liver organoids, lung organoids, kidney organoids, spleen organoids and intestinal organoids; Preferably, the vascular organoids include endothelial cells, fibroblasts and smooth muscle cells; Preferably, the brain organoids include brain stem cells, neuronal cells and glial cells; Preferably, the cardiac organoid comprises cardiomyocytes, endocardial cells, epicardial cells and cardiovascular cells.

7. A DNA protein binding analysis method, characterized in that: The method is implemented based on the microfluidic electronic chip according to any one of claims 1 to 4, and comprises: preparing a DNA template of a protein transcription factor, wherein the DNA template carries an antibody tag; Building a library based on the DNA template, adding a fluorescent marker, and obtaining a sample DNA dilution solution; Spotting the sample DNA dilution into the chamber of the microfluidic electronic chip; Surface patterning of the chamber based on biotinylated bovine serum albumin, phosphate buffered saline, and biotinylated antibodies; Loading an in vitro transcription / translation protein transcription factor mixture into the chamber, so that the in vitro transcription / translation protein transcription factor binds to the DNA template in the sample DNA dilution solution to obtain a binding molecule, and the binding molecule is captured and deposited by the biotinylated antibody covered in the chamber; washing the chamber with phosphate buffer to remove unbound molecules and capture the bound molecules; The microfluidic chip is scanned using a fluorescence microscope to quantify the binding molecules.

8. A method for analyzing RNA protein binding, characterized in that: The method is implemented based on the microfluidic electronic chip according to any one of claims 1 to 4, and comprises: Prepare protein and store it in a preset low temperature environment; Prepare RNA library transcription template dilutions based on DNA oligonucleotides; Spotting the RNA library transcription template dilution into the chamber of the microfluidic electronic chip; The chamber is surface patterned based on biotinylated bovine serum albumin, phosphate buffer, and capture probes containing fluorescent markers; Loading an in vitro transcription mixture into the chamber, so that the RNA library transcription template in the RNA library transcription template dilution solution is transcribed to obtain transcribed RNA, and the transcribed RNA is hybridized with the capture probe; Thawing the protein, mixing the thawed protein with an antibody to obtain a protein mixture; and loading the protein mixture into the chamber so that the transcribed RNA binds to the protein to obtain a binding molecule; washing the chamber with phosphate buffer to remove unbound molecules and capture the bound molecules; The microfluidic chip is scanned using a fluorescence microscope to quantify the binding molecules.

9. A protein binding analysis method, characterized in that: The method is implemented based on the microfluidic electronic chip according to any one of claims 1 to 4, and comprises: A DNA template for preparing a protein, wherein the protein includes a target protein and a binding protein; the target protein contains a first amino acid chain or molecule at its N-terminus and a second amino acid chain or molecule at its C-terminus; and the binding protein contains a third amino acid chain or molecule at its C-terminus; Spotting the DNA template into the chamber of the microfluidic electronic chip; patterning the surface of the chamber based on biotinylated bovine serum albumin, phosphate buffer, and anti-amino acid chain or molecule antibodies; Loading an in vitro transcription / translation protein transcription factor mixture into the chamber, so that the in vitro transcription / translation protein transcription factor binds to the DNA template to obtain a binding molecule, and the binding molecule is captured and deposited by the anti-amino acid chain or molecule antibody covered in the chamber; Loading an antibody solution into the chamber, so that the protein binds in the antibody solution to obtain a binding molecule; washing the chamber with phosphate buffer to remove unbound molecules and capture the bound molecules; The microfluidic chip is scanned using a fluorescence microscope to quantify the binding molecules.

10. A liposome transfection method, characterized in that: The method is implemented based on the microfluidic electronic chip according to any one of claims 1 to 4, and comprises: coating the fixed adhesion on a glass slide in the chamber of the microfluidic electronic chip; preparing a liposome sample, and transferring the liposome sample to the glass slide coated with the fixed adhesive; Plasma treatment of the microfluidic electronic chip loaded with the liposome sample; preparing a cell culture fluid, and injecting the cell culture fluid into the microfluidic electronic chip to culture and transfect cells in the cell culture fluid; Injecting culture medium into the microfluidic electronic chip to promote cell surface attachment; The microfluidic chip was scanned using a fluorescence microscope to measure the transfection efficiency.

11. An electroporation transfection method, characterized in that: The method is implemented based on the microfluidic electronic chip according to any one of claims 1 to 4, and comprises: Printing a plasmid containing a fluorescent protein onto the microfluidic electronic chip; preparing a cell culture fluid, and injecting the cell culture fluid into the microfluidic electronic chip to culture and transfect cells in the cell culture fluid; Stimulating the cells in the cell culture solution based on the electrode array of the microfluidic electronic chip, so that holes are temporarily formed in the cell membranes of the cells, and the plasmid enters the cells through the holes; Injecting culture medium into the microfluidic electronic chip to promote cell surface attachment; The microfluidic chip was scanned using a fluorescence microscope to measure the transfection efficiency.

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