WO2022257514A1 - Construction method of biosensing system for measuring physiological and pathological parameters of organ chip - Google Patents

Construction method of biosensing system for measuring physiological and pathological parameters of organ chip Download PDF

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WO2022257514A1
WO2022257514A1 PCT/CN2022/079828 CN2022079828W WO2022257514A1 WO 2022257514 A1 WO2022257514 A1 WO 2022257514A1 CN 2022079828 W CN2022079828 W CN 2022079828W WO 2022257514 A1 WO2022257514 A1 WO 2022257514A1
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chip
electrode
organ
sensor
cells
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PCT/CN2022/079828
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French (fr)
Chinese (zh)
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裴昊
余紫荆
李丽
万莹
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华东师范大学
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G01N27/28—Electrolytic cell components
    • G01N27/30—Electrodes, e.g. test electrodes; Half-cells
    • G01N27/327—Biochemical electrodes, e.g. electrical or mechanical details for in vitro measurements
    • G01N27/3275—Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction
    • G01N27/3278—Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction involving nanosized elements, e.g. nanogaps or nanoparticles

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  • the invention belongs to the technical field of 3D in vitro culture and electrochemical analysis of cells, and relates to a construction method of a biosensing system for detecting physiological and pathological parameters of an organ chip, which integrates an organ chip and a plurality of electrochemical biosensors.
  • Organ chip is a 3D in vitro tissue model that more realistically reflects the human body. During the construction of organ chip, corresponding human cell lines are cultivated in the chip model to simulate the structure and functional units of real human tissues or organs. As an emerging 3D in vitro disease model, organ chips make up for the shortcomings of existing 2D models and animal models, and have great application potential in biological development, drug development and precision medicine.
  • the purpose of the present invention is to provide a method for constructing a biosensing system for detecting the physiological and pathological parameters of the organ chip, which combines the organ chip technology with the electrochemical analysis technology, and connects the design at the outlet of the chip.
  • the organ-on-a-chip analysis platform is easy to construct, has a wide range of applications, and various analysis parameters; combining the organ-on-a-chip with electrochemical sensors, it can monitor the growth microenvironment of the three-dimensional in vitro blood vessel model in real time, and realize the real-time dynamic monitoring of the physiological status of the organ on the chip, which is convenient More accurate and rapid artificial adjustments to increase the success rate of preparing organ chips, more long-term dynamic monitoring of the growth of organ chips, and more real-time and convenient response to drug effects.
  • this integrated chip analysis platform facilitates researchers to more precisely control the microenvironment of chip growth, improve the success rate of organ chip in vitro culture, and more quickly monitor the microenvironment during the growth and development of 3D in vitro tissue models. Changes in clinical testing indicators for early diagnosis of diseases and detection of changes in physiological environment parameters.
  • FIG. 1 The design model of an electrochemical sensing system for monitoring the physiological indicators of the organ chip constructed by the present invention is shown in Figure 1, wherein the master plates of each layer of the blood vessel chip model are shown in Figure 2, and the single-layer detail diagram is shown in Figure 3 .
  • inject fibroblasts and human umbilical vein endothelial cells into the pre-prepared chip model combine the electrode sensor with the PDMS electrode chip with a liquid storage hole to obtain the electrode sensor chip; connect the organ chip and the electrode sensor chip in series , monitor five physiological environment parameters including pH, ATP, ROS, cholesterol and oxygen content in the metabolic solution of the chip.
  • the present invention provides a biosensing system for detecting physiological and pathological parameters of organ chips, said system comprising: organ chips, pH sensor chips, ROS sensor chips, ATP sensor chips, oxygen content sensor chips , Cholesterol sensor chip.
  • the organ-on-a-chip includes a heart organ-on-a-chip, a kidney organ-on-a-chip, a lung organ-on-a-chip, a brain organ-on-a-chip, a blood vessel organ-on-a-chip, and an intestinal organ-on-a-chip.
  • the invention provides a method for constructing a biosensing system for detecting physiological and pathological parameters of an organ chip, said method comprising the following steps:
  • Step (1) mix the polydimethylsiloxane main agent and the curing agent in a mass ratio of 10:1, and then pour it on the pre-designed chip motherboard for curing, and then combine the cured PDMS template with the master
  • the board is separated;
  • the main agent is Sylgard 184 polymer, and the curing agent is Sylgard 184 curing agent;
  • Step (2) bonding the PDMS template prepared in step (1) to obtain an organ chip
  • Step (3) injecting the cell mixture into the organ chip obtained in the above step (2), and culturing in a constant temperature incubator;
  • Step (4) modifying the electrochemical sensors that detect different parameters on the glass sheet
  • Step (5) mix the polydimethylsiloxane main agent and the curing agent in a mass ratio of 10:1, and then pour it on the pre-designed electrode chip motherboard for curing, and combine the PDMS template obtained after curing with the obtained
  • the electrode chip motherboard is separated;
  • the main agent is Sylgard 184 polymer, and the curing agent is Sylgard 184 curing agent;
  • Step (6) bonding the PDMS template prepared in step (5) on the glass sheet of the modified electrode sensor in the step (3) to obtain an electrode sensor chip;
  • Step (7) connecting the inlets of each electrode sensor chip in step (6) in series with the outlet of the organ chip in step (3) to form a liquid circulation pipeline, and the liquid in the blood vessel chip flows through the electrode chip through a peristaltic pump, Thus, a biosensing system for detecting physiological and pathological parameters of the organ chip is obtained.
  • step (1) the chip motherboard is divided into a cell culture layer and a culture medium layer;
  • the size of the main channel of the cell culture layer of the mother chip is 500-1000 ⁇ m in width and 100-200 ⁇ m in height; preferably, the size of the main channel is 500 ⁇ m in width and 200 ⁇ m in height.
  • the size of the branch channel of the cell culture layer of the motherboard chip is 200-250 ⁇ m in width and 400-500 ⁇ m in height; preferably, the size of the branch channel is 200 ⁇ m in width and 500 ⁇ m in height.
  • the size of the culture medium layer of the chip mother board is 17 mm in length, 7 mm in width, and 500-700 ⁇ m in height; preferably, it is 17 mm in length, 7 mm in width, and 500 ⁇ m in height.
  • the inlets and outlets of the chip motherboard are circular holes with a diameter of 200-500 ⁇ m; preferably, 500 ⁇ m.
  • the curing temperature is 60-80°C; preferably, 60°C.
  • the curing time ranges from 2 to 4 hours; preferably, it is 2 hours.
  • step (2) the number of bonded PDMS templates is 3.
  • the organ chip comprises medium outlet layer PDMS, PDMS porous membrane, cell culture layer PDMS, PDMS porous membrane, medium inlet layer PDMS; the order is the first layer of medium outlet layer PDMS, the second Two layers of PDMS porous membrane, the third layer of cell culture layer PDMS, the fourth layer of PDMS porous membrane, the fifth layer of medium inlet layer PDMS.
  • the bonding content is the PDMS template of the medium outlet layer, the PDMS porous membrane, the PDMS template of the cell culture layer, the PDMS porous membrane, and the PDMS template of the medium inlet layer.
  • the bonding conditions are that the radio frequency power is 400-600w, the processing time is 20-40s, and the oxygen flow rate is 50-400mL/min; preferably, the radio frequency power is 600w, the time is 40s, and the oxygen flow rate is 200mL/min. min;
  • the cell mixture is a cell mixture of fibroblasts and human umbilical vein endothelial cells, a cell mixture of human umbilical vessel endothelial cells and cardiomyocytes derived from human induced pluripotent stem cells;
  • the density of the human umbilical vein endothelial cells, fibroblasts, human umbilical vessel endothelial cells and human induced pluripotent stem cell-derived cardiomyocytes is 2 ⁇ (10 6 -10 7 ) cells/mL; preferably, 2 ⁇ 10 6 cells/mL.
  • the number ratio of the fibroblasts and the human umbilical vein endothelial cells is 1:1-1:5; preferably, it is 1:1.
  • the number ratio of the human umbilical vascular endothelial cells and the cardiomyocytes derived from human induced pluripotent stem cells is 1:1-1:3; preferably, it is 1:1.
  • step (3) the two kinds of cells are mixed in a matrigel solution with a concentration of 3-10 mg/mL; preferably, 5 mg/mL or 10 mg/mL.
  • the matrigel solution includes but not limited to matrigel, bovine fibrinogen solution, type I collagen, preferably, matrigel or type I collagen.
  • the fibroblasts and human umbilical vein endothelial cells are mixed in an 8-10 mg/mL matrigel solution; the matrigel solution includes matrigel, bovine fibrinogen solution, and type I collagen.
  • the human umbilical vessel endothelial cells and cardiomyocytes are mixed in a 3-10 mg/mL collagen solution;
  • the collagen solution includes matrigel, bovine fibrinogen solution, and type I collagen.
  • step (3) the culture condition of the cell mixture is: 37° C., 5% CO 2 .
  • the electrode sensor includes: pH electrode sensor, ROS electrode sensor, oxygen content electrode sensor, cholesterol electrode sensor, ATP electrode sensor;
  • the pH electrode sensor uses the carbon electrode modified polyaniline as the working electrode, the carbon electrode as the counter electrode, and Ag/AgCl as the reference electrode; by measuring the relationship between the potential difference between the working electrode and the reference electrode and the pH Calculate the pH of the solution.
  • the pH electrode sensor is a commercially prepared electrode purchased from Qingdao Wave Carbon Technology Co., Ltd.
  • the ROS electrode sensor uses a gold electrode modified with horseradish peroxidase as a working electrode, platinum as a counter electrode, and Ag/AgCl as a reference electrode; the ratio between the ROS concentration and the peak current is measured by cyclic voltammetry. Relationship to calculate the concentration of ROS in the solution to be tested.
  • the preparation method of the ROS electrode sensor is as follows: 1) Platinum nanoparticles and gold nanoparticles are respectively deposited on the pre-cleaned glass substrate, and the areas are successively 1mm ⁇ 3mm and 1mm ⁇ 1mm; 2) 0.5 ⁇ L horseradish The catalase polymer solution was added dropwise on the surface of the gold electrode, and placed in the dark overnight at 4°C. 3) Coating Ag/AgCl paste on the glass substrate.
  • the oxygen content electrode sensor uses platinum as the working electrode and the counter electrode, and Ag/AgCl as the reference electrode; the oxygen content in the solution to be measured is calculated by using the relationship between the oxygen content and the ampere current.
  • the preparation method of the oxygen content electrode sensor is as follows: 1) depositing platinum nanoparticles on a pre-cleaned glass substrate, and 2) coating Ag/AgCl paste on the glass substrate.
  • the cholesterol electrode sensor uses a gold electrode modified with horseradish peroxidase and cholesterol oxidase by DNA origami as a working electrode, platinum as a counter electrode, and Ag/AgCl as a reference electrode; Utilize cyclic voltammetry to measure cholesterol The relationship between concentration and peak current to calculate the concentration of cholesterol in the solution to be tested.
  • the preparation method of the cholesterol electrode sensor is as follows: 1) depositing gold nanoparticles on the pre-cleaned glass substrate; 2) dripping the peroxidase solution on the surface of the gold electrode, and drying naturally to obtain HOD/Au; 3) Add cholesterol oxidase dropwise on the surface of 1), and let it dry naturally to obtain a COD/HOD/Au electrode. 4) Deposit platinum nanoparticles and coat Ag/AgCl paste on the glass substrate.
  • the ATP electrode sensor uses a gold electrode modified with an ATP probe as a working electrode, platinum as a counter electrode, and Ag/AgCl as a reference electrode, and uses cyclic voltammetry to measure the relationship between the ATP concentration and the peak current. Calculate the ATP concentration in the solution to be tested.
  • the preparation method of the ATP electrode sensor is as follows: 1) depositing gold nanoparticles on a pre-washed glass substrate; 2) adding a DNA aptamer with a sulfhydryl group at the 3' end dropwise on the surface of the gold electrode, and incubating for 16 hours ; 3) Soak in 1 mol/L NaClO 4 containing 0.1 mol/L 2-mercaptoethanol for 10 min; 4) Rinse with 10 mol/L HEPES containing 50 mol/L NaClO 4 . 5) Deposit platinum nanoparticles and coat Ag/AgCl paste on the glass substrate.
  • the curing temperature is 60-80°C; preferably, 60°C.
  • the curing time ranges from 2 to 4 hours; preferably, it is 2 hours.
  • the flow channel of the electrode chip motherboard has a width of 500-1000 ⁇ m and a height of 200-500 ⁇ m; preferably, a width of 500 ⁇ m and a height of 500 ⁇ m.
  • the diameter of the hole on the electrode chip motherboard is 800-1000 ⁇ m, and the height is 500-1000 ⁇ m; preferably, the diameter of the hole is 800 ⁇ m, and the height is 500 ⁇ m.
  • the plasma bonding conditions are: radio frequency power 400-600w, time 20-40s, oxygen flow rate 50-200mL/min.
  • the radio frequency power is 600w
  • the time is 40s
  • the oxygen flow rate is 200mL/min.
  • step (7) the rotating speed of the peristaltic pump is 3rpm/min, and the transmission material of the peristaltic pump is: a 1:1 mixture of fibroblast culture medium and human umbilical vein endothelial cell culture medium or human umbilical vessel endothelial cell culture medium 1:1 mixture with cardiomyocyte culture medium;
  • the fibroblast culture medium is FGM-2;
  • the human umbilical vein endothelial cell culture medium is EGM-2;
  • the human umbilical vessel endothelial cell culture medium is EGM;
  • the cardiomyocyte culture medium is CCM.
  • the present invention also provides a biosensor system for monitoring organ-on-a-chip physiological and pathological indicators constructed by the above-mentioned method, and the system includes: organ-on-a-chip, pH, ATP, ROS, oxygen content and cholesterol sensor chips.
  • the method for constructing the biosensing system provided by the present invention can obtain a 3D artificial blood vessel model in vitro, and monitor the growth status of the organ chip dynamically in real time through an electrochemical sensor.
  • the biosensing system provided by the invention can realize automatic analysis, simplify manual operation, and reduce experimental error and experimental cost.
  • the monitoring objects of the biosensing system provided by the present invention include, but are not limited to, blood vessel organ-on-a-chip, heart organ-on-a-chip, kidney organ-on-a-chip, lung organ-on-a-chip, brain organ-on-a-chip, and intestinal organ-on-a-chip.
  • the present invention also provides the application of the above-mentioned biosensing system in monitoring the physiological state of the organ chip, disease process, simulating disease, drug efficacy testing process and predicting human drug response.
  • the present invention also provides a method of using the above-mentioned biosensor system, the method comprising the following steps:
  • Step 1 Inject the mixed cell liquid into the culture layer in the chip model for culturing; the chip outlet is connected to five electrode chip inlets; and the dynamic monitoring of the system is realized through a peristaltic pump.
  • Step 2 monitoring the electrochemical signal of each electrode chip
  • Step 3 According to the electrochemical signal, judge whether the microenvironment of cell growth in the chip is normal, such as: pH, oxygen content, cholesterol, etc.; judge whether the cells are subjected to external stimuli and cause stress reactions such as: ROS, ATP, etc.
  • the cells are fibroblasts, human umbilical vein endothelial cells, human umbilical vessel endothelial cells and cardiomyocytes derived from human induced pluripotent stem cells.
  • the density of human umbilical vein endothelial cells, fibroblasts, human umbilical vessel endothelial cells and cardiomyocytes is 2 ⁇ (10 6 -10 7 ) cells/mL; preferably, 2 ⁇ 10 6 cells/mL.
  • the number ratio of the fibroblasts and the human umbilical vein endothelial cells is 1:1-1:5; preferably, it is 1:1.
  • the number ratio of the human umbilical vascular endothelial cells and the cardiomyocytes derived from human induced pluripotent stem cells is 1:1-1:3; preferably, it is 1:1.
  • Described human umbilical vein endothelial cells and fibroblasts are mixed in matrigel solution, and the concentration of described matrigel solution is 8-10mg/mL; Preferably, is 10mg/mL; Described matrigel solution comprises matrigel, bovine Fibrinogen solution, type I collagen, etc.
  • the human umbilical vessel endothelial cells and cardiomyocytes are mixed in a 3-10 mg/mL collagen solution; the collagen solution includes Matrigel, bovine fibrinogen solution, type I collagen, etc.; preferably, 5 mg/mL of I type collagen.
  • step 1 the culture condition of the organ chip is: 37° C., 5% CO 2 .
  • the cell culture medium in the culture layer is an equal mixture of fibroblast culture medium FGM-2 and human umbilical vein endothelial cell culture medium EGM-2 or cardiomyocyte culture medium CCM and human umbilical vein endothelial cell culture medium An equal mixture of EGM.
  • step 1 the speed of the peristaltic pump is 3rpm/min.
  • the electrochemical signals monitored are: the pH sensor measures the potential difference, the oxygen content sensor measures the peak current in cyclic voltammetry, the ROS sensor measures the peak current in cyclic voltammetry, and the ATP sensor measures the peak value in cyclic voltammetry Current, the cholesterol sensor measures the peak current in cyclic voltammetry.
  • the beneficial effects of the present invention include: in the present invention, human umbilical vein endothelial cells and fibroblasts and/or human umbilical vessel endothelial cells and cardiomyocytes are mixed and cultured in a chip model to obtain an organ chip, and the organ chip is combined with a plurality of electrochemical sensors
  • a chip analysis platform is integrated to realize real-time dynamic monitoring of the growth status of the 3D in vitro model and the physiological microenvironment, reduce research costs and speed up research, and can also provide new monitoring parameters and detection methods for clinical sample analysis.
  • Fig. 1 is the schematic diagram of the blood vessel chip model proposed by the present invention
  • Fig. 2 is a schematic diagram of each layer of the motherboard of the blood vessel chip proposed by the present invention.
  • Fig. 3 is a detailed view of a single layer of the blood vessel chip proposed by the present invention.
  • Fig. 4 is a schematic diagram of a pH sensing electrode in the present invention.
  • Fig. 5 is a schematic diagram of the ATP sensing electrode in the present invention.
  • Fig. 6 is a schematic diagram of a ROS sensing electrode in the present invention.
  • Fig. 7 is a schematic diagram of a cholesterol sensing electrode in the present invention.
  • Fig. 8 is a schematic diagram of an oxygen content sensing electrode in the present invention.
  • Fig. 9 is a schematic diagram of a biosensing system.
  • Fig. 10 is a physical diagram of the blood vessel chip.
  • Figure 11 is a physical diagram of the biosensing system.
  • Fig. 12 is a physical diagram of the ATP sensor chip.
  • Fig. 13 is a physical diagram of the oxygen content sensing chip.
  • Figure 14 is a physical diagram of the ROS sensor chip.
  • Fig. 15 is a physical diagram of a cholesterol sensing chip.
  • Fig. 16 is a physical diagram of the pH sensor chip.
  • step (3) The three PDMS templates prepared in step (1) were bonded layer by layer through a plasma cleaner to obtain a blood vessel chip model.
  • the bonding conditions were: RF power 600w, time 40s, oxygen flow rate 200mL/min.
  • the electrode for electrochemical sensing of pH is a commercially prepared electrode, purchased from Qingdao Wave Carbon Technology Co., Ltd.
  • the formed pH sensor chip is shown in FIG. 16 .
  • (11) Mix the polydimethylsiloxane main agent and the curing agent evenly at a mass ratio of 10:1, and after removing air bubbles in a vacuum, pour it on the pre-designed electrode chip motherboard, and cure the obtained product after two hours of curing.
  • the PDMS template is separated from the electrode chip motherboard; the main agent is Sylgard 184 polymer, and the curing agent is Sylgard 184 curing agent.
  • the electrode sensor includes: pH electrode sensor, ROS electrode sensor, oxygen content electrode sensor, cholesterol electrode sensor, ATP electrode sensor;
  • Step (14) Plasma bond the PDMS template prepared in step (13) with the electrode sensor glass sheet to obtain an electrode sensor chip; the bonding conditions are: RF power 600w, time 40s, oxygen flow rate 200mL/min.
  • the peristaltic pump continuously feeds the 1:1 mixture of endothelial cell culture medium and fibroblast culture medium to the vascular organ chip at a speed of 3 rpm/min.
  • step (2) Perforate the inlet and outlet of the PDMS template obtained in step (1), wherein the media outlet and inlet layers each have six 500 ⁇ m round holes, and the cell culture layer has three 500 ⁇ m round holes.
  • step (3) The three PDMS templates prepared in step (1) were bonded layer by layer through a plasma cleaner to obtain a blood vessel chip model.
  • the bonding conditions were: RF power 600w, time 40s, oxygen flow rate 200mL/min.
  • step (4) The heart-vascular chip containing cells obtained in step (4) was cultured at 37° C. under 5% CO 2 .
  • the electrode for electrochemical sensing of pH is a commercially prepared electrode, purchased from Qingdao Wave Carbon Technology Co., Ltd.
  • the formed pH sensor chip is shown in FIG. 16 .
  • (11) Mix the polydimethylsiloxane main agent and the curing agent evenly at a mass ratio of 10:1, and after removing air bubbles in a vacuum, pour it on the pre-designed electrode chip motherboard, and cure the obtained product after two hours of curing.
  • the PDMS template is separated from the electrode chip motherboard; the main agent is Sylgard 184 polymer, and the curing agent is Sylgard 184 curing agent.
  • the electrode sensor includes: pH electrode sensor, ROS electrode sensor, oxygen content electrode sensor, cholesterol electrode sensor, ATP electrode sensor;
  • Step (14) Plasma bond the PDMS template prepared in step (13) with the electrode sensor glass sheet to obtain an electrode sensor chip; the bonding conditions are: RF power 600w, time 40s, oxygen flow rate 200mL/min.
  • the peristaltic pump continuously feeds the 1:1 mixture of endothelial cell culture medium EGM and cardiomyocyte culture medium CCM to the heart-vascular organ chip at a speed of 3 rpm/min.

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Abstract

A construction method of a biosensing system for measuring physiological and pathological parameters of an organ chip, comprising the following steps: injecting a cell mixed solution into a pre-designed chip model, and continuously culturing to form the organ chip; and then connecting an electrochemical sensing chip in which a micro-flow control pipeline is designed and an outlet of the organ chip for specifically monitoring the oxygen content, ATP, pH, ROS, and cholesterol in metabolites of the organ chip. The construction method is simple and convenient, wide in application range, and diversified in analysis parameters; the organ chip is combined with an electrochemical sensor, such that the growth micro-environment of a three-dimensional in-vitro blood vessel/heart model can be dynamically monitored in real time; the model culture condition can be adjusted in real time, such that the model culture condition better conforms to a real living body condition; and the biosensing system can be used for exploring the physiological state change in a disease process and can also be used for online monitoring of the response of the organ chip to drugs.

Description

一种检测器官芯片生理病理参数的生物传感系统的构建方法Construction method of a biosensing system for detecting physiological and pathological parameters of organ chips 技术领域technical field
本发明属于细胞3D体外培养及电化学分析技术领域，涉及一种检测器官芯片生理病理参数的生物传感系统的构建方法，将器官芯片和多个电化学生物传感器进行集成。The invention belongs to the technical field of 3D in vitro culture and electrochemical analysis of cells, and relates to a construction method of a biosensing system for detecting physiological and pathological parameters of an organ chip, which integrates an organ chip and a plurality of electrochemical biosensors.
背景技术Background technique
器官芯片作为一个更真实反映人体的3D体外组织模型，在器官芯片的构建过程中，通过在芯片模型中培养相应的人源细胞系以模拟人体真实组织或者器官的结构和功能单元。器官芯片作为一种新兴的3D体外疾病模型，弥补了现有2D模型和动物模型的缺陷，在生物发育学、药物研发和精准医疗方面具有巨大的应用潜力。Organ chip is a 3D in vitro tissue model that more realistically reflects the human body. During the construction of organ chip, corresponding human cell lines are cultivated in the chip model to simulate the structure and functional units of real human tissues or organs. As an emerging 3D in vitro disease model, organ chips make up for the shortcomings of existing 2D models and animal models, and have great application potential in biological development, drug development and precision medicine.
常规的血管芯片在用于发育生物学和疾病病理学研究时，通常采用免疫荧光染色法，借助荧光染料和光学成像手段进行芯片的观察，该方法操作复杂，且受限于操作者的操作技术，检测耗时长，成本高昂，只能对特定参数的分析，无法实现实时动态监测。When conventional blood vessel chips are used in the research of developmental biology and disease pathology, immunofluorescence staining is usually used to observe the chip with the help of fluorescent dyes and optical imaging methods. This method is complicated to operate and is limited by the operator's operating skills , the detection takes a long time and the cost is high. It can only analyze specific parameters and cannot realize real-time dynamic monitoring.
发明内容Contents of the invention
为了解决现有技术存在的不足，本发明的目的是提供一种检测器官芯片生理病理参数的生物传感系统的构建方法，将器官芯片技术与电化学分析技术相结合，在芯片的出口连接设计有微流控管路的电化学传感器，用于特异性监测器官芯片代谢物中的氧含量、ATP、pH、ROS、胆固醇。该器官芯片分析平台构建简便，适用范围广泛，分析参数多样；将器官芯片与电化学传感器相结合，可以实时动态监测三维体外血管模型的生长微环境，实现实时动态监测器官芯片的生理状况，便于更加准确快速的人为调整以增大制备器官芯片的成功率、更加长期动态的监测器官芯片的生长状况、更加实时简便的响应药物效果。In order to solve the deficiencies in the prior art, the purpose of the present invention is to provide a method for constructing a biosensing system for detecting the physiological and pathological parameters of the organ chip, which combines the organ chip technology with the electrochemical analysis technology, and connects the design at the outlet of the chip. Electrochemical sensors with microfluidic circuits for specific monitoring of oxygen content, ATP, pH, ROS, cholesterol in organ-on-a-chip metabolites. The organ-on-a-chip analysis platform is easy to construct, has a wide range of applications, and various analysis parameters; combining the organ-on-a-chip with electrochemical sensors, it can monitor the growth microenvironment of the three-dimensional in vitro blood vessel model in real time, and realize the real-time dynamic monitoring of the physiological status of the organ on the chip, which is convenient More accurate and rapid artificial adjustments to increase the success rate of preparing organ chips, more long-term dynamic monitoring of the growth of organ chips, and more real-time and convenient response to drug effects.
其潜在应用价值在于这种集成式的芯片分析平台便于研究者对芯片生长的微环境实施更加精确地控制，提高器官芯片体外培养的成功率，更加快速监测3D体外组织模型生长发育过程中微环境的变化，为疾病早期诊断及生理环境参数的变化检测增加临床检验指标。Its potential application value lies in that this integrated chip analysis platform facilitates researchers to more precisely control the microenvironment of chip growth, improve the success rate of organ chip in vitro culture, and more quickly monitor the microenvironment during the growth and development of 3D in vitro tissue models. Changes in clinical testing indicators for early diagnosis of diseases and detection of changes in physiological environment parameters.
本发明构建的一种监测器官芯片生理指标的电化学传感系统的设计模型如图1所示，其中血管芯片模型的各层母版如图2所示，单层细节图如图3所示。具体为：将成纤维细胞和人脐静脉内皮细胞混合注入预先制备芯片模型中；将电极传感器与具有储液孔的PDMS电极芯片相结合，得到电极传感芯片；将器官芯片与电极传感芯片串联，监测芯片代谢溶液中的pH、ATP、ROS、胆固醇、氧含量五项生理环境参数。The design model of an electrochemical sensing system for monitoring the physiological indicators of the organ chip constructed by the present invention is shown in Figure 1, wherein the master plates of each layer of the blood vessel chip model are shown in Figure 2, and the single-layer detail diagram is shown in Figure 3 . Specifically: inject fibroblasts and human umbilical vein endothelial cells into the pre-prepared chip model; combine the electrode sensor with the PDMS electrode chip with a liquid storage hole to obtain the electrode sensor chip; connect the organ chip and the electrode sensor chip in series , monitor five physiological environment parameters including pH, ATP, ROS, cholesterol and oxygen content in the metabolic solution of the chip.
根据上述原理，本发明提供了一种检测器官芯片生理病理参数的生物传感系统，所述系统包括：器官芯片，pH传感芯片、ROS传感芯片、ATP传感芯片、氧含量传感芯片、胆 固醇传感芯片。According to the above principles, the present invention provides a biosensing system for detecting physiological and pathological parameters of organ chips, said system comprising: organ chips, pH sensor chips, ROS sensor chips, ATP sensor chips, oxygen content sensor chips , Cholesterol sensor chip.
所述器官芯片包括心脏器官芯片、肾器官芯片、肺器官芯片、脑器官芯片、血管器官芯片、肠器官芯片。The organ-on-a-chip includes a heart organ-on-a-chip, a kidney organ-on-a-chip, a lung organ-on-a-chip, a brain organ-on-a-chip, a blood vessel organ-on-a-chip, and an intestinal organ-on-a-chip.
本发明提供了一种检测器官芯片生理病理参数的生物传感系统的构建方法，所述方法包括如下步骤：The invention provides a method for constructing a biosensing system for detecting physiological and pathological parameters of an organ chip, said method comprising the following steps:
步骤(1)、将聚二甲基硅氧烷主剂和固化剂按质量比10:1的比例混合，然后浇筑在预先设计的芯片母板上固化，然后将固化后获得的PDMS模板与母板分开；所述主剂为Sylgard 184聚合体，所述固化剂为Sylgard 184固化剂；Step (1), mix the polydimethylsiloxane main agent and the curing agent in a mass ratio of 10:1, and then pour it on the pre-designed chip motherboard for curing, and then combine the cured PDMS template with the master The board is separated; the main agent is Sylgard 184 polymer, and the curing agent is Sylgard 184 curing agent;
步骤(2)、将步骤(1)中制备的PDMS模板进行键合，获得器官芯片；Step (2), bonding the PDMS template prepared in step (1) to obtain an organ chip;
步骤(3)、向上述步骤(2)获得的器官芯片中注入细胞混合液，于恒温培养箱中培养；Step (3), injecting the cell mixture into the organ chip obtained in the above step (2), and culturing in a constant temperature incubator;
步骤(4)、在玻璃片上修饰上检测不同参数的电化学传感器；Step (4), modifying the electrochemical sensors that detect different parameters on the glass sheet;
步骤(5)、将聚二甲基硅氧烷主剂和固化剂按质量比10:1的比例混合，然后浇筑在预先设计的电极芯片母板上固化，将固化后获得的PDMS模板与所述电极芯片母板分开；所述主剂为Sylgard 184聚合体，所述固化剂为Sylgard 184固化剂；Step (5), mix the polydimethylsiloxane main agent and the curing agent in a mass ratio of 10:1, and then pour it on the pre-designed electrode chip motherboard for curing, and combine the PDMS template obtained after curing with the obtained The electrode chip motherboard is separated; the main agent is Sylgard 184 polymer, and the curing agent is Sylgard 184 curing agent;
步骤(6)、将步骤(5)中制备好的PDMS模板键合在所述步骤(3)修饰电极传感器的玻璃片上，获得电极传感芯片；Step (6), bonding the PDMS template prepared in step (5) on the glass sheet of the modified electrode sensor in the step (3) to obtain an electrode sensor chip;
步骤(7)、将步骤(6)中的各个电极传感芯片入口与步骤(3)中的器官芯片出口进行串联，形成液体流通管道，通过蠕动泵使得血管芯片中的液体流经电极芯片，从而获得检测器官芯片生理病理参数的生物传感系统。Step (7), connecting the inlets of each electrode sensor chip in step (6) in series with the outlet of the organ chip in step (3) to form a liquid circulation pipeline, and the liquid in the blood vessel chip flows through the electrode chip through a peristaltic pump, Thus, a biosensing system for detecting physiological and pathological parameters of the organ chip is obtained.
步骤(1)中，所述芯片母板分为细胞培养层和培养基层；In step (1), the chip motherboard is divided into a cell culture layer and a culture medium layer;
所述芯片母板的细胞培养层的主流道尺寸为宽500-1000μm，高100-200μm；优选地，主流道尺寸宽500μm，高200μm。The size of the main channel of the cell culture layer of the mother chip is 500-1000 μm in width and 100-200 μm in height; preferably, the size of the main channel is 500 μm in width and 200 μm in height.
所述芯片母板的细胞培养层的支流道尺寸为宽200-250μm，高400-500μm；优选地，支流道的尺寸为宽200μm，高500μm。The size of the branch channel of the cell culture layer of the motherboard chip is 200-250 μm in width and 400-500 μm in height; preferably, the size of the branch channel is 200 μm in width and 500 μm in height.
所述芯片母板的培养基层尺寸为长17mm，宽7mm，高500-700μm；优选地，长17mm，宽7mm，高500μm。The size of the culture medium layer of the chip mother board is 17 mm in length, 7 mm in width, and 500-700 μm in height; preferably, it is 17 mm in length, 7 mm in width, and 500 μm in height.
所述芯片母板的出入口均为直径为200-500μm的圆孔；优选地，为500μm。The inlets and outlets of the chip motherboard are circular holes with a diameter of 200-500 μm; preferably, 500 μm.
步骤(1)中，所述固化的温度为60-80℃；优选地，为60℃。In step (1), the curing temperature is 60-80°C; preferably, 60°C.
步骤(1)中，所述固化的时间范围为2-4h；优选地，为2h。In step (1), the curing time ranges from 2 to 4 hours; preferably, it is 2 hours.
步骤(2)中，所述PDMS模板键合的个数为3个。In step (2), the number of bonded PDMS templates is 3.
步骤(2)中，所述器官芯片包括培养基出口层PDMS，PDMS多孔膜，细胞培养层PDMS， PDMS多孔膜，培养基入口层PDMS；所述顺序为第一层培养基出口层PDMS，第二层PDMS多孔膜，第三层细胞培养层PDMS，第四层PDMS多孔膜，第五层培养基入口层PDMS。In step (2), the organ chip comprises medium outlet layer PDMS, PDMS porous membrane, cell culture layer PDMS, PDMS porous membrane, medium inlet layer PDMS; the order is the first layer of medium outlet layer PDMS, the second Two layers of PDMS porous membrane, the third layer of cell culture layer PDMS, the fourth layer of PDMS porous membrane, the fifth layer of medium inlet layer PDMS.
步骤(2)中，所述键合的内容为培养基出口层PDMS模板，PDMS多孔膜，细胞培养层PDMS模板，PDMS多孔膜，培养基入口层PDMS模板。In step (2), the bonding content is the PDMS template of the medium outlet layer, the PDMS porous membrane, the PDMS template of the cell culture layer, the PDMS porous membrane, and the PDMS template of the medium inlet layer.
步骤(2)中，所述键合的条件为射频功率为400-600w，处理时间为20-40s，氧气流量为50-400mL/min；优选地，射频功率600w，时间40s，氧气流量200mL/min；In step (2), the bonding conditions are that the radio frequency power is 400-600w, the processing time is 20-40s, and the oxygen flow rate is 50-400mL/min; preferably, the radio frequency power is 600w, the time is 40s, and the oxygen flow rate is 200mL/min. min;
步骤(3)中，所述细胞混合液为成纤维细胞和人脐静脉内皮细胞的细胞混合液、人脐血管内皮细胞和人诱导多功能干细胞来源的心肌细胞的细胞混合液；In step (3), the cell mixture is a cell mixture of fibroblasts and human umbilical vein endothelial cells, a cell mixture of human umbilical vessel endothelial cells and cardiomyocytes derived from human induced pluripotent stem cells;
其中，所述人脐静脉内皮细胞、成纤维细胞、人脐血管内皮细胞和人诱导多功能干细胞来源的心肌细胞的密度均为2×(10 6-10 7)个/mL；优选地，为2×10 6个/mL。 Wherein, the density of the human umbilical vein endothelial cells, fibroblasts, human umbilical vessel endothelial cells and human induced pluripotent stem cell-derived cardiomyocytes is 2×(10 6 -10 7 ) cells/mL; preferably, 2×10 6 cells/mL.
其中，所述成纤维细胞和人脐静脉内皮细胞两种细胞的数量比为1:1～1:5；优选地，为1:1。Wherein, the number ratio of the fibroblasts and the human umbilical vein endothelial cells is 1:1-1:5; preferably, it is 1:1.
其中，所述人脐血管内皮细胞和人诱导多功能干细胞来源的心肌细胞两种细胞的数量比为1:1～1:3；优选地，为1:1。Wherein, the number ratio of the human umbilical vascular endothelial cells and the cardiomyocytes derived from human induced pluripotent stem cells is 1:1-1:3; preferably, it is 1:1.
步骤(3)中，所述两种细胞混合在浓度为3-10mg/mL的基质胶溶液中；优选地，为5mg/mL或10mg/mL。In step (3), the two kinds of cells are mixed in a matrigel solution with a concentration of 3-10 mg/mL; preferably, 5 mg/mL or 10 mg/mL.
步骤(3)中，所述基质胶溶液包括但不限于基质胶，牛纤维蛋白原溶液，Ⅰ型胶原，优选地为，基质胶或I型胶原。In step (3), the matrigel solution includes but not limited to matrigel, bovine fibrinogen solution, type I collagen, preferably, matrigel or type I collagen.
在一个具体实施方式中，所述成纤维细胞和人脐静脉内皮细胞混合在8-10mg/mL的基质胶溶液中；所述基质胶溶液包括基质胶，牛纤维蛋白原溶液，I型胶原。In a specific embodiment, the fibroblasts and human umbilical vein endothelial cells are mixed in an 8-10 mg/mL matrigel solution; the matrigel solution includes matrigel, bovine fibrinogen solution, and type I collagen.
在另一个具体实施方式中，所述人脐血管内皮细胞和心肌细胞混合在3-10mg/mL的胶原溶液中；所述胶原溶液包括基质胶，牛纤维蛋白原溶液，I型胶原。In another specific embodiment, the human umbilical vessel endothelial cells and cardiomyocytes are mixed in a 3-10 mg/mL collagen solution; the collagen solution includes matrigel, bovine fibrinogen solution, and type I collagen.
步骤(3)中，所述细胞混合液的培养条件为：37℃，5％CO 2。 In step (3), the culture condition of the cell mixture is: 37° C., 5% CO 2 .
步骤(4)中，所述电极传感器包括：pH电极传感器、ROS电极传感器、氧含量电极传感器、胆固醇电极传感器、ATP电极传感器；In step (4), the electrode sensor includes: pH electrode sensor, ROS electrode sensor, oxygen content electrode sensor, cholesterol electrode sensor, ATP electrode sensor;
其中，所述pH电极传感器以修饰聚苯胺的碳电极为工作电极，碳电极为对电极，Ag/AgCl作为参比电极；通过测量工作电极和参比电极之间的电势差与pH之间的关系计算溶液的pH值。Wherein, the pH electrode sensor uses the carbon electrode modified polyaniline as the working electrode, the carbon electrode as the counter electrode, and Ag/AgCl as the reference electrode; by measuring the relationship between the potential difference between the working electrode and the reference electrode and the pH Calculate the pH of the solution.
在一个优选的实施方案中，所述pH电极传感器为商业制备电极，购买自青岛波碳科技有限公司。In a preferred embodiment, the pH electrode sensor is a commercially prepared electrode purchased from Qingdao Wave Carbon Technology Co., Ltd.
其中，所述ROS电极传感器以修饰辣根过氧化物酶的金电极为工作电极，铂为对电极，Ag/AgCl作为参比电极；利用循环伏安法测得ROS浓度和峰值电流之间的关系从而计算待测 溶液中ROS的浓度。Wherein, the ROS electrode sensor uses a gold electrode modified with horseradish peroxidase as a working electrode, platinum as a counter electrode, and Ag/AgCl as a reference electrode; the ratio between the ROS concentration and the peak current is measured by cyclic voltammetry. Relationship to calculate the concentration of ROS in the solution to be tested.
所述ROS电极传感器的制备方法为：1)在预先洗净的玻璃基底上分别沉积上铂纳米颗粒和金纳米颗粒，其面积依次为1mm×3mm和1mm×1mm；2)将0.5μL辣根过氧化氢酶聚合物溶液滴加在金电极表面，置于4℃条件下黑暗中过夜。3)在玻璃片基底上涂覆Ag/AgCl糊。The preparation method of the ROS electrode sensor is as follows: 1) Platinum nanoparticles and gold nanoparticles are respectively deposited on the pre-cleaned glass substrate, and the areas are successively 1mm×3mm and 1mm×1mm; 2) 0.5 μL horseradish The catalase polymer solution was added dropwise on the surface of the gold electrode, and placed in the dark overnight at 4°C. 3) Coating Ag/AgCl paste on the glass substrate.
其中，所述氧含量电极传感器以铂为工作电极和对电极，Ag/AgCl作为参比电极；利用氧含量和安培电流之间的关系计算待测溶液中的氧含量。Wherein, the oxygen content electrode sensor uses platinum as the working electrode and the counter electrode, and Ag/AgCl as the reference electrode; the oxygen content in the solution to be measured is calculated by using the relationship between the oxygen content and the ampere current.
所述氧含量电极传感器的制备方法为：1)在预先洗净的玻璃基底上沉积上铂纳米颗粒，2)在玻璃基底上涂覆Ag/AgCl糊。The preparation method of the oxygen content electrode sensor is as follows: 1) depositing platinum nanoparticles on a pre-cleaned glass substrate, and 2) coating Ag/AgCl paste on the glass substrate.
其中，所述胆固醇电极传感器以通过DNAorigami修饰有辣根过氧化物酶和胆固醇氧化酶的金电极作为工作电极，铂为对电极，Ag/AgCl作为参比电极；利用循环伏安法测得胆固醇浓度和峰值电流之间的关系从而计算待测溶液中胆固醇的浓度。Wherein, the cholesterol electrode sensor uses a gold electrode modified with horseradish peroxidase and cholesterol oxidase by DNA origami as a working electrode, platinum as a counter electrode, and Ag/AgCl as a reference electrode; Utilize cyclic voltammetry to measure cholesterol The relationship between concentration and peak current to calculate the concentration of cholesterol in the solution to be tested.
所述胆固醇电极传感器的制备方法为：1)在预先洗净的玻璃基底上沉积上金纳米颗粒；2)将过氧化物酶溶液滴加在金电极的表面，自然风干，得到HOD/Au；3)将胆固醇氧化酶滴加在1)的表面，自然风干，得到COD/HOD/Au电极。4)在玻璃片基底上沉积上铂纳米颗粒和涂覆Ag/AgCl糊。The preparation method of the cholesterol electrode sensor is as follows: 1) depositing gold nanoparticles on the pre-cleaned glass substrate; 2) dripping the peroxidase solution on the surface of the gold electrode, and drying naturally to obtain HOD/Au; 3) Add cholesterol oxidase dropwise on the surface of 1), and let it dry naturally to obtain a COD/HOD/Au electrode. 4) Deposit platinum nanoparticles and coat Ag/AgCl paste on the glass substrate.
其中，所述ATP电极传感器以修饰有ATP探针的金电极作为工作电极，铂为对电极，Ag/AgCl作为参比电极，利用循环伏安法测得ATP浓度和峰值电流之间的关系从而计算待测溶液中ATP浓度。Wherein, the ATP electrode sensor uses a gold electrode modified with an ATP probe as a working electrode, platinum as a counter electrode, and Ag/AgCl as a reference electrode, and uses cyclic voltammetry to measure the relationship between the ATP concentration and the peak current. Calculate the ATP concentration in the solution to be tested.
所述ATP电极传感器的制备方法为：1)在预先洗净的玻璃基底上沉积上金纳米颗粒；2)将3’端修饰巯基的DNA适配体滴加在金电极表面，共孵育16小时；3)在含有0.1mol/L 2-巯基乙醇的1mol/LNaClO 4浸泡10min；4)用含有50mol/LNaClO 4的10mol/LHEPES冲洗。5)在玻璃片基底上沉积上铂纳米颗粒和涂覆Ag/AgCl糊。 The preparation method of the ATP electrode sensor is as follows: 1) depositing gold nanoparticles on a pre-washed glass substrate; 2) adding a DNA aptamer with a sulfhydryl group at the 3' end dropwise on the surface of the gold electrode, and incubating for 16 hours ; 3) Soak in 1 mol/L NaClO 4 containing 0.1 mol/L 2-mercaptoethanol for 10 min; 4) Rinse with 10 mol/L HEPES containing 50 mol/L NaClO 4 . 5) Deposit platinum nanoparticles and coat Ag/AgCl paste on the glass substrate.
步骤(5)中，所述固化的温度为60-80℃；优选地，为60℃。In step (5), the curing temperature is 60-80°C; preferably, 60°C.
步骤(5)中，所述固化的时间范围为2–4h；优选地，为2h。In step (5), the curing time ranges from 2 to 4 hours; preferably, it is 2 hours.
步骤(5)中，所述电极芯片母板的流道宽500-1000μm，高200-500μm；优选地，宽500μm，高500μm。In step (5), the flow channel of the electrode chip motherboard has a width of 500-1000 μm and a height of 200-500 μm; preferably, a width of 500 μm and a height of 500 μm.
步骤(5)中，所述电极芯片母板的圆孔直径800-1000μm，高500-1000μm；优选地，圆孔直径800μm，高500μm。In step (5), the diameter of the hole on the electrode chip motherboard is 800-1000 μm, and the height is 500-1000 μm; preferably, the diameter of the hole is 800 μm, and the height is 500 μm.
步骤(6)中，所述等离子键合条件为：射频功率400-600w，时间20-40s，氧气流量50-200mL/min。优选地，射频功率600w，时间40s，氧气流量200mL/min。In step (6), the plasma bonding conditions are: radio frequency power 400-600w, time 20-40s, oxygen flow rate 50-200mL/min. Preferably, the radio frequency power is 600w, the time is 40s, and the oxygen flow rate is 200mL/min.
步骤(7)中，所述蠕动泵的转速为3rpm/min，蠕动泵的传输物质为：成纤维细胞培养基和人脐静脉内皮细胞培养基的1:1混合物或人脐血管内皮细胞培养基和心肌细胞培养基的1：1混合物；In step (7), the rotating speed of the peristaltic pump is 3rpm/min, and the transmission material of the peristaltic pump is: a 1:1 mixture of fibroblast culture medium and human umbilical vein endothelial cell culture medium or human umbilical vessel endothelial cell culture medium 1:1 mixture with cardiomyocyte culture medium;
所述成纤维细胞培养基为FGM-2；The fibroblast culture medium is FGM-2;
所述人脐静脉内皮细胞培养基为EGM-2；The human umbilical vein endothelial cell culture medium is EGM-2;
所述人脐血管内皮细胞培养基为EGM；The human umbilical vessel endothelial cell culture medium is EGM;
所述心肌细胞培养基为CCM。The cardiomyocyte culture medium is CCM.
本发明还提供了由上述方法构建获得的监测器官芯片生理病理指标生物传感器系统，所述系统包括：器官芯片，pH，ATP、ROS、氧含量和胆固醇传感芯片。The present invention also provides a biosensor system for monitoring organ-on-a-chip physiological and pathological indicators constructed by the above-mentioned method, and the system includes: organ-on-a-chip, pH, ATP, ROS, oxygen content and cholesterol sensor chips.
本发明所提供的构建生物传感系统的方法可以在体外得到3D人工血管模型，并通过电化学传感器实时动态监测器官芯片的生长状况。The method for constructing the biosensing system provided by the present invention can obtain a 3D artificial blood vessel model in vitro, and monitor the growth status of the organ chip dynamically in real time through an electrochemical sensor.
本发明所提供的生物传感系统可以实现自动化的分析，简化人工操作，降低实验误差和实验成本。The biosensing system provided by the invention can realize automatic analysis, simplify manual operation, and reduce experimental error and experimental cost.
本发明所提供的生物传感系统的监测对象包括但不限于血管器官芯片、心脏器官芯片、肾器官芯片、肺器官芯片、脑器官芯片、肠器官芯片等。The monitoring objects of the biosensing system provided by the present invention include, but are not limited to, blood vessel organ-on-a-chip, heart organ-on-a-chip, kidney organ-on-a-chip, lung organ-on-a-chip, brain organ-on-a-chip, and intestinal organ-on-a-chip.
本发明还提供了上述生物传感系统在监测器官芯片的生理状况、病变过程、模拟疾病、药效测试过程和预测人体药物反应中的应用。The present invention also provides the application of the above-mentioned biosensing system in monitoring the physiological state of the organ chip, disease process, simulating disease, drug efficacy testing process and predicting human drug response.
本发明还提供了一种使用上述生物传感器系统的方法，所述方法包括如下步骤：The present invention also provides a method of using the above-mentioned biosensor system, the method comprising the following steps:
步骤一、将混合细胞液注入芯片模型中的培养层进行培养；所述芯片出口连接到5个电极芯片入口；通过蠕动泵实现系统的动态监控。Step 1: Inject the mixed cell liquid into the culture layer in the chip model for culturing; the chip outlet is connected to five electrode chip inlets; and the dynamic monitoring of the system is realized through a peristaltic pump.
步骤二、监测每个电极芯片的电化学信号；Step 2, monitoring the electrochemical signal of each electrode chip;
步骤三、根据电化学信号，判断芯片中细胞生长的微环境是否正常如：pH、氧含量、胆固醇等；判断细胞是否受到外界刺激而发生应激反应如：ROS、ATP等。Step 3. According to the electrochemical signal, judge whether the microenvironment of cell growth in the chip is normal, such as: pH, oxygen content, cholesterol, etc.; judge whether the cells are subjected to external stimuli and cause stress reactions such as: ROS, ATP, etc.
步骤一中，所述细胞为成纤维细胞、人脐静脉内皮细胞、人脐血管内皮细胞和人诱导多功能干细胞来源的心肌细胞。In step 1, the cells are fibroblasts, human umbilical vein endothelial cells, human umbilical vessel endothelial cells and cardiomyocytes derived from human induced pluripotent stem cells.
人脐静脉内皮细胞、成纤维细胞、人脐血管内皮细胞和心肌细胞的密度均为2×(10 6-10 7)个/mL；优选地，为2×10 6个/mL。 The density of human umbilical vein endothelial cells, fibroblasts, human umbilical vessel endothelial cells and cardiomyocytes is 2×(10 6 -10 7 ) cells/mL; preferably, 2×10 6 cells/mL.
其中，所述成纤维细胞和人脐静脉内皮细胞两种细胞的数量比为1:1～1:5；优选地，为1:1。Wherein, the number ratio of the fibroblasts and the human umbilical vein endothelial cells is 1:1-1:5; preferably, it is 1:1.
其中，所述人脐血管内皮细胞和人诱导多功能干细胞来源的心肌细胞两种细胞的数量比为1:1～1:3；优选地，为1:1。Wherein, the number ratio of the human umbilical vascular endothelial cells and the cardiomyocytes derived from human induced pluripotent stem cells is 1:1-1:3; preferably, it is 1:1.
所述人脐静脉内皮细胞和成纤维细胞混合在基质胶溶液中，所述基质胶溶液的浓度为8 -10mg/mL；优选地，为10mg/mL；所述基质胶溶液包括基质胶，牛纤维蛋白原溶液，I型胶原等。Described human umbilical vein endothelial cells and fibroblasts are mixed in matrigel solution, and the concentration of described matrigel solution is 8-10mg/mL; Preferably, is 10mg/mL; Described matrigel solution comprises matrigel, bovine Fibrinogen solution, type I collagen, etc.
所述人脐血管内皮细胞和心肌细胞混合在3-10mg/mL的胶原溶液中；所述胶原溶液包括基质胶，牛纤维蛋白原溶液，Ⅰ型胶原等；优选地，为5mg/mL的Ⅰ型胶原。The human umbilical vessel endothelial cells and cardiomyocytes are mixed in a 3-10 mg/mL collagen solution; the collagen solution includes Matrigel, bovine fibrinogen solution, type I collagen, etc.; preferably, 5 mg/mL of I type collagen.
步骤一中，所述器官芯片的培养条件为：37℃，5％CO 2。 In step 1, the culture condition of the organ chip is: 37° C., 5% CO 2 .
步骤一中，所述培养层中细胞培养基为成纤维细胞培养基FGM-2和人脐静脉内皮细胞培养基EGM-2的等量混合物或心肌细胞培养基CCM和人脐血管内皮细胞培养基EGM的等量混合物。In step one, the cell culture medium in the culture layer is an equal mixture of fibroblast culture medium FGM-2 and human umbilical vein endothelial cell culture medium EGM-2 or cardiomyocyte culture medium CCM and human umbilical vein endothelial cell culture medium An equal mixture of EGM.
步骤一中，所述蠕动泵的转速为3rpm/min。In step 1, the speed of the peristaltic pump is 3rpm/min.
步骤二中，所述监测的电化学信号为：pH传感器测电势差，氧含量传感器测循环伏安中的峰值电流，ROS传感器测循环伏安中的峰值电流，ATP传感器测循环伏安中的峰值电流，胆固醇传感器测循环伏安中的峰值电流。In step 2, the electrochemical signals monitored are: the pH sensor measures the potential difference, the oxygen content sensor measures the peak current in cyclic voltammetry, the ROS sensor measures the peak current in cyclic voltammetry, and the ATP sensor measures the peak value in cyclic voltammetry Current, the cholesterol sensor measures the peak current in cyclic voltammetry.
本发明的有益效果包括：本发明将人脐静脉内皮细胞与成纤维细胞和/或人脐血管内皮细胞与心肌细胞在芯片模型中混合培养得到器官芯片，并将器官芯片与多个电化学传感器集成得到一个芯片分析平台，实现对3D体外模型的生长状况和生理微环境实时动态的监测，降低研究成本并加快研究速度，也可以为临床样本分析提供新的监测参数和检测手段。The beneficial effects of the present invention include: in the present invention, human umbilical vein endothelial cells and fibroblasts and/or human umbilical vessel endothelial cells and cardiomyocytes are mixed and cultured in a chip model to obtain an organ chip, and the organ chip is combined with a plurality of electrochemical sensors A chip analysis platform is integrated to realize real-time dynamic monitoring of the growth status of the 3D in vitro model and the physiological microenvironment, reduce research costs and speed up research, and can also provide new monitoring parameters and detection methods for clinical sample analysis.
附图说明Description of drawings
图1为本发明所提出的血管芯片模型的示意图Fig. 1 is the schematic diagram of the blood vessel chip model proposed by the present invention
图2为本发明所提出的血管芯片各层母板示意图。Fig. 2 is a schematic diagram of each layer of the motherboard of the blood vessel chip proposed by the present invention.
图3为本发明所提出的血管芯片单层的细节图。Fig. 3 is a detailed view of a single layer of the blood vessel chip proposed by the present invention.
图4为本发明中pH传感电极的示意图。Fig. 4 is a schematic diagram of a pH sensing electrode in the present invention.
图5为本发明中ATP传感电极的示意图。Fig. 5 is a schematic diagram of the ATP sensing electrode in the present invention.
图6为本发明中ROS传感电极的示意图。Fig. 6 is a schematic diagram of a ROS sensing electrode in the present invention.
图7为本发明中胆固醇传感电极的示意图。Fig. 7 is a schematic diagram of a cholesterol sensing electrode in the present invention.
图8为本发明中氧含量传感电极的示意图。Fig. 8 is a schematic diagram of an oxygen content sensing electrode in the present invention.
图9为生物传感系统示意图。Fig. 9 is a schematic diagram of a biosensing system.
图10为血管芯片实物图。Fig. 10 is a physical diagram of the blood vessel chip.
图11为生物传感系统实物图。Figure 11 is a physical diagram of the biosensing system.
图12为ATP传感芯片实物图。Fig. 12 is a physical diagram of the ATP sensor chip.
图13为氧含量传感芯片实物图。Fig. 13 is a physical diagram of the oxygen content sensing chip.
图14为ROS传感芯片实物图。Figure 14 is a physical diagram of the ROS sensor chip.
图15为胆固醇传感芯片实物图。Fig. 15 is a physical diagram of a cholesterol sensing chip.
图16为pH传感芯片实物图。Fig. 16 is a physical diagram of the pH sensor chip.
具体实施方式Detailed ways
结合以下具体实施例和附图，对本发明作进一步的详细说明。实施本发明的过程、条件、实验方法等，除以下专门提及的内容之外，均为本领域的普遍知识和公知常识，本发明没有特别限制内容。The present invention will be further described in detail in conjunction with the following specific embodiments and accompanying drawings. The process, conditions, experimental methods, etc. for implementing the present invention, except for the content specifically mentioned below, are common knowledge and common knowledge in this field, and the present invention has no special limitation content.
实施例1Example 1
(1)将聚二甲基硅氧烷主剂和固化剂按质量比10:1混合均匀，真空除气泡之后，浇筑在预先设计好的血管芯片母板上，固化两小时后将固化后获得的PDMS模板与母版分开；所述主剂为Sylgard 184聚合体，所述固化剂为Sylgard 184固化剂。(1) Mix the polydimethylsiloxane main agent and the curing agent evenly at a mass ratio of 10:1, and after removing air bubbles in vacuum, cast it on the pre-designed blood vessel chip motherboard, and after curing for two hours, obtain The PDMS template is separated from the master plate; the main agent is Sylgard 184 polymer, and the curing agent is Sylgard 184 curing agent.
(2)对步骤(1)得到的PDMS模板的出入口进行打孔，其中培养基出口和入口层各有6个500μm的圆孔，细胞培养曾有3个500μm的圆孔。(2) Holes were punched at the inlet and outlet of the PDMS template obtained in step (1), wherein the medium outlet and inlet layers each had six 500 μm round holes, and the cell culture had three 500 μm round holes.
(3)将步骤(1)制备得到的3个PDMS模板通过等离子清洗机进行逐层键合，得到血管芯片模型，键合条件为：射频功率600w，时间40s，氧气流量200mL/min。(3) The three PDMS templates prepared in step (1) were bonded layer by layer through a plasma cleaner to obtain a blood vessel chip model. The bonding conditions were: RF power 600w, time 40s, oxygen flow rate 200mL/min.
(4)将2×10 6个人脐静脉内皮细胞与2×10 6个成纤维细胞混合在10mg/mL的基质胶溶液中，将基质胶溶液注入步骤(3)所得的血管芯片模型中。 (4) Mix 2×10 6 human umbilical vein endothelial cells and 2×10 6 fibroblasts in a 10 mg/mL matrigel solution, and inject the matrigel solution into the blood vessel chip model obtained in step (3).
(5)将步骤(4)得到的包含细胞的血管芯片在37℃，5％CO 2下培养。 (5) Culture the blood vessel chip containing cells obtained in step (4) at 37° C. under 5% CO 2 .
(6)制备检测ATP浓度的电化学传感电极：1)在预先洗净的玻璃基底上沉积上金纳米颗粒；2)将3′端修饰巯基的DNA滴加在金电极表面，共孵育16小时；3)在含有0.1mol/L2-巯基乙醇的1mol/LNaClO 4浸泡10min；4)用含有0.05mol/LNaClO 4的0.01mol/LHEPES冲洗。5)在玻璃片基底上沉积上铂纳米颗粒和涂覆Ag/AgCl糊。形成的ATP传感芯片如图12所示。 (6) Prepare an electrochemical sensing electrode for detecting ATP concentration: 1) Deposit gold nanoparticles on a pre-cleaned glass substrate; 2) Drop the DNA modified with thiol at the 3' end on the surface of the gold electrode, and incubate for 16 3) soak in 1 mol/L NaClO 4 containing 0.1 mol/L 2-mercaptoethanol for 10 min; 4) rinse with 0.01 mol/L HEPES containing 0.05 mol/L NaClO 4 . 5) Deposit platinum nanoparticles and coat Ag/AgCl paste on the glass substrate. The formed ATP sensor chip is shown in FIG. 12 .
(7)制备检测氧含量的电化学传感电极：1)在预先洗净的玻璃基底上沉积上铂纳米颗粒，2)在玻璃基底上涂覆Ag/AgCl糊。形成的氧含量传感芯片如图13所示。(7) Preparation of an electrochemical sensing electrode for detecting oxygen content: 1) Platinum nanoparticles were deposited on a pre-cleaned glass substrate, and 2) Ag/AgCl paste was coated on the glass substrate. The formed oxygen content sensor chip is shown in FIG. 13 .
(8)制备检测ROS浓度的电化学传感电极：1)在预先洗净的玻璃基底上分别沉积上铂纳米颗粒和金纳米颗粒，其面积依次为1mm×3mm和1mm×1mm；2)将0.5μL辣根过氧化氢酶聚合物溶液滴加在金电极表面，置于4℃条件下黑暗中过夜。3)在玻璃片基底上涂覆Ag/AgCl糊。形成的ROS传感芯片如图14所示。(8) Preparation of electrochemical sensing electrodes for detecting ROS concentration: 1) Platinum nanoparticles and gold nanoparticles were respectively deposited on pre-cleaned glass substrates, the areas of which were 1mm×3mm and 1mm×1mm; 2) 0.5 μL of horseradish catalase polymer solution was dropped on the surface of the gold electrode, and placed in the dark overnight at 4°C. 3) Coating Ag/AgCl paste on the glass substrate. The formed ROS sensor chip is shown in Figure 14.
(9)制备检测胆固醇浓度的电化学传感电极：1)在预先洗净的玻璃基底上沉积上金纳米颗粒；2)将辣根过氧化氢酶溶液滴加在金电极的表面，自然风干，得到HOD/Au；3)将 胆固醇氧化酶滴加在1)的表面，自然风干，得到COD/HOD/Au电极。4)在玻璃片基底上沉积上铂纳米颗粒和涂覆Ag/AgCl糊。形成的胆固醇传感芯片如图15所示。(9) Prepare an electrochemical sensing electrode for detecting cholesterol concentration: 1) deposit gold nanoparticles on a pre-cleaned glass substrate; 2) add horseradish catalase solution dropwise on the surface of the gold electrode, and let it dry naturally , to obtain HOD/Au; 3) Add cholesterol oxidase dropwise on the surface of 1), and let it dry naturally to obtain a COD/HOD/Au electrode. 4) Deposit platinum nanoparticles and coat Ag/AgCl paste on the glass substrate. The formed cholesterol sensor chip is shown in FIG. 15 .
(10)pH的电化学传感电极为商业制备电极，购买自青岛波碳科技有限公司。形成的pH传感芯片如图16所示。(10) The electrode for electrochemical sensing of pH is a commercially prepared electrode, purchased from Qingdao Wave Carbon Technology Co., Ltd. The formed pH sensor chip is shown in FIG. 16 .
(11)将聚二甲基硅氧烷主剂和固化剂按质量比10:1混合均匀，真空除气泡之后，浇筑在预先设计好的电极芯片母板上，固化两小时后将固化获得的PDMS模板与所述电极芯片母板分开；所述主剂为Sylgard 184聚合体，所述固化剂为Sylgard 184固化剂。(11) Mix the polydimethylsiloxane main agent and the curing agent evenly at a mass ratio of 10:1, and after removing air bubbles in a vacuum, pour it on the pre-designed electrode chip motherboard, and cure the obtained product after two hours of curing. The PDMS template is separated from the electrode chip motherboard; the main agent is Sylgard 184 polymer, and the curing agent is Sylgard 184 curing agent.
(12)对步骤(11)得到的PDMS模板的出入口进行打孔，共2个800μm的圆孔。(12) Hole is punched at the entrance and exit of the PDMS template obtained in step (11), a total of two 800 μm circular holes.
(13)在玻璃片上修饰电极传感器；所述电极传感器包括：pH电极传感器、ROS电极传感器、氧含量电极传感器、胆固醇电极传感器、ATP电极传感器；(13) modify the electrode sensor on the glass sheet; the electrode sensor includes: pH electrode sensor, ROS electrode sensor, oxygen content electrode sensor, cholesterol electrode sensor, ATP electrode sensor;
(14)将步骤(13)制备得到的PDMS模板与电极传感器玻璃片等离子进行键合，获得电极传感芯片；所述键合条件为：射频功率600w，时间40s，氧气流量200mL/min。(14) Plasma bond the PDMS template prepared in step (13) with the electrode sensor glass sheet to obtain an electrode sensor chip; the bonding conditions are: RF power 600w, time 40s, oxygen flow rate 200mL/min.
(16)将所述血管器官芯片的培养基出口与所述电极芯片入口连接。构建如图11所示的传感系统。(16) Connecting the culture medium outlet of the vascular organ chip to the electrode chip inlet. Build the sensing system as shown in Figure 11.
(17)蠕动泵以3rpm/min的转速持续向血管器官芯片通入内皮细胞培养基和成纤维细胞培养基的1:1混合物。(17) The peristaltic pump continuously feeds the 1:1 mixture of endothelial cell culture medium and fibroblast culture medium to the vascular organ chip at a speed of 3 rpm/min.
实施例2Example 2
(1)将聚二甲基硅氧烷主剂和固化剂按质量比10:1混合均匀，真空除气泡之后，浇筑在预先设计好的血管芯片母板上，固化两小时后将固化后获得的PDMS模板与母版分开；所述主剂为Sylgard 184聚合体，所述固化剂为Sylgard 184固化剂。(1) Mix the polydimethylsiloxane main agent and the curing agent evenly at a mass ratio of 10:1, and after removing air bubbles in vacuum, cast it on the pre-designed blood vessel chip motherboard, and after curing for two hours, obtain The PDMS template is separated from the master plate; the main agent is Sylgard 184 polymer, and the curing agent is Sylgard 184 curing agent.
(2)对步骤(1)得到的PDMS模板的出入口进行打孔，其中培养基出口和入口层各有6个500μm的圆孔，细胞培养层有3个500μm的圆孔。(2) Perforate the inlet and outlet of the PDMS template obtained in step (1), wherein the media outlet and inlet layers each have six 500 μm round holes, and the cell culture layer has three 500 μm round holes.
(3)将步骤(1)制备得到的3个PDMS模板通过等离子清洗机进行逐层键合，得到血管芯片模型，键合条件为：射频功率600w，时间40s，氧气流量200mL/min。(3) The three PDMS templates prepared in step (1) were bonded layer by layer through a plasma cleaner to obtain a blood vessel chip model. The bonding conditions were: RF power 600w, time 40s, oxygen flow rate 200mL/min.
(4)将2×10 6个人脐血管内皮细胞与2×10 6个人诱导多功能干细胞来源的心肌细胞混合在5mg/mL的Ⅰ型胶原溶液中，将Ⅰ型胶原溶液注入步骤(3)所得的血管芯片模型中。 (4) Mix 2× 106 human umbilical endothelial cells and 2× 106 human induced pluripotent stem cell-derived cardiomyocytes in a 5 mg/mL type I collagen solution, and inject the type I collagen solution into the result of step (3) in the vascular chip model.
(5)将步骤(4)得到的包含细胞的心脏-血管芯片在37℃，5％CO 2下培养。 (5) The heart-vascular chip containing cells obtained in step (4) was cultured at 37° C. under 5% CO 2 .
(6)制备检测ATP浓度的电化学传感电极：1)在预先洗净的玻璃基底上沉积上金纳米颗粒；2)将3′端修饰巯基的DNA滴加在金电极表面，共孵育16小时；3)在含有0.1mol/L2-巯基乙醇的1mol/LNaClO 4浸泡10min；4)用含有0.05mol/LNaClO 4的0.01mol/LHEPES冲洗。5)在玻璃片基底上沉积上铂纳米颗粒和涂覆Ag/AgCl糊。形成的ATP传感芯片如图 12所示。 (6) Prepare an electrochemical sensing electrode for detecting ATP concentration: 1) Deposit gold nanoparticles on a pre-cleaned glass substrate; 2) Drop the DNA modified with thiol at the 3' end on the surface of the gold electrode, and incubate for 16 3) soak in 1 mol/L NaClO 4 containing 0.1 mol/L 2-mercaptoethanol for 10 min; 4) rinse with 0.01 mol/L HEPES containing 0.05 mol/L NaClO 4 . 5) Deposit platinum nanoparticles and coat Ag/AgCl paste on the glass substrate. The formed ATP sensor chip is shown in FIG. 12 .
(7)制备检测氧含量的电化学传感电极：1)在预先洗净的玻璃基底上沉积上铂纳米颗粒，2)在玻璃基底上涂覆Ag/AgCl糊。形成的氧含量传感芯片如图13所示。(7) Preparation of an electrochemical sensing electrode for detecting oxygen content: 1) Platinum nanoparticles were deposited on a pre-cleaned glass substrate, and 2) Ag/AgCl paste was coated on the glass substrate. The formed oxygen content sensor chip is shown in FIG. 13 .
(8)制备检测ROS浓度的电化学传感电极：1)在预先洗净的玻璃基底上分别沉积上铂纳米颗粒和金纳米颗粒，其面积依次为1mm×3mm和1mm×1mm；2)将0.5μL辣根过氧化氢酶聚合物溶液滴加在金电极表面，置于4℃条件下黑暗中过夜。3)在玻璃片基底上涂覆Ag/AgCl糊。形成的ROS传感芯片如图14所示。(8) Preparation of electrochemical sensing electrodes for detecting ROS concentration: 1) Platinum nanoparticles and gold nanoparticles were respectively deposited on pre-cleaned glass substrates, the areas of which were 1mm×3mm and 1mm×1mm; 2) 0.5 μL of horseradish catalase polymer solution was dropped on the surface of the gold electrode, and placed in the dark overnight at 4°C. 3) Coating Ag/AgCl paste on the glass substrate. The formed ROS sensor chip is shown in Figure 14.
(9)制备检测胆固醇浓度的电化学传感电极：1)在预先洗净的玻璃基底上沉积上金纳米颗粒；2)将辣根过氧化氢酶溶液滴加在金电极的表面，自然风干，得到HOD/Au；3)将胆固醇氧化酶滴加在1)的表面，自然风干，得到COD/HOD/Au电极。4)在玻璃片基底上沉积上铂纳米颗粒和涂覆Ag/AgCl糊。形成的胆固醇传感芯片如图15所示。(9) Prepare an electrochemical sensing electrode for detecting cholesterol concentration: 1) deposit gold nanoparticles on a pre-cleaned glass substrate; 2) add horseradish catalase solution dropwise on the surface of the gold electrode, and let it dry naturally , to obtain HOD/Au; 3) Add cholesterol oxidase dropwise on the surface of 1), and let it dry naturally to obtain a COD/HOD/Au electrode. 4) Deposit platinum nanoparticles and coat Ag/AgCl paste on the glass substrate. The formed cholesterol sensor chip is shown in FIG. 15 .
(10)pH的电化学传感电极为商业制备电极，购买自青岛波碳科技有限公司。形成的pH传感芯片如图16所示。(10) The electrode for electrochemical sensing of pH is a commercially prepared electrode, purchased from Qingdao Wave Carbon Technology Co., Ltd. The formed pH sensor chip is shown in FIG. 16 .
(11)将聚二甲基硅氧烷主剂和固化剂按质量比10:1混合均匀，真空除气泡之后，浇筑在预先设计好的电极芯片母板上，固化两小时后将固化获得的PDMS模板与所述电极芯片母板分开；所述主剂为Sylgard 184聚合体，所述固化剂为Sylgard 184固化剂。(11) Mix the polydimethylsiloxane main agent and the curing agent evenly at a mass ratio of 10:1, and after removing air bubbles in a vacuum, pour it on the pre-designed electrode chip motherboard, and cure the obtained product after two hours of curing. The PDMS template is separated from the electrode chip motherboard; the main agent is Sylgard 184 polymer, and the curing agent is Sylgard 184 curing agent.
(12)对步骤(11)得到的PDMS模板的出入口进行打孔，共2个800μm的圆孔。(12) Hole is punched at the entrance and exit of the PDMS template obtained in step (11), a total of two 800 μm circular holes.
(13)在玻璃片上修饰电极传感器；所述电极传感器包括：pH电极传感器、ROS电极传感器、氧含量电极传感器、胆固醇电极传感器、ATP电极传感器；(13) modify the electrode sensor on the glass sheet; the electrode sensor includes: pH electrode sensor, ROS electrode sensor, oxygen content electrode sensor, cholesterol electrode sensor, ATP electrode sensor;
(14)将步骤(13)制备得到的PDMS模板与电极传感器玻璃片等离子进行键合，获得电极传感芯片；所述键合条件为：射频功率600w，时间40s，氧气流量200mL/min。(14) Plasma bond the PDMS template prepared in step (13) with the electrode sensor glass sheet to obtain an electrode sensor chip; the bonding conditions are: RF power 600w, time 40s, oxygen flow rate 200mL/min.
(16)将所述心脏-血管器官芯片的培养基出口与所述电极芯片入口连接。构建如图11所示的传感系统。(16) Connecting the medium outlet of the heart-vascular organ chip to the electrode chip inlet. Build the sensing system as shown in Figure 11.
(17)蠕动泵以3rpm/min的转速持续向心脏-血管器官芯片通入内皮细胞培养基EGM和心肌细胞培养基CCM的1:1混合物。(17) The peristaltic pump continuously feeds the 1:1 mixture of endothelial cell culture medium EGM and cardiomyocyte culture medium CCM to the heart-vascular organ chip at a speed of 3 rpm/min.
本发明的保护内容不局限于以上实施例。在不背离本发明构思的精神和范围下，本领域技术人员能够想到的变化和优点都被包括在本发明中，并且以所附的权利要求书为保护范围。The protection content of the present invention is not limited to the above embodiments. Without departing from the spirit and scope of the concept of the present invention, changes and advantages conceivable by those skilled in the art are all included in the present invention, and the appended claims are the protection scope.

Claims (14)

  1. 一种检测器官芯片生理病理参数的生物传感系统，其特征在于，所述系统包括：器官芯片，pH传感芯片、ROS传感芯片、ATP传感芯片、氧含量传感芯片、胆固醇传感芯片。A biosensing system for detecting physiological and pathological parameters of an organ chip, characterized in that the system includes: an organ chip, a pH sensor chip, a ROS sensor chip, an ATP sensor chip, an oxygen content sensor chip, a cholesterol sensor chip chip.
  2. 如权利要求1所述的生物传感系统，其特征在于，所述器官芯片包括心脏器官芯片、肾器官芯片、肺器官芯片、脑器官芯片、血管器官芯片、肠器官芯片。The biosensing system according to claim 1, wherein the organ-on-a-chip includes a heart organ-on-a-chip, a kidney organ-on-a-chip, a brain organ-on-a-chip, a blood vessel organ-on-a-chip, and an intestinal organ-on-a-chip.
  3. 一种检测器官芯片生理病理参数的生物传感系统的构建方法，其特征在于，所述构建方法包括如下步骤：A construction method of a biosensing system for detecting physiological and pathological parameters of an organ chip, characterized in that the construction method comprises the following steps:
    步骤(1)、将聚二甲基硅氧烷主剂和固化剂以质量比10:1的比例混合，并浇筑在预先设计的芯片母板上固化，然后将固化后获得的聚二甲基硅氧烷PDMS模板与所述芯片母板分开；所述主剂为Sylgard 184聚合体，所述固化剂为Sylgard 184固化剂；Step (1), mix the polydimethylsiloxane main agent and the curing agent with a mass ratio of 10:1, and pour it on the pre-designed chip motherboard for curing, and then the polydimethylsiloxane obtained after curing The siloxane PDMS template is separated from the chip motherboard; the main agent is Sylgard 184 polymer, and the curing agent is Sylgard 184 curing agent;
    步骤(2)、将上述步骤(1)制备的PDMS模板进行键合，获得器官芯片；Step (2), bonding the PDMS template prepared in the above step (1) to obtain an organ chip;
    步骤(3)、向上述步骤(2)获得的器官芯片中注入细胞混合液，于恒温培养箱中培养；Step (3), injecting the cell mixture into the organ chip obtained in the above step (2), and culturing in a constant temperature incubator;
    步骤(4)、在玻璃片上修饰电极传感器；Step (4), modifying the electrode sensor on the glass sheet;
    步骤(5)、将聚二甲基硅氧烷主剂和固化剂按照质量比10:1的比例混合，然后浇筑在预先设计的电极芯片母板上固化，然后将固化后获得的PDMS模板与所述电极芯片母板分开；所述主剂为Sylgard 184聚合体，所述固化剂为Sylgard 184固化剂；Step (5), mix the polydimethylsiloxane main agent and the curing agent according to the mass ratio of 10:1, and then pour it on the pre-designed electrode chip motherboard for curing, and then combine the PDMS template obtained after curing with The electrode chip motherboard is separated; the main agent is Sylgard 184 polymer, and the curing agent is Sylgard 184 curing agent;
    步骤(6)、将上述步骤(5)制备获得的PDMS模板键合在所述步骤(4)修饰电极传感器的玻璃片上，获得电极传感芯片；Step (6), bonding the PDMS template prepared in the above step (5) on the glass sheet of the modified electrode sensor in the step (4) to obtain an electrode sensor chip;
    步骤(7)、将步骤(6)中的电极传感芯片入口与步骤(3)中的器官芯片出口连接，形成液体流通管道，即获得检测器官芯片生理病理参数的生物传感系统。Step (7), connecting the inlet of the electrode sensor chip in step (6) to the outlet of the organ chip in step (3) to form a liquid flow channel, that is, to obtain a biosensing system for detecting physiological and pathological parameters of the organ chip.
  4. 如权利要求3所述的构建方法，其特征在于，步骤(1)中，所述芯片母板分为细胞培养层和培养基层；The construction method according to claim 3, characterized in that, in step (1), the chip motherboard is divided into a cell culture layer and a culture medium layer;
    所述芯片母板的细胞培养层的主流道尺寸为宽500-1000μm，高100-200μm；The size of the main channel of the cell culture layer of the motherboard chip is 500-1000 μm in width and 100-200 μm in height;
    所述芯片母板的细胞培养层的支流道的尺寸为宽200-250μm，高400-500μm；The size of the branch channel of the cell culture layer of the chip motherboard is 200-250 μm in width and 400-500 μm in height;
    所述芯片母板的培养基层尺寸为长17mm，宽7mm，高500-700μm；The size of the medium layer of the chip motherboard is 17mm long, 7mm wide, and 500-700 μm high;
    所述芯片母板的出入口均为直径为200-500μm的圆孔；The entrances and exits of the chip motherboard are circular holes with a diameter of 200-500 μm;
    所述固化的温度为60-80℃；The curing temperature is 60-80°C;
    所述固化的时间范围为2-4h。The curing time range is 2-4h.
  5. 如权利要求3所述的构建方法，其特征在于，步骤(2)中，所述键合的内容为培养基出口层PDMS、PDMS多孔膜、细胞培养层PDMS，PDMS多孔膜，培养基出口层PDMS；The construction method according to claim 3, characterized in that, in step (2), the content of the bonding is medium outlet layer PDMS, PDMS porous membrane, cell culture layer PDMS, PDMS porous membrane, medium outlet layer PDMS;
    和/或，所述键合的条件为：射频功率400-600w，时间20-40s，氧气流量50-400mL/min。And/or, the bonding conditions are: radio frequency power 400-600w, time 20-40s, oxygen flow rate 50-400mL/min.
  6. 如权利要求3所述的构建方法，其特征在于，步骤(3)中，所述细胞混合液为成纤 维细胞和人脐静脉内皮细胞的细胞混合液、人脐血管内皮细胞和人诱导多功能干细胞来源的心肌细胞混合液；所述细胞混合在浓度为3-10mg/mL的基质胶溶液中；The construction method according to claim 3, wherein in step (3), the cell mixture is a cell mixture of fibroblasts and human umbilical vein endothelial cells, human umbilical vessel endothelial cells and human induced multifunctional Stem cell-derived cardiomyocyte mixture; the cells are mixed in a matrigel solution with a concentration of 3-10 mg/mL;
    其中，所述成纤维细胞和人脐静脉内皮细胞的密度为2×(10 6-10 7)个/mL，两种细胞的数量比为1:1～1:5；所述成纤维细胞和人脐静脉内皮细胞混合在8-10mg/mL的基质胶溶液中；所述基质胶溶液包括基质胶，牛纤维蛋白原溶液，I型胶原； Wherein, the density of the fibroblasts and human umbilical vein endothelial cells is 2×(10 6 -10 7 ) cells/mL, and the ratio of the two cells is 1:1-1:5; the fibroblasts and human umbilical vein endothelial cells are Human umbilical vein endothelial cells are mixed in a matrigel solution of 8-10 mg/mL; the matrigel solution includes matrigel, bovine fibrinogen solution, and type I collagen;
    所述人脐血管内皮细胞和人诱导多功能干细胞来源的心肌细胞的密度为2×(10 6-10 7)个/mL，两种细胞的数量比为1:1～1:3；所述人脐血管内皮细胞和心肌细胞混合在3-10mg/mL的胶原溶液中；所述胶原溶液包括基质胶，牛纤维蛋白原溶液，I型胶原。 The density of the human umbilical endothelial cells and the cardiomyocytes derived from human induced pluripotent stem cells is 2×(10 6 -10 7 ) cells/mL, and the ratio of the two cells is 1:1 to 1:3; the Human umbilical vessel endothelial cells and cardiomyocytes are mixed in a 3-10 mg/mL collagen solution; the collagen solution includes matrigel, bovine fibrinogen solution, and type I collagen.
  7. 如权利要求3所述的构建方法，其特征在于，步骤(4)中，所述电极传感器包括：pH电极传感器、ROS电极传感器、氧含量电极传感器、胆固醇电极传感器、ATP电极传感器；The construction method according to claim 3, wherein, in step (4), the electrode sensors include: pH electrode sensors, ROS electrode sensors, oxygen content electrode sensors, cholesterol electrode sensors, ATP electrode sensors;
    其中，所述pH电极传感器以修饰聚苯胺的碳电极为工作电极，碳电极为对电极，Ag/AgCl作为参比电极；Wherein, the pH electrode sensor uses a polyaniline-modified carbon electrode as a working electrode, the carbon electrode as a counter electrode, and Ag/AgCl as a reference electrode;
    所述ROS电极传感器以修饰辣根过氧化物酶的金电极为工作电极，铂为对电极，Ag/AgCl作为参比电极；The ROS electrode sensor uses a gold electrode modified with horseradish peroxidase as a working electrode, platinum as a counter electrode, and Ag/AgCl as a reference electrode;
    所述氧含量电极传感器以铂为工作电极和对电极，Ag/AgCl作为参比电极；The oxygen content electrode sensor uses platinum as a working electrode and a counter electrode, and Ag/AgCl as a reference electrode;
    所述胆固醇电极传感器以通过DNA origami修饰有辣根过氧化物酶和胆固醇氧化酶的金电极作为工作电极，铂为对电极，Ag/AgCl作为参比电极；The cholesterol electrode sensor uses a gold electrode modified with horseradish peroxidase and cholesterol oxidase by DNA origami as a working electrode, platinum as a counter electrode, and Ag/AgCl as a reference electrode;
    所述ATP电极传感器以修饰有ATP探针的金电极作为工作电极，铂为对电极，Ag/AgCl作为参比电极。The ATP electrode sensor uses a gold electrode modified with an ATP probe as a working electrode, platinum as a counter electrode, and Ag/AgCl as a reference electrode.
  8. 如权利要求3所述的构建方法，其特征在于，步骤(5)中，所述电极芯片母板的流道宽500-1000μm，高200-500μm；所述电极芯片母板的圆孔直径800-1000μm，高500-1000μm；The construction method according to claim 3, wherein in step (5), the flow channel of the electrode chip motherboard is 500-1000 μm wide and 200-500 μm high; the diameter of the hole of the electrode chip motherboard is 800 μm. -1000μm, height 500-1000μm;
    所述固化的温度为60-80℃；The curing temperature is 60-80°C;
    所述固化的时间范围为2-4h。The curing time range is 2-4h.
    所述键合的条件为射频功率400-600w，时间20-40s，氧气流量50-200mL/min。The bonding conditions are radio frequency power 400-600w, time 20-40s, oxygen flow rate 50-200mL/min.
  9. 如权利要求3所述的构建方法，其特征在于，所述器官芯片包括心脏器官芯片、肾器官芯片、肺器官芯片、脑器官芯片、血管器官芯片、肠器官芯片。The construction method according to claim 3, wherein the organ-on-a-chip includes heart organ-on-a-chip, kidney organ-on-a-chip, lung organ-on-a-chip, brain organ-on-a-chip, blood vessel organ-on-a-chip, and intestinal organ-on-a-chip.
  10. 如权利要求3所述的构建方法，其特征在于，步骤(7)中，所述系统中的液体通过蠕动泵的转速为3rpm/min；所述蠕动泵的传输物质为成纤维细胞培养基和人脐静脉内皮细胞培养基1：1的混合物或人脐血管内皮细胞培养基和心肌细胞培养基的1：1混合物。The construction method according to claim 3, wherein in step (7), the liquid in the system passes through the rotating speed of the peristaltic pump at 3rpm/min; the transmission material of the peristaltic pump is fibroblast culture medium and Human umbilical vein endothelial cell medium 1:1 mixture or human umbilical vessel endothelial cell medium and cardiomyocyte medium 1:1 mixture.
  11. 一种如权利要求3-10之任一项所述方法构建获得的监测器官芯片生理病理指标生物传感器系统，其特征在于，所述系统包括：器官芯片，pH传感芯片、ROS传感芯片、ATP传感芯片、氧含量传感芯片、胆固醇传感芯片。A biosensor system for monitoring organ-on-a-chip physiological and pathological indicators constructed by the method according to any one of claims 3-10, characterized in that the system comprises: an organ chip, a pH sensor chip, a ROS sensor chip, ATP sensor chip, oxygen content sensor chip, cholesterol sensor chip.
  12. 如权利要求1或11所述的监测器官芯片生理病理指标的电化学传感系统在监测器官芯片的生理状况、病变过程、模拟疾病、药效测试过程和预测人体药物反应中的应用。The application of the electrochemical sensing system for monitoring the physiological and pathological indicators of the organ chip as claimed in claim 1 or 11 in monitoring the physiological state of the organ chip, disease process, simulated disease, drug efficacy test process and predicting human drug response.
  13. 一种使用如权利要求1或11所述的生物传感器系统的方法，其特征在于，所述方法包括如下步骤：A method of using the biosensor system according to claim 1 or 11, wherein the method comprises the steps of:
    步骤一、将细胞混合并注入器官芯片中，将器官芯片与电极芯片连接，利用蠕动泵使得器官芯片中代谢物流经电极芯片；Step 1. The cells are mixed and injected into the organ chip, the organ chip is connected to the electrode chip, and the metabolites in the organ chip flow through the electrode chip by using a peristaltic pump;
    步骤二、监测每个电极芯片的电化学信号；Step 2, monitoring the electrochemical signal of each electrode chip;
    步骤三、根据电化学信号，判断芯片中细胞生长的微环境是否正常以及判断细胞是否受到外界刺激而发生应激反应。Step 3. According to the electrochemical signal, it is judged whether the microenvironment of cell growth in the chip is normal, and whether the cells are subjected to external stimuli to cause stress response.
  14. 如权利要求13所述的方法，其特征在于，步骤一中，所述细胞为成纤维细胞、人脐静脉内皮细胞、人脐血管内皮细胞、人诱导多功能干细胞来源的心肌细胞；The method according to claim 13, wherein in step one, the cells are fibroblasts, human umbilical vein endothelial cells, human umbilical vessel endothelial cells, and cardiomyocytes derived from human induced pluripotent stem cells;
    所述人脐静脉内皮细胞、成纤维细胞、人脐血管内皮细胞和人诱导多功能干细胞来源的心肌细胞的密度均为2×(10 6-10 7)个/mL； The density of the human umbilical vein endothelial cells, fibroblasts, human umbilical vessel endothelial cells and cardiomyocytes derived from human induced pluripotent stem cells is 2×(10 6 -10 7 ) cells/mL;
    步骤一中，所述蠕动泵的转速设置为：3rpm/min；所述器官芯片的培养条件为：37℃，5％CO 2；和/或， In step 1, the speed of the peristaltic pump is set to 3rpm/min; the culture condition of the organ chip is: 37°C, 5% CO 2 ; and/or,
    步骤二中，所述监测的电化学信号为：pH传感器测电势差，氧含量传感器测循环伏安中的峰值电流，ROS传感器测循环伏安中的峰值电流，ATP传感器测循环伏安中的峰值电流，胆固醇传感器测循环伏安中的峰值电流。In step 2, the electrochemical signals monitored are: the pH sensor measures the potential difference, the oxygen content sensor measures the peak current in cyclic voltammetry, the ROS sensor measures the peak current in cyclic voltammetry, and the ATP sensor measures the peak value in cyclic voltammetry Current, the cholesterol sensor measures the peak current in cyclic voltammetry.
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