CN116410860A - Human retina-like chip and its microelectrode array sensing system - Google Patents

Abstract

Translated fromChinese

一种类人视网膜芯片及其微电极阵列传感系统，该芯片系统由六个独立的对比系统组成，每个系统包括上层芯片、下层芯片和中间多孔膜，上层腔室提供毛细血管内皮细胞的生长环境，中间多孔膜分隔上层芯片和下层芯片，下层腔室提供神经细胞的生长环境。上层腔室和下层腔室分别有微电极阵列，用来采集神经元场电位信号和跨内皮细胞电阻信号。本发明模拟人视网膜神经血管单元组织结构，构建类人视网膜芯片，可采集光照影响下多细胞体系的数据信息，为视觉认知和视力神经保护与修复相关研究及智能‑智慧结构网络整体建模与机制揭示提供有效平台。

A human retina-like chip and its microelectrode array sensing system, the chip system consists of six independent contrast systems, each system includes an upper chip, a lower chip and a porous membrane in the middle, and the upper chamber provides the growth of capillary endothelial cells environment, the middle porous membrane separates the upper chip and the lower chip, and the lower chamber provides a growth environment for nerve cells. The upper chamber and the lower chamber respectively have microelectrode arrays, which are used to collect neuron field potential signals and transendothelial cell electrical resistance signals. The invention simulates the organizational structure of the human retinal neurovascular unit and constructs a human-like retinal chip, which can collect data information of a multi-cell system under the influence of light, and provide research on visual cognition, vision nerve protection and repair, and overall modeling of an intelligent-smart structure network Provide an effective platform with mechanism revealing.

Description

Translated fromChinese

类人视网膜芯片及其微电极阵列传感系统Human retina-like chip and its microelectrode array sensing system

技术领域technical field

本发明涉及类人视网膜芯片及其微电极阵列传感系统技术领域，属于器官芯片、视觉与视力、神经认知、生物医学及人工智能领域。The invention relates to the technical field of a human retina chip and a microelectrode array sensing system thereof, and belongs to the fields of organ chips, vision and vision, neurocognition, biomedicine and artificial intelligence.

背景技术Background technique

器官芯片(Organ-on-a-chip)系统是一种微流控3D细胞培养模型，采用微纳加工工艺与技术制作，通常包含一个连续灌流的微型腔室，可以模拟生理条件，在腔室内可培养人体组织和细胞。在器官芯片上引入多细胞的关键功能单元，通过微电极等传感技术，可以在体外模拟评价其功能参数。在生物医学方面，器官芯片技术有广泛的应用前景。通过器官芯片技术，可以研究疾病的发病机理和治疗方法，加速药物的筛选和研发，同时也有望为个性化医疗提供更加准确和有效的治疗方案。器官芯片技术对于生物医学领域的发展和进步具有重要的意义。Organ-on-a-chip (Organ-on-a-chip) system is a microfluidic 3D cell culture model, which is manufactured by micro-nano processing technology and technology, and usually includes a continuous perfusion micro-chamber, which can simulate physiological conditions. Human tissues and cells can be cultured. The key functional units of multi-cells are introduced on the organ chip, and its functional parameters can be simulated and evaluated in vitro through sensing technologies such as microelectrodes. In biomedicine, organ-on-a-chip technology has broad application prospects. Through organ-on-a-chip technology, the pathogenesis and treatment methods of diseases can be studied, and the screening and development of drugs can be accelerated. At the same time, it is also expected to provide more accurate and effective treatment plans for personalized medicine. Organ chip technology is of great significance to the development and progress of the biomedical field.

神经血管单元是由血管和与之相邻的神经元以及其它细胞组成的结构单元。人视网膜与脑等器官组织中包含丰富的神经血管单元，由内皮细胞、神经元、星形胶质细胞、肌细胞、周细胞和细胞外基质成分组成。内皮细胞和神经元是神经血管单元的关键构成要素，它们通过解剖和化学关系紧密相连。这种联系使它们能够监测细胞间的供应需求，引发必要的血管扩张或血管收缩反应，并在建立功能完整的整体中发挥关键作用，形成高效的脑血流调节系统。The neurovascular unit is a structural unit composed of blood vessels and adjacent neurons and other cells. Organs and tissues such as the human retina and brain contain abundant neurovascular units, which are composed of endothelial cells, neurons, astrocytes, myocytes, pericytes and extracellular matrix components. Endothelial cells and neurons are key building blocks of the neurovascular unit, closely connected by anatomical and chemical relationships. This connection allows them to monitor intercellular supply demands, trigger the necessary vasodilation or vasoconstriction responses, and play a key role in establishing a fully functioning whole, resulting in an efficient cerebral blood flow regulation system.

人视觉系统是中枢神经系统的一部分，由视网膜、视交叉、视束、外侧膝状体、视放射及视皮质等组成，可以在380nm-740nm波长的可见光光谱内检测到超过1000万种颜色。视网膜作为视觉系统的入口，可以感受到光线并将其转化为神经信号，然后通过视神经传送到大脑中进行进一步的处理和解读。神经认知是大脑处理信息和进行认知活动的过程，包括知觉、思考、注意力和记忆等方面。视网膜所传递的信息是视觉系统的基础，对于神经认知的形成和发展起着至关重要的作用。视网膜是一个层叠结构，由三层相互连接的细胞组成，跨越两个丛状层进行通信，以进行信号传输。视杆和视锥感光层含有大量平行排列的视杆细胞和视锥细胞，这些感光细胞被称为初级神经元，实现了光的转导。视杆细胞和视锥细胞吸收光线，并改变它们的膜电位，通过突触将这些信号传递到次级神经元或中间神经元、神经节神经元。视网膜内的神经元受到Müller细胞的高度调节和支持。Müller细胞属于神经胶质细胞，这些神经胶质细胞的胞体位于视网膜内核层，但整个细胞延伸跨越整个视网膜。这些细胞通过摄取和调节神经递质(如谷氨酸、γ-氨基丁酸)、细胞外离子、生长因子和pH，在视网膜中提供基本的支持和保护功能。Müller细胞也是与视网膜血管系统相互作用的主要神经细胞，它们参与保护视网膜神经元免受损伤或将功能回路中受损或退化的细胞中分离出来。视网膜中各个细胞在信号传递过程中发挥不同的作用，对视觉认知和视力神经保护与修复具有重要意义。但是目前仍然缺乏有效的系统和平台，对视网膜中多细胞信息传递和交流，进行实时、稳定的监测。The human visual system is a part of the central nervous system, consisting of the retina, optic chiasm, optic tract, lateral geniculate body, optic radiation, and visual cortex. It can detect more than 10 million colors in the visible light spectrum with wavelengths of 380nm-740nm. As the entrance of the visual system, the retina can sense light and convert it into nerve signals, which are then sent to the brain through the optic nerve for further processing and interpretation. Neurocognition is the process by which the brain processes information and performs cognitive activities, including perception, thinking, attention, and memory. The information transmitted by the retina is the basis of the visual system and plays a vital role in the formation and development of neurocognition. The retina is a layered structure consisting of three layers of interconnected cells that communicate across two plexiform layers for signal transmission. The rod and cone photoreceptor layer contains a large number of parallel rods and cones. These photoreceptor cells are called primary neurons and realize light transduction. Rods and cones absorb light, alter their membrane potential, and transmit these signals via synapses to secondary or interneurons, ganglion neurons. Neurons within the retina are highly regulated and supported by Müller cells. Müller cells belong to the group of glial cells whose cell bodies are located in the inner nuclear layer of the retina, but whose entire cells extend across the entire retina. These cells provide essential support and protective functions in the retina through the uptake and regulation of neurotransmitters (eg, glutamate, GABA), extracellular ions, growth factors, and pH. Müller cells are also the main neurons that interact with the retinal vasculature, and they are involved in protecting retinal neurons from damage or separating damaged or degenerated cells from functional circuits. Each cell in the retina plays a different role in the process of signal transmission, which is of great significance to visual cognition and visual nerve protection and repair. However, there is still a lack of effective systems and platforms for real-time and stable monitoring of multicellular information transmission and communication in the retina.

视网膜中的神经元网络对于人工神经网络的设计也具有重要意义。人工神经网络是一种模拟生物神经网络的技术，其结构和功能与视网膜中的神经元网络类似。通过研究视网膜中的神经元网络，可以优化人工神经网络的设计和训练算法，提高其性能和效率。The network of neurons in the retina is also important for the design of artificial neural networks. Artificial neural network is a technology that simulates biological neural network, and its structure and function are similar to the neuronal network in the retina. By studying the neuronal network in the retina, the design and training algorithms of artificial neural networks can be optimized to improve their performance and efficiency.

血视网膜屏障(Blood-Retinal Barrier,BRB)控制血管腔和视网膜之间的营养物质与代谢废物之间的交换，由内皮细胞、色素上皮细胞、视网膜胶质细胞与周细胞相互作用而形成。BRB可以限制血液中物质的渗透，同时防止视网膜内部的代谢产物、细胞和液体外渗到血液中。BRB的破坏可能会导致眼部疾病的发生，例如视网膜毛细血管瘤、糖尿病视网膜病变等。The blood-retinal barrier (Blood-Retinal Barrier, BRB) controls the exchange of nutrients and metabolic waste between the vascular lumen and the retina, and is formed by the interaction of endothelial cells, pigment epithelial cells, retinal glial cells, and pericytes. BRB can limit the penetration of substances in the blood, while preventing the extravasation of metabolites, cells and fluids inside the retina into the blood. The destruction of BRB may lead to the occurrence of eye diseases, such as retinal capillary hemangioma, diabetic retinopathy, etc.

跨内/上皮电阻(Transendothelial/Transepithelial Electrical Resistance,TEER)是一种被广泛接受的定量技术，用于测量内/上皮细胞培养模型中紧密连接动力学的完整性。在对药物或化学元素的运输进行评估之前，TEER值是细胞屏障完整性的有力指标。TEER测量可以在不损坏细胞的情况下实时进行，通常是基于测量欧姆电阻或测量宽频段的阻抗。Transendothelial/Transepithelial Electrical Resistance (TEER) is a widely accepted quantitative technique for measuring the integrity of tight junction dynamics in endothelial/epithelial cell culture models. TEER values are a strong indicator of cellular barrier integrity prior to assessment of drug or chemical element transport. TEER measurements can be performed in real time without damaging cells and are usually based on measuring ohmic resistance or measuring impedance over a broad frequency band.

神经元场电位是神经细胞集群活动在时间和空间上的叠加，反映了神经元网络中各个神经元的协同作用。通过多个微电极检测位点，可观测到各个区域场电位的相关性，用于揭示神经信号的传输和编码方式。微电极阵列是一种至少一维度尺寸为微米或纳米级的电极，用于测量生物细胞的活动，其主要优势是能够同时记录多个神经元活动，并通过空间发布分析神经元之间相互作用关系，从而更深入地了解神经网络的功能和行为。Neuronal field potential is the superposition of neuronal cluster activities in time and space, reflecting the synergy of individual neurons in a neuronal network. Through multiple microelectrode detection sites, the correlation of field potentials in various regions can be observed, which can be used to reveal the transmission and encoding methods of neural signals. A microelectrode array is an electrode with at least one dimension of micron or nanometer scale, which is used to measure the activity of biological cells. Its main advantage is that it can record the activities of multiple neurons at the same time, and analyze the interaction between neurons through spatial distribution. relationships to gain a deeper understanding of the function and behavior of neural networks.

人工智能和生物细胞神经元密切相关，人工神经网络中的设计灵感来自于生物细胞神经元的功能和结构，它们具有类似的工作原理。人工神经网络中的神经元节点通常包括一个输入层、一个或多个隐层和一个输出层，这类似于生物细胞神经元的树突、轴突和轴突末梢。同时，人工神经网络中的神经元之间的连接权重可以通过训练算法进行调整，类似于生物细胞神经元之间的突触可塑性。人类大脑的神经网络与任何其他动物的脑模型和现有的人工智能系统截然不同，其中人类的视觉与整个人类大脑和整个人体内的大规模连接的神经网络相互协同合作。我们尝试将这些独特的人类神经网络命名为真实人脑神经网络(real human brain neural networks,rhBNN)和真实人体神经网络(real human bodyneural networks,rhBNN⁺)，将新的生物启发式神经网络命名为新一代人工神经网络(innovative artificial neural networks,iANN)。Artificial intelligence and biological cell neurons are closely related. The design in artificial neural network is inspired by the function and structure of biological cell neurons, and they have similar working principles. Neuron nodes in artificial neural networks usually include an input layer, one or more hidden layers, and an output layer, which are similar to dendrites, axons, and axon terminals of biological cell neurons. At the same time, the connection weights between neurons in the artificial neural network can be adjusted by training algorithms, similar to the synaptic plasticity between neurons in biological cells. The neural network of the human brain is very different from any other animal brain model and existing artificial intelligence systems, in which human vision cooperates with a large-scale connected neural network throughout the human brain and throughout the human body. We try to name these unique human neural networks as real human brain neural networks (rhBNN) and real human body neural networks (rhBNN⁺ ), and name the new biologically inspired neural networks as A new generation of artificial neural networks (innovative artificial neural networks, iANN).

发明内容Contents of the invention

为了达到上述目的，本发明采用的技术方案为：一种类人视网膜芯片的PDMS腔室中包括六个独立的系统组成，每个系统均包括上层芯片、下层芯片和中间多孔膜，所述上层芯片包括上层入口、上层腔室、上层出口，上层入口和上层出口通过上层腔室连通，所述上层入口和上层出口分别位于上层腔室两侧，所述下层芯片包括下层入口、下层腔室、下层出口，下层入口和下层出口通过下层腔室连通，所述下层入口和下层出口分别位于下层腔室两侧，上层入口位置高于上层出口位置，下层入口位置高于下层出口位置，上层入口、上层出口和下层入口、下层出口呈十字交叉，上层入口、上层出口和下层入口、下层出口通道直径均大于等于100μm，上层腔室和下层腔室内的代谢产物通过渗透作用穿过中间多孔膜，实现细胞非接触式信息交流。In order to achieve the above object, the technical solution adopted in the present invention is: a PDMS chamber of a human retina chip comprises six independent systems, each system includes an upper chip, a lower chip and a middle porous membrane, the upper chip It includes an upper layer inlet, an upper layer chamber, and an upper layer outlet. The upper layer inlet and the upper layer outlet are connected through the upper layer chamber. The upper layer inlet and the upper layer outlet are respectively located on both sides of the upper layer chamber. Outlet, the lower layer entrance and the lower layer outlet are connected through the lower layer chamber, the lower layer inlet and the lower layer outlet are respectively located on both sides of the lower layer chamber, the position of the upper layer entrance is higher than the position of the upper layer exit, the position of the lower layer entrance is higher than the position of the lower layer exit, the upper layer entrance, the upper layer The outlet, the lower layer inlet, and the lower layer outlet form a cross, and the diameters of the upper layer inlet, the upper layer outlet, the lower layer inlet, and the lower layer outlet channel are all greater than or equal to 100 μm. The metabolites in the upper layer chamber and the lower layer chamber pass through the middle porous membrane through osmosis, realizing the Contactless information exchange.

进一步地，所述上层腔室的高度大于等于120μm、长度大于等于350μm、宽度大于等于350μm。Further, the height of the upper chamber is greater than or equal to 120 μm, the length is greater than or equal to 350 μm, and the width is greater than or equal to 350 μm.

进一步地，所述下层腔室的高度大于等于120μm、长度大于等于320μm、宽度大于等于320μm。Further, the lower chamber has a height greater than or equal to 120 μm, a length greater than or equal to 320 μm, and a width greater than or equal to 320 μm.

进一步地，所述中间多孔膜的厚度为10μm，长度大于等于350μm、宽度大于等于350μm，孔径为0.4、1、3或10μm，相邻孔间距为5μm，孔密度为4×106/cm²，中间多孔膜为聚对苯二甲酸乙二醇酯PET，中间多孔膜尺寸的设定与上层腔室的尺寸对应。Further, the thickness of the intermediate porous membrane is 10 μm, the length is greater than or equal to 350 μm, the width is greater than or equal to 350 μm, the pore diameter is 0.4, 1, 3 or 10 μm, the distance between adjacent pores is 5 μm, and the pore density is 4×106/cm² , The middle porous membrane is polyethylene terephthalate PET, and the setting of the size of the middle porous membrane corresponds to the size of the upper chamber.

进一步地，所述六个独立的系统整个核心部件面积≥1070×1820μm²，其中相邻两个系统的间距≥400μm。Further, the entire core component area of the six independent systems is ≥1070×1820 μm² , and the distance between two adjacent systems is ≥400 μm.

一种微电极阵列传感系统，所述上层芯片和下层芯片上分别嵌入微电极，实现细胞屏障TEER和神经元场电位的测量，包括用于测量TEER的3对微电极，每一个系统的下层腔室出口正上方放置一个下层TEER电极，下层TEER电极位于中间多孔膜下侧表面、下层腔室对侧顶角各放置一个下层TEER电极，共三个下层TEER电极，上层腔室放置的三个上层TEER电极与下层腔室的三个下层TEER电极位置对应，用于测量神经元场电位信号，采用三电极体系，每个下层芯片的硅基衬底集成11个微电极，包括1个Pt对电极、1个Ag/AgCl参比电极和9个Pt工作电极，每个独立的系统中上层腔室、下层腔室的总电极数量为17，整个芯片系统总电极数为102个，Pt工作电极与电极导线连接。A microelectrode array sensing system, the upper chip and the lower chip are respectively embedded with microelectrodes to realize the measurement of cell barrier TEER and neuron field potential, including 3 pairs of microelectrodes for measuring TEER, and the lower layer of each system A lower TEER electrode is placed directly above the chamber outlet. The lower TEER electrode is located on the lower surface of the middle porous membrane, and one lower TEER electrode is placed at the top corner of the opposite side of the lower chamber. There are three lower TEER electrodes in total, and three in the upper chamber. The upper TEER electrode corresponds to the position of the three lower TEER electrodes in the lower chamber, and is used to measure the neuron field potential signal. A three-electrode system is adopted. The silicon-based substrate of each lower chip integrates 11 microelectrodes, including a Pt pair. electrode, 1 Ag/AgCl reference electrode and 9 Pt working electrodes, the total number of electrodes in the upper chamber and lower chamber in each independent system is 17, the total number of electrodes in the whole chip system is 102, and the Pt working electrode Connect with electrode leads.

进一步地，所述的用于细胞屏障测量的上层TEER电极、下层TEER电极，上层TEER电极、下层TEER电极的尺寸均为20μm*30μm，电极材料为Ti/Pt，电极厚度为230nm，电极材料Pt与外界直接接触，三对电极的设置在不同位置进行多点数据测量，通过平均计算获取TEER值。Further, the upper layer TEER electrode and the lower layer TEER electrode used for cell barrier measurement, the size of the upper layer TEER electrode and the lower layer TEER electrode are both 20 μm*30 μm, the electrode material is Ti/Pt, the electrode thickness is 230nm, and the electrode material is Pt In direct contact with the outside world, three pairs of electrodes are set at different positions for multi-point data measurement, and the TEER value is obtained through average calculation.

进一步地，所述用于测量神经元电场电位的微电极，Pt工作电极尺寸为20μm*20μm，电极材料为Ti/Pt，厚度为230nm，Pt对电极和Ag/AgCl参比电极位于Pt工作电极两侧，Pt对电极和Ag/AgCl参比电极的尺寸均为100μm*20μm，Pt对电极材料为Ti/Pt，厚度为230nm，Ag/AgCl参比电极材料为Ti/Pt,Ag/AgCl，厚度为430nm。Further, the microelectrode used for measuring the electric field potential of neurons, the Pt working electrode has a size of 20 μm*20 μm, the electrode material is Ti/Pt, and the thickness is 230 nm, and the Pt counter electrode and Ag/AgCl reference electrode are located on the Pt working electrode On both sides, the size of Pt counter electrode and Ag/AgCl reference electrode is 100μm*20μm, the material of Pt counter electrode is Ti/Pt, the thickness is 230nm, the material of Ag/AgCl reference electrode is Ti/Pt, Ag/AgCl, The thickness is 430nm.

与现有技术相比，本发明的有益效果是：Compared with prior art, the beneficial effect of the present invention is:

类人视网膜芯片模拟人视网膜神经血管单元组织结构，借助器官芯片技术，体外构建由人视网膜毛细血管内皮细胞、视锥细胞、视杆细胞和米勒细胞组成的多细胞相互作用网络。毛细血管内皮细胞可以形成BRB，并与其它三种神经细胞进行交流，形成一个简单有序的rhBNN-rhBNN⁺神经网络。利用微电极阵列，在单细胞尺度的类人视网膜芯片中可以实时监测神经元场电位，探究多细胞信息传递与相互作用机理。此外，毛细血管内皮细胞生长连接的血视网膜屏障的生长连接状态也可以通过微电极进行TEER的量化评价。类人视网膜芯片及其微电极阵列传感系统为人体组织器官感受、感觉、感知、认知机制的揭示以及人体组织器官稳态、保护、维持和损失修复提供研究平台。The human retina chip simulates the organizational structure of the human retinal neurovascular unit. With the help of organ chip technology, a multicellular interaction network composed of human retinal capillary endothelial cells, cone cells, rod cells and Müller cells is constructed in vitro. Capillary endothelial cells can form BRB and communicate with other three types of nerve cells to form a simple and orderly rhBNN-rhBNN⁺ neural network. Using microelectrode arrays, the field potential of neurons can be monitored in real time in a single-cell-scale human retina chip, and the mechanism of multi-cellular information transmission and interaction can be explored. In addition, the growth junction status of the blood-retinal barrier connected by capillary endothelial cells can also be quantitatively evaluated by microelectrodes for TEER. The human retina-like chip and its microelectrode array sensing system provide a research platform for the revelation of human tissue organ perception, sensation, perception, and cognitive mechanisms, as well as the homeostasis, protection, maintenance, and loss repair of human tissue organs.

本发明利用微流控技术，构造六个结构相同的对比系统，控制变量因素，将毛细血管内皮细胞和神经细胞引入腔室，实现了对比参照。The invention utilizes the microfluidic technology to construct six comparison systems with the same structure, controls variable factors, introduces capillary endothelial cells and nerve cells into the chamber, and realizes comparison and reference.

本发明通过引入微电极阵列，构建类人视网膜芯片及其微电极阵列传感系统，实现了TEER和神经元场电位的实时监测。The invention realizes the real-time monitoring of TEER and neuron field potential by introducing the micro-electrode array, constructing the human retina chip and its micro-electrode array sensing system.

附图说明Description of drawings

图1为本发明的整体结构示意图；Fig. 1 is the overall structure schematic diagram of the present invention;

图2为本发明的整体结构剖面图；Fig. 2 is a sectional view of the overall structure of the present invention;

图3微电极阵列制备流程示意图；The schematic diagram of the preparation process of the microelectrode array in Fig. 3;

图4为本发明的TEER微电极阵列结构示意图；Fig. 4 is the structural representation of TEER microelectrode array of the present invention;

图5为本发明的神经元场电位微电极阵列结构示意图。Fig. 5 is a schematic diagram of the structure of the neuron field potential microelectrode array of the present invention.

图中：1、PDMS腔室，2、上层腔室，3、上层入口，4、上层出口，5、中间多孔膜，6、下层腔室，7、下层入口，8、下层出口，9、上层TEER电极，10、下层TEER电极，11、Pt工作电极，12、Pt对电极，13、Ag/AgCl参比电极，14、电极导线，15、硅基衬底。In the figure: 1. PDMS chamber, 2. Upper chamber, 3. Upper inlet, 4. Upper outlet, 5. Middle porous membrane, 6. Lower chamber, 7. Lower inlet, 8. Lower outlet, 9. Upper TEER electrode, 10, lower TEER electrode, 11, Pt working electrode, 12, Pt counter electrode, 13, Ag/AgCl reference electrode, 14, electrode wire, 15, silicon-based substrate.

具体实施方式Detailed ways

以下结合具体实施方式对本专利的技术方案作进一步详细说明，但并不因此而限制于本发明。The technical solution of this patent will be described in further detail below in conjunction with specific embodiments, but the present invention is not limited thereto.

实施例1Example 1

类人视网膜芯片，如图1，图2所示，该系统包括六个独立的对比系统。六个独立的培养系统中，可以注入不同细胞组合，实现不同细胞组合的对比参照。微电极阵列可实现细胞屏障TEER和神经元场电位的实时采集。Human retinal chip, as shown in Figure 1 and Figure 2, the system includes six independent comparison systems. In the six independent culture systems, different cell combinations can be injected to achieve comparison and reference of different cell combinations. Microelectrode arrays enable real-time acquisition of cell barrier TEER and neuronal field potentials.

所述的六个独立的对比系统内部构造相同，分为上层芯片、下层芯片和中间多孔膜5。所述的上层芯片包括上层入口3、上层腔室2、上层出口4，上层入口3和上层出口4通过上层腔室2连通；所述的上层腔室2高≥120μm、长≥350μm、宽≥350μm。所述的下层芯片包括下层入口7、下层腔室6、下层出口8，下层入口7和下层出口8通过下层腔室6连通；所述的下层腔室6高≥120μm、长≥320μm、宽≥320μm。上层腔室2和下层腔室6尺寸的设定，为匹配神经细胞尺寸，尽可能在体外实现真实的细胞生长环境。上层腔室2的长、宽略大于下层腔室6的长、宽。所述的上层入口3和上层出口4分别位于上层腔室2两侧；上层入口3和上层出口4通道直径≥100μm，上层入口3略高于上层出口4。所述的下层入口7和下层出口8分别位于下层腔室6两侧；下层入口7和下层出口8通道直径≥100μm，下层入口7位置高于下层出口8位置。上层入口3、出口和下层入口7、出口呈十字交叉。入口和出口尺寸和位置的设定，为细胞和营养液充分进入腔室，细胞和营养液不会在入口和出口产生阻塞。所述的中间多孔膜5厚度为10μm，长≥350μm、宽≥350μm，孔径为0.4、1、3或10μm，相邻孔间距为5μm，孔密度为4×10⁶/cm²。该尺寸的设定与上层腔室2尺寸对应，可以保证不同的信号分子通过中间多孔膜5实现上层腔室2和下层腔室6不同的信息交流。上层腔室2和下层腔室6所用材料为聚二甲基硅氧烷(Polydimethylsiloxane,PDMS)，中间多孔膜5所用材料为聚对苯二甲酸乙二醇酯(Polyethylene Terephthalate,PET)。上层腔室2和下层腔室6内的代谢产物可通过渗透作用穿过中间多孔膜5，实现细胞非接触式信息交流。The six independent comparison systems have the same internal structure, and are divided into upper chip, lower chip and middle porous membrane 5 . The upper layer chip includes anupper layer inlet 3, anupper layer chamber 2, and an upper layer outlet 4, and theupper layer inlet 3 and the upper layer outlet 4 are connected through theupper layer chamber 2; theupper layer chamber 2 is high ≥ 120 μm, long ≥ 350 μm, and wide ≥ 350 μm. The lower layer chip includes a lower layer inlet 7, a lower layer chamber 6, and a lower layer outlet 8, and the lower layer inlet 7 and the lower layer outlet 8 are connected through the lower layer chamber 6; the lower layer chamber 6 is high ≥ 120 μm, long ≥ 320 μm, and wide ≥ 320 μm. The dimensions of theupper chamber 2 and the lower chamber 6 are set to match the size of the nerve cells and to achieve a real cell growth environment in vitro as much as possible. The length and width of theupper chamber 2 are slightly greater than the length and width of the lower chamber 6 . Theupper inlet 3 and the upper outlet 4 are located on both sides of theupper chamber 2 respectively; The lower inlet 7 and the lower outlet 8 are respectively located on both sides of the lower chamber 6; the lower inlet 7 and the lower outlet 8 have channel diameters greater than or equal to 100 μm, and the position of the lower inlet 7 is higher than that of the lower outlet 8. Theentrance 3 and the exit of the upper floor and the entrance 7 and the exit of the lower floor are in a cross. The size and position of the inlet and outlet are set so that the cells and nutrient solution can fully enter the chamber, and the cells and nutrient solution will not be blocked at the inlet and outlet. The intermediate porous membrane 5 has a thickness of 10 μm, a length ≥ 350 μm, a width ≥ 350 μm, a pore diameter of 0.4, 1, 3 or 10 μm, a distance between adjacent pores of 5 μm, and a pore density of 4×10⁶ /cm² . The setting of this size corresponds to the size of theupper chamber 2 , which can ensure that different signal molecules pass through the middle porous membrane 5 to realize different information exchanges between theupper chamber 2 and the lower chamber 6 . Theupper chamber 2 and the lower chamber 6 are made of polydimethylsiloxane (Polydimethylsiloxane, PDMS), and the material of the middle porous membrane 5 is polyethylene terephthalate (PET). Metabolites in theupper chamber 2 and the lower chamber 6 can pass through the middle porous membrane 5 through osmosis, realizing non-contact information exchange between cells.

六个独立的对比系统整个核心部件面积≥1070×1820μm，其中相邻两个系统的间距≥400μm。该尺寸的设定，可以确保各个系统互不干扰。The area of the entire core components of the six independent comparison systems is ≥1070×1820 μm, and the distance between two adjacent systems is ≥400 μm. The setting of this size can ensure that each system does not interfere with each other.

一种微电极阵列传感系统，所述的微电极阵列如图4，图5所示，包括用于测量细胞屏障TEER的微电极和神经元场电位的微电极。所述的用于测量细胞屏障TEER的微电极，在六个独立的系统中，每一组下层出口8正上方放置一下层TEER电极10，电极位于多孔膜下侧表面、下层腔室6对侧顶角各放置一个下层TEER电极10，共三个微电极；上层腔室2也放置三个电极，为上层TEER电极9，上层TEER电极9的位置与下层腔室6的下层TEER电极10位置相对应，电极位于上层腔室2正上方，上层TEER电极9、下层TEER电极10的电极尺寸为20μm*30μm，电极材料为Ti/Pt，电极厚度为230nm，电极材料Pt与外界直接接触。三对电极的设置可在不同位置进行多点数据测量，通过平均计算获取TEER。所述的用于采集神经元场电位信号的微电极，位于硅基衬底15上方，硅基衬底15与下层腔室6紧密封接。每个下层腔室6内用于采集神经元场电位信号的微电极采用三电极体系，共11个微电极，其中包括1个Pt对电极12，1个Ag/AgCl参比电极13和9个Pt工作电极11。Pt对电极12和Pt工作电极11的材料为Ti/Pt，电极厚度为230nm，电极材料Pt与外界直接接触，Ag/AgCl参比电极13材料为Ti/Pt,Ag/AgCl，电极厚度为430nm，电极材料Ag/AgCl与外界直接接触。所述的Pt工作电极11尺寸为20μm*20μm。该尺寸的设定，符合神经元大小。所述的Pt对电极12和Ag/AgCl参比电极13位于工作电极11两侧，电极尺寸为100μm*20μm。该尺寸的设定，可保证获得稳定的参考电压。电极导线14材料为Ti/Pt，最小线宽为10μm，厚度为230nm。六个独立的系统每一组上下腔室总电极数量为17，整个芯片系统总电极数为102个。A microelectrode array sensing system, the microelectrode array is shown in Figure 4 and Figure 5, including microelectrodes for measuring cell barrier TEER and neuron field potential microelectrodes. For the microelectrode used to measure the cell barrier TEER, in six independent systems, the lowerlayer TEER electrode 10 is placed directly above each set of lower layer outlets 8, and the electrode is located on the lower surface of the porous membrane and opposite to the lower layer chamber 6 Alower TEER electrode 10 is placed in each of the top corners, and there are three microelectrodes in total; theupper chamber 2 also places three electrodes, which are theupper TEER electrodes 9, and the position of theupper TEER electrode 9 is the same as that of thelower TEER electrode 10 in the lower chamber 6. Correspondingly, the electrode is located directly above theupper chamber 2, the electrode size of theupper TEER electrode 9 and thelower TEER electrode 10 is 20 μm*30 μm, the electrode material is Ti/Pt, the electrode thickness is 230 nm, and the electrode material Pt is in direct contact with the outside world. The setting of three pairs of electrodes can be used for multi-point data measurement at different positions, and the TEER can be obtained through average calculation. The microelectrodes for collecting neuron field potential signals are located above the silicon-basedsubstrate 15 , and the silicon-basedsubstrate 15 is tightly sealed with the lower chamber 6 . The microelectrodes used to collect neuron field potential signals in each lower chamber 6 adopt a three-electrode system, with a total of 11 microelectrodes, including 1Pt counter electrode 12, 1 Ag/AgCl reference electrode 13 and 9Pt working electrode 11 . The material of thePt counter electrode 12 and thePt working electrode 11 is Ti/Pt, the electrode thickness is 230nm, the electrode material Pt is in direct contact with the outside world, the material of the Ag/AgCl reference electrode 13 is Ti/Pt, Ag/AgCl, and the electrode thickness is 430nm , the electrode material Ag/AgCl is in direct contact with the outside world. The size of thePt working electrode 11 is 20 μm*20 μm. The setting of this size corresponds to the size of the neuron. ThePt counter electrode 12 and the Ag/AgCl reference electrode 13 are located on both sides of the workingelectrode 11, and the size of the electrodes is 100 μm*20 μm. The setting of this dimension can ensure a stable reference voltage. The material of theelectrode lead 14 is Ti/Pt, the minimum line width is 10 μm, and the thickness is 230 nm. The total number of electrodes in each group of the upper and lower chambers of the six independent systems is 17, and the total number of electrodes in the whole chip system is 102.

PDMS腔室1是整个类人视网膜芯片微电极阵列传感系统的一个大腔室；PDMS腔室1包含六个独立的类人视网膜芯片微电极阵列传感系统，类人视网膜芯片微电极阵列传感系统中包含上层芯片、下层芯片和中间多孔膜5PDMS chamber 1 is a large chamber of the whole human retina chip microelectrode array sensing system; PDMS chamber 1 contains six independent human retina chip microelectrode array sensing systems, human retina chip microelectrode array sensor The sensing system includes the upper chip, the lower chip and the middle porous membrane5

上层芯片包括上层腔室2、上层入口3、上层出口4，通过上层入口3灌入内皮细胞组织液，内皮细胞组织液从上层出口4灌出，上层腔室2提供毛细血管内皮细胞的生长环境；The upper layer chip includes anupper layer chamber 2, anupper layer inlet 3, and an upper layer outlet 4, through which the endothelial cell tissue fluid is poured into theupper layer inlet 3, and the endothelial cell tissue fluid is poured out from the upper layer outlet 4, and theupper layer chamber 2 provides a growth environment for capillary endothelial cells;

下层芯片包括下层腔室6、下层入口7、下层出口8，通过上层入口7灌入神经细胞组织液，神经细胞组织液从上层出口8灌出，下层腔室6提供神经细胞的生长环境；The lower layer chip includes a lower layer chamber 6, a lower layer inlet 7, and a lower layer outlet 8. Nerve cell tissue fluid is poured through the upper layer inlet 7, and the nerve cell tissue fluid is poured out from the upper layer outlet 8. The lower layer chamber 6 provides a growth environment for nerve cells;

上层TEER电极9、下层TEER电极10，用于测量TEER值，评价细胞屏障完整性；Theupper TEER electrode 9 and thelower TEER electrode 10 are used to measure the TEER value and evaluate the integrity of the cell barrier;

Pt工作电极11、Pt对电极12、Ag/AgCl参比电极13，三电极体系用于采集神经元场电位，电极导线14连接引出Pt工作电极11、Pt对电极12、Ag/AgCl参比电极13，硅基衬底15作为基底平台用于集成Pt工作电极11、Pt对电极12、Ag/AgCl参比电极13、电极导线14。Pt working electrode 11,Pt counter electrode 12, Ag/AgCl reference electrode 13, the three-electrode system is used to collect neuron field potential,electrode lead 14 is connected to leadPt working electrode 11,Pt counter electrode 12, Ag/AgCl reference electrode 13. The silicon-basedsubstrate 15 is used as a base platform for integrating thePt working electrode 11, thePt counter electrode 12, the Ag/AgCl reference electrode 13, and theelectrode wire 14.

上述的类人视网膜芯片及其微电极阵列传感系统利用氧等离子体进行各个部分的封接，形成封闭的类人视网膜芯片。适当密度内皮细胞和神经细胞引入类人视网膜芯片的培养腔室内，可以长时间稳定采集到评价细胞屏障特性的TEER和神经元场电位。The aforementioned human retinal chip and its microelectrode array sensing system use oxygen plasma to seal various parts to form a closed human retinal chip. Endothelial cells and nerve cells at appropriate density are introduced into the culture chamber of the human retina chip, and the TEER and neuron field potential for evaluating the cell barrier properties can be collected stably for a long time.

一种类人视网膜芯片及其微电极阵列传感系统，微电极阵列制备过程如图3详述如下：A human retina-like chip and its microelectrode array sensing system, the preparation process of the microelectrode array is described in detail as shown in Figure 3 as follows:

1.将2英寸硅圆晶片样品放置HMDS预处理系统中，通过加温发生反应，将硅片表面由亲水变为疏水，增强光刻胶与基底的粘附力电极层结构；1. Place the 2-inch silicon wafer sample in the HMDS pretreatment system, and react by heating to change the surface of the silicon wafer from hydrophilic to hydrophobic, and enhance the adhesion between the photoresist and the substrate electrode layer structure;

2.在样品表面旋涂一层正性光刻胶AZ4620，厚度为7μm，经光刻、显影形成微电极阵列图案；2. Spin-coat a layer of positive photoresist AZ4620 on the surface of the sample, with a thickness of 7 μm, and form a micro-electrode array pattern through photolithography and development;

3.显影后的表面依旧会存留几纳米的光刻胶，这些残胶会对后续金属薄膜沉积产生影响，导致金属薄膜脱落。使用去胶机对样品进行去胶，参数选择，传输功率为300W，等离子体反应时间为120s，氧气流量为200sccm，压强为110mTorr；3. A few nanometers of photoresist will still remain on the surface after development. These residual adhesives will affect the subsequent metal film deposition and cause the metal film to fall off. Use the degumming machine to degumming the sample, parameter selection, the transmission power is 300W, the plasma reaction time is 120s, the oxygen flow rate is 200sccm, and the pressure is 110mTorr;

4.样品经电子束蒸发在微电极阵列图案表面沉积一层Ti/Pt金属薄膜，Ti的厚度为30nm，可增加Pt导电薄膜与衬底的粘附性，Pt的厚度为200nm，可用于电信号数据传导；4. The sample is evaporated by electron beam to deposit a layer of Ti/Pt metal film on the surface of the microelectrode array pattern. The thickness of Ti is 30nm, which can increase the adhesion between the Pt conductive film and the substrate. The thickness of Pt is 200nm, which can be used for electric signal data transmission;

5.样品通过丙酮浸泡，剥离光刻胶和其上的多余金属薄膜，在样品表面形成裸露的微电极阵列；5. The sample is soaked in acetone, the photoresist and the excess metal film on it are peeled off, and a bare microelectrode array is formed on the surface of the sample;

6.利用等离子体增强化学气相沉积法(PECVD)在样品表面形成一层Si₃N₄薄膜绝缘层，覆盖样品表面,样品表面被厚度为400nmSi₃N₄绝缘层覆盖，形成对电极引线的绝缘保护；6. Use plasma enhanced chemical vapor deposition (PECVD) to form a layer of Si₃ N₄ thin film insulating layer on the surface of the sample to cover the surface of the sample. The surface of the sample is covered with a Si₃ N₄ insulating layer with a thickness of 400nm to form insulation for the electrode leads Protect;

7.样品经反应离子刻蚀，去除微电极阵列检测位点处的电极，使电极接触位点裸露；7. The sample is subjected to reactive ion etching to remove the electrode at the detection site of the microelectrode array, so that the electrode contact site is exposed;

8.经1-7步骤可完成TEER微电极阵列制备，神经元场电位微电极阵列制备需额外进行金属Ag薄膜的沉积在1-7步骤的基础上，重复进行1-5步骤，4步骤中电子束蒸发的金属为厚度200nm的Ag，Ag薄膜经氯化处理形成Ag/AgCl电极。8. The preparation of TEER microelectrode arrays can be completed through steps 1-7. The preparation of neuron field potential microelectrode arrays requires additional deposition of metal Ag thin films. On the basis of steps 1-7, steps 1-5 are repeated, and steps 4 The metal evaporated by the electron beam is Ag with a thickness of 200nm, and the Ag film is chlorinated to form an Ag/AgCl electrode.

实施例2Example 2

一种类人视网膜芯片及其微电极阵列传感系统，有六个独立的系统分别为S1、S2、S3、S4、S5和S6，其细胞培养方式为：A human retina-like chip and its microelectrode array sensing system, there are six independent systems respectively S1, S2, S3, S4, S5 and S6, and the cell culture method is as follows:

S1中上层腔室2仅进行培养基的灌注，下层腔室6进行视锥细胞和培养基的灌注；S2中上层腔室2仅进行培养基的灌注，下层腔室6进行视杆细胞和培养基的灌注；S3中上层腔室2进行毛细血管内皮细胞和培养基的灌注，下层腔室6进行视锥细胞和培养基的灌注；S4中上层腔室2进行毛细血管内皮细胞和培养基的灌注，下层腔室6进行视杆细胞和培养基的灌注；S5中上层腔室2进行毛细血管内皮细胞和培养基的灌注，下层腔室6进行视锥细胞、视杆细胞和培养基的灌注；S6中上层腔室2进行毛细血管内皮细胞和培养基的灌注，下层腔室6进行视锥细胞、视杆细胞、Müller细胞和培养基的灌注；In S1, theupper chamber 2 is only perfused with medium, and the lower chamber 6 is used for perfusion of cone cells and medium; in S2, theupper chamber 2 is only perfused with medium, and the lower chamber 6 is used for rod cells and culture In S3,upper chamber 2 is used for perfusion of capillary endothelial cells and medium, and lower chamber 6 is used for perfusion of cone cells and medium; in S4,upper chamber 2 is used for perfusion of capillary endothelial cells and medium Perfusion, the lower chamber 6 is used for perfusion of rod cells and medium;upper chamber 2 of S5 is used for perfusion of capillary endothelial cells and medium, and the lower chamber 6 is used for perfusion of cone cells, rod cells and medium ; In S6, theupper chamber 2 is used for perfusion of capillary endothelial cells and medium, and the lower chamber 6 is used for perfusion of cone cells, rod cells, Müller cells and medium;

上层芯片引入光照，光线通过毛细血管内皮细胞，进入下层芯片；毛细血管内皮细胞的生长连接程度会影响进入下层芯片的光线强度；毛细血管内皮细胞的生长连接程度可通过TEER来刻画，当人视网膜内皮细胞生长良好、连接紧密时，TEER较大，反之亦然；当光线进入下层芯片，会引起人视网膜神经元场电位的变化，衬底上的微电极可实时检测电位变化。The upper chip introduces light, and the light passes through the capillary endothelial cells and enters the lower chip; the growth and connection degree of the capillary endothelial cells will affect the light intensity entering the lower layer chip; the growth and connection degree of the capillary endothelial cells can be described by TEER, when the human retina When the endothelial cells grow well and connect tightly, the TEER is larger, and vice versa; when the light enters the underlying chip, it will cause changes in the field potential of human retinal neurons, and the microelectrodes on the substrate can detect the potential changes in real time.

本发明模拟人视网膜神经血管单元组织结构，构建类人视网膜芯片，可采集光照影响下多细胞体系的数据信息，可能会在非线性映射(从不同的特征绑定照明和细胞屏障图形输入到四细胞rhBNN-rhBNN⁺网络的诱发场电位输出)、生成性对抗网络(从视杆细胞暗视觉到视锥细胞明视觉、神经胶质到神经元、抗炎到促炎)与信息和人体安全、网络结构自动组织与演化、从神经元到模块功能特异化、小/弱标记/无标记样本学习(与相关脑底物的比较或联系)和/或可解释性(特别是对于可解释的人工智能)方面获得相应认识。The present invention simulates the organizational structure of the human retinal neurovascular unit, constructs a human-like retinal chip, and can collect data information of a multicellular system under the influence of light, which may be processed in nonlinear mapping (from different features bound to illumination and cell barrier graphics input to four evoked field potential output of cellular rhBNN-rhBNN⁺ network), generative adversarial network (from rod scotopic to cone photopic, glial to neuron, anti-inflammatory to pro-inflammatory) and information and human safety, Automatic organization and evolution of network structures, functional specialization from neurons to modules, learning from small/weakly labeled/unlabeled samples (comparison or connection with relevant brain substrates) and/or interpretability (especially for interpretable artificial Intelligence) to gain corresponding understanding.

以上所述实施例仅表达本发明的实施方式，但并不能因此而理解为对本发明专利的范围的限制，应当指出，对于本领域的技术人员来说，在不脱离本发明构思的前提下，还可以做出若干变形和改进，这些均属于本发明的保护范围。The above-mentioned embodiment only expresses the implementation mode of the present invention, but can not therefore be interpreted as the limitation of the scope of the patent of the present invention, it should be pointed out that, for those skilled in the art, under the premise of not departing from the concept of the present invention, Several modifications and improvements can also be made, all of which belong to the protection scope of the present invention.

Claims (8)

Translated fromChinese

1.一种类人视网膜芯片，其特征在于，类人视网膜芯片的PDMS腔室(1)中包括六个独立的系统组成，每个系统均包括上层芯片、下层芯片和中间多孔膜(5)，所述上层芯片包括上层入口(3)、上层腔室(2)、上层出口(4)，上层入口(3)和上层出口(4)通过上层腔室(2)连通，所述上层入口(3)和上层出口(4)分别位于上层腔室(2)两侧，所述下层芯片包括下层入口(7)、下层腔室(6)、下层出口(8)，下层入口(7)和下层出口(8)通过下层腔室(6)连通，所述下层入口(7)和下层出口(8)分别位于下层腔室(6)两侧，上层入口(3)位置高于上层出口(4)位置，下层入口(7)位置高于下层出口(8)位置，上层入口(3)、上层出口(4)和下层入口(7)、下层出口(8)呈十字交叉，上层入口(3)、上层出口(4)和下层入口(7)、下层出口(8)通道直径均大于等于100μm，上层腔室(2)和下层腔室(6)内的代谢产物通过渗透作用穿过中间多孔膜(5)，实现细胞非接触式信息交流。1. A human retinal chip is characterized in that the PDMS chamber (1) of the human retinal chip comprises six independent systems, each system comprising an upper chip, a lower chip and a middle porous membrane (5), The upper layer chip comprises an upper layer inlet (3), an upper layer chamber (2), an upper layer outlet (4), and the upper layer inlet (3) and the upper layer outlet (4) communicate through the upper layer chamber (2), and the upper layer inlet (3 ) and the upper layer outlet (4) are respectively located at the upper layer chamber (2) both sides, and the lower layer chip includes the lower layer inlet (7), the lower layer chamber (6), the lower layer outlet (8), the lower layer inlet (7) and the lower layer outlet (8) communicated through the lower chamber (6), the lower inlet (7) and the lower outlet (8) are respectively located on both sides of the lower chamber (6), and the upper inlet (3) is higher than the upper outlet (4) , the position of the lower entrance (7) is higher than that of the lower exit (8), the upper entrance (3), the upper exit (4) and the lower entrance (7), the lower exit (8) form a cross, the upper entrance (3), the upper The channel diameters of the outlet (4), the lower inlet (7), and the lower outlet (8) are all greater than or equal to 100 μm, and the metabolites in the upper chamber (2) and the lower chamber (6) pass through the middle porous membrane (5) through osmosis. ), realizing non-contact information exchange between cells.2.根据权利要求1所述的类人视网膜芯片，其特征在于，所述上层腔室(2)的高度大于等于120μm、长度大于等于350μm、宽度大于等于350μm。2. The human retinal chip according to claim 1, characterized in that, the height of the upper chamber (2) is greater than or equal to 120 μm, the length is greater than or equal to 350 μm, and the width is greater than or equal to 350 μm.3.根据权利要求1所述的类人视网膜芯片，其特征在于，所述下层腔室(6)的高度大于等于120μm、长度大于等于320μm、宽度大于等于320μm。3. The human retinal chip according to claim 1, characterized in that, the height of the lower chamber (6) is greater than or equal to 120 μm, the length is greater than or equal to 320 μm, and the width is greater than or equal to 320 μm.4.根据权利要求1所述的类人视网膜芯片，其特征在于，所述中间多孔膜(5)的厚度为10μm，长度大于等于350μm、宽度大于等于350μm，孔径为0.4、1、3或10μm，相邻孔间距为5μm，孔密度为4×10⁶/cm²，中间多孔膜(5)为聚对苯二甲酸乙二醇酯PET，中间多孔膜(5)尺寸的设定与上层腔室(2)的尺寸对应。4. The human retina chip according to claim 1, characterized in that, the thickness of the intermediate porous membrane (5) is 10 μm, the length is greater than or equal to 350 μm, the width is greater than or equal to 350 μm, and the aperture is 0.4, 1, 3 or 10 μm , the distance between adjacent holes is 5 μm, the hole density is 4×10⁶ /cm² , the middle porous membrane (5) is polyethylene terephthalate PET, the size setting of the middle porous membrane (5) is the same as that of the upper chamber The dimensions of the chamber (2) correspond.5.根据权利要求1所述的类人视网膜芯片，其特征在于，所述六个独立的系统整个核心部件面积≥1070×1820μm²，其中相邻两个系统的间距≥400μm。5. The human retinal chip according to claim 1, wherein the area of the entire core components of the six independent systems is ≥1070×1820 μm² , and the distance between two adjacent systems is ≥400 μm.6.一种微电极阵列传感系统，采用权利要求1-5任一项权利要求所述的类人视网膜芯片，其特征在于，所述上层芯片和下层芯片上分别嵌入微电极，实现细胞屏障TEER和神经元场电位的测量，包括用于测量TEER的3对微电极，每一个系统的下层出口(8)正上方放置一个下层TEER电极(10)，下层TEER电极(10)位于中间多孔膜(5)下侧表面、下层腔室(6)对侧顶角各放置一个下层TEER电极(10)，共三个下层TEER电极(10)，上层腔室(2)放置的三个上层TEER电极(9)与下层腔室(6)的三个下层TEER电极(10)位置对应，用于测量神经元场电位信号，采用三电极体系，每个下层芯片的硅基衬底(15)集成11个微电极，包括1个Pt对电极(12)、1个Ag/AgCl参比电极(13)和9个Pt工作电极(11)，每个独立的系统中上层腔室(2)、下层腔室(6)的总电极数量为17，整个芯片系统总电极数为102个，Pt工作电极(11)与电极导线(14)连接。6. A microelectrode array sensing system, adopting the human retina chip described in any one of claims 1-5, characterized in that microelectrodes are respectively embedded on the upper chip and the lower chip to realize cell barrier Measurement of TEER and neuron field potential, including 3 pairs of microelectrodes for measuring TEER, a lower TEER electrode (10) is placed directly above the lower outlet (8) of each system, and the lower TEER electrode (10) is located in the middle porous membrane (5) One lower TEER electrode (10) is placed on the lower side surface and the upper corner of the lower chamber (6), and there are three lower TEER electrodes (10) in total, and three upper TEER electrodes placed in the upper chamber (2) (9) corresponds to the positions of the three lower TEER electrodes (10) of the lower chamber (6), and is used to measure neuron field potential signals. A three-electrode system is adopted, and the silicon-based substrate (15) of each lower chip is integrated with 11 Microelectrodes, including 1 Pt counter electrode (12), 1 Ag/AgCl reference electrode (13) and 9 Pt working electrodes (11), in each independent system, the upper chamber (2), the lower chamber The total number of electrodes in the chamber (6) is 17, the total number of electrodes in the whole chip system is 102, and the Pt working electrode (11) is connected with the electrode wire (14).7.根据权利要求6所述的微电极阵列传感系统，其特征在于，所述的用于细胞屏障测量的上层TEER电极(9)、下层TEER电极(10)，上层TEER电极(9)、下层TEER电极(10)的尺寸均为20μm*30μm，电极材料为Ti/Pt，电极厚度为230nm，电极材料Pt与外界直接接触，三对电极的设置在不同位置进行多点数据测量，通过平均计算获取TEER值。7. microelectrode array sensing system according to claim 6, is characterized in that, described upper strata TEER electrode (9), lower strata TEER electrode (10) that is used for cell barrier measurement, upper strata TEER electrode (9), The size of the lower TEER electrode (10) is 20 μm*30 μm, the electrode material is Ti/Pt, the electrode thickness is 230nm, the electrode material Pt is in direct contact with the outside world, and the three pairs of electrodes are set at different positions for multi-point data measurement. Calculate and obtain the TEER value.8.根据权利要求6所述的微电极阵列传感系统，其特征在于，所述用于测量神经元电场电位的微电极，Pt工作电极(11)尺寸为20μm*20μm，电极材料为Ti/Pt，厚度为230nm，Pt对电极(12)和Ag/AgCl参比电极(13)位于Pt工作电极(11)两侧，Pt对电极(12)和Ag/AgCl参比电极(13)的尺寸均为100μm*20μm，Pt对电极(12)材料为Ti/Pt，厚度为230nm，Ag/AgCl参比电极(13)材料为Ti/Pt,Ag/AgCl，厚度为430nm。8. microelectrode array sensing system according to claim 6, is characterized in that, described microelectrode that is used to measure neuronal electric field potential, Pt working electrode (11) size is 20 μ m*20 μ m, and electrode material is Ti/ Pt, the thickness is 230nm, the Pt counter electrode (12) and the Ag/AgCl reference electrode (13) are located on both sides of the Pt working electrode (11), the size of the Pt counter electrode (12) and the Ag/AgCl reference electrode (13) Both are 100 μm*20 μm, the material of the Pt counter electrode (12) is Ti/Pt, and the thickness is 230nm, and the material of the Ag/AgCl reference electrode (13) is Ti/Pt, Ag/AgCl, and the thickness is 430nm.

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