WO2003056321A1 - Dispositif pour stimuler une cellule animale et enregistrer son signal physiologique, procedes de production et d'utilisation correspondants - Google Patents

Dispositif pour stimuler une cellule animale et enregistrer son signal physiologique, procedes de production et d'utilisation correspondants Download PDF

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Publication number
WO2003056321A1
WO2003056321A1 PCT/CN2002/000856 CN0200856W WO03056321A1 WO 2003056321 A1 WO2003056321 A1 WO 2003056321A1 CN 0200856 W CN0200856 W CN 0200856W WO 03056321 A1 WO03056321 A1 WO 03056321A1
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Prior art keywords
recording
conductive polymer
physiological signals
polymer layer
animal cells
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PCT/CN2002/000856
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English (en)
French (fr)
Inventor
Wanli Xing
Zhe Yu
Guangxin Xiang
Liangbin Pan
Jing Cheng
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Capital Biochip Company, Ltd.
Tsinghua University
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Application filed by Capital Biochip Company, Ltd., Tsinghua University filed Critical Capital Biochip Company, Ltd.
Priority to EP02785015A priority Critical patent/EP1464952B1/en
Priority to AU2002354331A priority patent/AU2002354331A1/en
Priority to JP2003556795A priority patent/JP2005514019A/ja
Priority to US10/499,098 priority patent/US7632674B2/en
Priority to DE60220947T priority patent/DE60220947T2/de
Publication of WO2003056321A1 publication Critical patent/WO2003056321A1/zh

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54366—Apparatus specially adapted for solid-phase testing
    • G01N33/54373—Apparatus specially adapted for solid-phase testing involving physiochemical end-point determination, e.g. wave-guides, FETS, gratings
    • G01N33/5438—Electrodes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M35/00—Means for application of stress for stimulating the growth of microorganisms or the generation of fermentation or metabolic products; Means for electroporation or cell fusion
    • C12M35/02—Electrical or electromagnetic means, e.g. for electroporation or for cell fusion
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/10—Screening for compounds of potential therapeutic value involving cells

Definitions

  • the invention relates to a device capable of stimulating animal cells and recording their physiological signals, and a method for producing and using the device, and particularly to a device capable of electrically stimulating animal cells and recording their physiological signals, and the device. Production method and use method of the device.
  • Controlling the growth and differentiation of nerve cells is conducive to in-depth study of the mechanism of the nervous system, treatment of certain diseases of the nervous system, and effective repair of nerve tissue damage. Therefore, it has been a research subject for a long time.
  • Conductive polymers are a new class of materials whose optoelectronic properties can be adjusted over a wide range and can be reversibly adjusted. Conductive polymers are stable in nature and can function for long periods in cell physiological culture fluids or body fluids, and have good biocompatibility with nerve cells [CE Schmidt, VR Shastri, JP Vacant i, R. Langer, Stimu lation of neurite outgrowth using an electrically conducting polymer, Proc. Natl. Acad. Sci. USA, 1997, 94, 8948-8953] 0 In recent years, studies have shown that conducting electrical stimulation with conductive polymers as the energizing medium can enhance the growth of nerve processes. [A.
  • microelectrodes and their arrays to record the electrophysiological signals of nerve cells and their networks extracellularly has been studied since the late 1970s, and has developed more rapidly in recent years.
  • One way is to insert multiple microelectrodes into living cells. Extracellular measurements were performed in vivo, and the electrode shapes were each needle-like and multiple electrodes were shaped as needle clusters [E. Ferna ndez, JM Ferrandez, J. Ammermuller, RA Normann, Population coding in spike trains of simultaneously recorded retinal ganglion cells, Brain Research, 2000, 887, 222-229; DJ Warren, E.
  • Jimbo Activity-dependent enhancement in the reliability of correlated spike timings in cultured cortical neurons, Biological Cybernetics, 1999, 80, 45-55; MP Maher, J. Pine, J. Wright, YC Tai, The neurochip: a new multielectrode device for stimulating and recording from cultured neurons, Jounal of Neuroscience Methods, 1999, 87, 45 -56.];
  • the third way is to make the overall shape of the microelectrode array with embryonic nerve cells of the second way into a needle shape, insert it into the living body, and observe the integration of the cells on the electrode array with the host cells and Information exchange situation [J. Pine, M. Maher, S. Potter, YC Tai, S. Tatic-Lucic, J.
  • the advantage of performing measurements in the body is that the position and culture conditions of the cells can be controlled artificially, so various functions of nerve cells can be clearly and conveniently observed and studied. However, it is difficult to control the growth direction of neurites, and it is often allowed to grow randomly. This is often not conducive to forming an effective neural network between multiple neural cells cultured in vitro to record the transmission of electrophysiological signals, and it is not conducive to implantation Integration and exchange of information between nerve cells and the main nervous system. Some researchers have used mechanical restraints to make neurites grow in the channels, but this restriction will affect the normal growth of the neurites.
  • the object of the present invention is to provide a device capable of effectively, conveniently and accurately stimulating animal cells and recording their physiological signals.
  • the technical solution adopted by the present invention is: a device capable of stimulating animal cells and recording its physiological signals, which includes a substrate of a non-conducting conductor, and at least one unit of at least one side of the substrate is provided with a polymer substrate.
  • an intermediate layer is provided between the substrate and the conductive polymer layer.
  • the intermediate layer is made of gold or platinum.
  • One of its functions is to function as a carrier in electropolymerization, and the other function is that the conductive polymer can be attached to it well.
  • the matrix may be a two-dimensional plane, or a pit for cell positioning may be provided on the matrix. . '
  • the matrix material can be rigid or elastic, and can be selected from silicon, glass, polymers or metal oxides.
  • the role of the conductive polymer layer is mainly to guide the growth of neurites, so its pattern is usually a grid of broken nodes, and the nodes of the grid place nerve cells, so that the protrusions of nerve cells can run along the conductive polymer layer. Patterns grow and exchange information with each other, because the diameter of neurites is usually 1-2 microns, so the width of each strip of conductive polymer is usually less than 5 microns, and the strips are too wide or too narrow to provide good guidance.
  • the principle of the conductive polymer pattern is to facilitate the connection of the protrusions of each nerve cell to form a neural network.
  • the conductive polymer layer is a plurality of units, and each unit may be connected to each other and share a pair of power-on electrodes, or they may not be connected to each other, each having a pair of power-on electrodes. If they are not connected to each other, the power-on parameters of the conductive polymer layer units (such as the current intensity, the length of time, the range of change, and the frequency) may be the same or different from each other.
  • the thickness of the conductive polymer layer may be uniform or non-uniform.
  • the thickness of the conductive polymer layer can be between tens of nanometers and several millimeters. In order to facilitate observation with an inverted microscope in vitro, it is better to control the thickness of the conductive polymer to less than 500 nanometers, which not only can ensure high light transmittance, At the same time, it can ensure good adhesion between the polymer and the matrix.
  • the raw material for manufacturing the conductive polymer layer is one of a family of polyaniline, polypyrrole, polythiophene, and derivatives, copolymers or mixtures thereof.
  • the conductive properties of conductive polymers can be adjusted in space and time as needed.
  • the conductive polymer layer can be adjusted to a non-conductive state or maintained in a conductive state, provided that it does not adversely affect the measurement of the electrophysiological signals.
  • micro-electrodes There are several micro-electrodes, and they form a micro-electrode array.
  • the microelectrode is made of gold, platinum or indium tin oxide ( ⁇ 0).
  • the conductive polymer layer and the microelectrode may be connected or may not be connected. When not connected, there is a distance between 1 micrometer and 50 micrometers between the conductive polymer layer and the microelectrode. The distance between the conductive polymer of each unit and the microelectrode may be the same or inconsistent.
  • the part of the substrate other than the conductive polymer and non-cellular body growing and microelectrode measurement is plated with a layer of insulating material.
  • the isolation material layer is made of a non-conductive material (such as polyimide and other materials with good biocompatibility) that has no toxic effect on cells, and the thickness is generally 5 to 100 microns.
  • a method for producing a device for stimulating animal cells and recording their physiological signals is to deposit a good conductive microelectrode and its lead on a substrate by evaporation or sputtering, and then pass on the substrate
  • PVD, CVD, electropolymerization, or macromolecular self-assembly methods are used to deposit at least one unit of a conductive polymer layer with a design pattern required; a portion of the metal lead is deposited under the conductive polymer layer to be connected to the power electrode.
  • the method for using a device for stimulating animal cells and recording their physiological signals is to continuously or intermittently energize a conductive polymer.
  • the electrical stimulation can be direct current or alternating current, and the current intensity is in the range of pA to mA.
  • its stimulation frequency is L-10 6 Hz; through the microelectrode, measure the physiological signal generated in the measured cell by means of current, potential or impedance electrical signal.
  • the device for stimulating animal cells and recording its physiological signals may have a shape of two-dimensional square, circle, and various irregular shapes, or a three-dimensional hollow cylinder, sphere, cube, or cuboid. , Cone and any of a variety of irregular solids.
  • the invention is further described below with reference to the drawings and specific embodiments.
  • FIG. 1 is a schematic partial structure diagram of the device of the present invention.
  • Fig. 2 is an external view of a device according to the present invention.
  • Fig. 3 is a partially enlarged view of a microelectrode array.
  • Figure 4 is a cross-sectional view of a single culture unit used for studying neural networks in vitro.
  • Figure 5 is a perspective view of a device of the present invention for implantation in a body.
  • a layer of 300 micrometers in thickness with a design required by design is processed with gold using conventional photolithography methods. Partially connected) and leads.
  • the electrified electrode was energized, and a layer of 100-nm-thick polyaniline conductive polymer was polymerized on the gold surface connected to the electrified electrode by electrochemical polymerization to form a desired pattern.
  • An isolation layer is formed on the surface by a spin coating method, and a desired structure is formed by a photolithography method.
  • a device of the tritium agonist cell of the present invention and a physiological signal can be obtained.
  • the range of selecting a substrate is wide, as long as it is a poor conductor, the thickness and shape of the substrate can be determined according to actual needs. In order to effectively use space, the above fabrication can be performed on multiple faces of the substrate.
  • the electrode layer can also be made of a good electrical conductor such as platinum or IT0, and the thickness is preferably between 30 and 400 nanometers.
  • the choice of the conductive polymer layer material is also large, and it may be one of polyaniline, polypyrrole, polythiophene and its derivatives, copolymers or mixtures depending on the needs.
  • the processing of electrodes and conductive polymer layers, as well as the formation of cabinets, can also use other coating and photolithographic methods.
  • the device for stimulating animal cells and recording their physiological signals includes a square silicon wafer substrate 4 having a thickness of 400 micrometers; and a 100-nm-thick polypyrrole conductive polymer disposed on the substrate 4
  • the object layer 3 and the conductive polymer layer 3 have two electrode lead-out ends (not shown in the figure); sixteen platinum microelectrodes 2 provided at a thickness of 60 nanometers on the substrate 4, and each microelectrode 2 is provided with an output end ( Not shown in the figure); there is a distance of 25 micrometers between the conductive polymer layer 3 and the microelectrode 2; the portion of the substrate 4 other than the conductive polymer layer 3 and the non-cellular growth and microelectrode measurement are provided with polyimide isolation Material layer 1, which has a thickness of 10 micrometers and forms grooves with a width of 3-7 micrometers to prevent cells from escaping.
  • a small culture cavity 5 made of a material with good biocompatibility (such as polyimide) is combined on the base layer 4 to hold a culture liquid, and the height of the culture cavity 5 is usually greater than 100. Millimeters, the area of which depends on the number and distribution of electrodes contained in the microelectrode array in the device.
  • an intermediate layer may be provided between the substrate and the conductive polymer layer (not shown in the figure).
  • the shape and size of the device are determined according to actual needs, and a conductive polymer layer and microelectrodes are provided on the specific surface or surfaces of the substrate.
  • a conductive polymer layer and microelectrodes are provided on the specific surface or surfaces of the substrate.
  • conductive polymer layers 3 and microelectrodes 2 are provided on both sides of the substrate 4, and nerve cells can be placed on both sides to increase utilization and cells.
  • Implant density As shown in FIG. 5, the surface of the implantable device of the present invention is provided with a covering layer 6. Its function is to prevent cells from being damaged during the insertion into the body, and to prevent the cells from escaping in the body.
  • the material is biocompatible, Non-conductive materials with good stability (such as polyimide) are preferred.
  • the nerve cells 7 are placed on the device of the present invention, and the conductive polymer 3 is intermittently energized.
  • the electrical stimulation can be direct current or alternating current, and the excitation frequency is 1 to 10 3 ⁇ 4z;
  • the growth of the cells in the energized group and the control group were compared, such as cell size, protrusion length and other indicators.
  • the microelectrodes were used to measure changes in the extracellular membrane potential of the cells to study the response of the cells to various stimuli.
  • Example 4 Research on neural network implanted in vivo using the device of the present invention
  • the implantable device of the present invention as shown in FIG. 5 is implanted in the body.
  • the damaged nerve fibers are connected through the neural cell network cultured in the device, and the device measures the response of neurons in local neural tissues to external stimuli in vivo ( (Including stimulations such as electricity and drugs); at the same time, the device can be used to perform electrical stimulation of local nerve tissues to stimulate the repair and regeneration of damaged nerves.
  • the present invention cleverly applies the latest developments in the fields of conductive polymers and microelectronic processing technology to neuroscience, and achieves effective control of the growth direction and growth rate of neurites through electrical stimulation based on conductive polymers, and can more effectively record neural networks in vitro Electrophysiological signals make it possible to better understand the information transmission and response mechanisms of neural networks.
  • the implanted nerve cells can be more effectively integrated with the main nervous system, and then repair the damaged nerve tissue. Important application value.
  • the device can also be applied to treat neurological diseases such as urinary incontinence and retinal damage.
  • the device of the present invention is not only suitable for researching cells taken from different parts of various animal nerve tissues, but also for researching cells derived from non-neural cell differentiation.

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Description

剌激动物细胞并记录其生理信号的装置及其生产使用方法
技术领域
本发明涉及一种能够对动物细胞进行剌激并记录其生理信号的装置以 及该装置的生产方法和使用方法， 特别是涉及一种能够对动物细胞进行电 刺激并记录其生理信号的装置以及该装置的生产方法和使用方法。
背景技术
对神经细胞的生长和分化进行控制有利于对神经系统的机理进行深入的 研究， 对某些神经系统的疾病进行治疗， 对神经组织的损伤进行有效的修 复。 因此长期以来一直是人们致力研究的课题。
经过多年的探索， 人们发现电刺激可在体外增强神经突起的伸展， 在 体内能够增强神经细胞的再生。 有研究表明神经突起的伸展可以在压电材 料表面得至 lj增强 [Aebis cher, et al， Piezoelectric guidance channels enhance regeneration in the mouse sciatic nerve after axotomy, Brain Research, 1987， 436 (1) , 165-168]， 其机理是由于当 材料受到微小的机械压力时， 材料中产生表面束缚电荷。 这些现象的机理 尚不很清楚， 一种推理是， 某些对神经突起生长起重要作用的蛋白质或其 它分子在电场作用下重新分布， 或这些蛋白质的构型发生有利于神经突起 生长的变化。
导电聚合物是一类新型材料， 它的光电性质可以在很大范围内进行调 节， 且可以可逆地调节。 导电聚合物的性质稳定， 可以在细胞生理培养液 或体液中长期作用， 与神经细胞的生物兼容性好 [C. E. Schmidt ， V. R. Shastri, J. P. Vacant i, R. Langer， Stimu lation of neurite outgrowth using an electrically conducting polymer, Proc. Natl. Acad. Sci. USA, 1997, 94, 8948-8953] 0 近年来， 有研究表明以导电聚 合物为通电介质进疔电刺激， 可以对神经突起的生长起到增强作用 [A. Kotwal, C. E. Schmidt, Electrical stimulation alters protein adsorption and nerve cell interactions with electrically conducting bi ornate rials, Bioraaterials, 2001， 22, 1055-1064 ; C. E. Schmidt , V. R. Shastri, J. P. Vacant i, R. Langer， Stimulation of neurite outgrowth using an electrically conducting polymer, Proc. Natl. Acad. Sci. USA, 1997, 94, 8948- 8953 ] 。 已有学者研究将其作为支架植入体内， 通过电剌激使断开的神经 组织在此支架的导引下连接在一起 [V. R. Shastri, C. E. Schmidt, R. S. Langer, J. P. Vacant i, US Patent, 6095148, 2000, August 1]。 利用微电极及其阵列在胞外记录神经细胞及其网络的电生理信号自二十世 纪 70年代末就有人开始研究， 近年来得到更快速的发展， 一种方式是将多 个微电极插入活体内进行胞外测量， 其电极形状既有每个电极呈针状， 多 个电极形为针簇 [E. Ferna ndez, J. M. Ferrandez, J. Ammermuller, R. A. Normann, Population coding in spike trains of simultaneously recorded retinal ganglion cells, Brain Research, 2000, 887， 222-229 ; D. J. Warren, E. Fernandez, R. A. Normann, High-resolution two-dimensional spatial mapping of cat striate cortex using a 100-raicroelectrode array, Neuroscience, 2001, 105 (1)， 19-31 ; P. J. Rousche, R. S. Petersen, S. Battiston, S. Giannotta, M. E. Diamond, Examination of the spatial and temporal distribution of sensory cortical activity using a 100- electrode array, Journal of Neuralscience Methods, 1999, 90, 57- 66] , 也有整体制成扁平的锥形， 每个电极是锥形平面上的一个平面电极 [G. Ensell, D. J. Banks, P. R. Richards, W. Balachan dran, D. J. Ewins, Silicon-based microelectrodes for neurophysiology, micromachined form silicon- on- insulator wafers ， Medical & Biological Engineering & Computing, 2000, 38， 175-179]； 另一种方 式是制成二维的微电极阵列芯片在体外培养细胞进行多个神经细胞的同时 测量 [Y. Jimbo, A. Kawana, P. Parodi, V. Torre, The dynamics of a neuronal culture of dissociated cortical neurons of neonatal rats, Biological Cybernetics, 2000, 83, 1-20 ; T. Tateno, Y. Jimbo, Activity-dependent enhancement in the reliability of correlated spike timings in cultured cortical neurons, Biological Cybernetics, 1999， 80, 45-55 ; M. P. Maher, J. Pine, J. Wright, Y. C. Tai, The neurochip : a new multielectrode device for stimulating and recording from cultured neurons, Jounal of Neuroscience Methods, 1999, 87, 45-56. ] ; 第三种方式是将带有胚胎 神经细胞的第二种方式的微电极阵列的整体外形制成针状， 插入活体内， 观察电极阵列上的细胞与主体细胞的整合情况和信息交流情况 [J. Pine, M. Maher, S. Potter, Y. C. Tai, S. Tatic-Lucic, J. Wright, A cultured neuron probe, Proceedings of IEEE- EMBS Annual Meeting, Araersterdam, The Netherlands, 1996， Nov.， paper #421]。 在体夕卜进 行测量的优点是可以人为地控制细胞的位置和培养条件， 因此可以清晰、 方便地对神经细胞的各种功能进行观察和研究。 但是， 对于神经突起的生 长方向却较难控制， 常常任其随机生长， 这往往不利于在多个体外培养的 神经细胞间形成有效的神经网络以记录电生理信号的传导， 也不利于植入 神经细胞与主体神经系统的整合和信息交流。 有研究者采用机械限制的方 法， 使神经突起在沟道内生长， 但这种限制会影响突起的正常生长。 还有 研究者利用诸如金属氧化物的材料构成的特定图案来研究这些材料对神经 细胞突起生长的导向性作用 [Yasuhiko Jimbo, P. C. Robinson, Akio Kawana, Simultaneous Measurement of Intracellar Calcium and Electrical Activity form Patterned Neural Networks in Culture, IEEE transaction on biomedical engineering, 1993, 40 (8)， 804- 810 ]。
发明公开
本发明的目的是提供一种能够有效、 方便、 准确地对动物细胞进行剌激 并记录其生理信号的装置。
为实现上述目的， 本发明采用的技术方案是： 一种能够刺激动物细胞 并记录其生理信号的装置， 它包括非良导体的基质， 在所述基质的至少一 面上设有至少一个单元具有加电功能的导电聚合物层和至少一个良导体微 电极。 为了使导电聚合物在培养液或体液长期浸泡的环境中不至于脱落， 在 所述基质与导电聚合物层之间设有中间层。 所述中间层为金或铂制成， 其 作用之一是在电聚合中起载体作用， 另一个作用是导电聚合物可以很好地 贴附其上。
所述基质可以是二维平面， 也可以在基质上设有用于细胞定位的凹 坑。 . '
制作基质材料可以是刚性的也可以是弹性的， 可以选自硅、 玻璃、 聚 合物或金属氧化物。
导电聚合物层的作用主要是为神经突起的生长起导向作用， 所以其图 案通常呈节点断开的网格状， 网格的节点放置神经细胞， 这样神经细胞的 突起可以沿导电聚合物层的图案生长、 彼此进行信息交流， 因为神经突起 的直径通常为 1-2微米， 因此导电聚合物的每根条带的宽度通常小于 5微 米， 条带太宽或太窄均不能起到良好的导向作用， 导电聚合物图案的原则 是便于各神经细胞的突起相连形成神经网络。
所述导电聚合物层为若干个单元， 各单元可以彼此相连共用一对加电 电极， 也可以彼此不相连， 各自拥有一对加电电极。 如果彼此不相连， 各 导电聚合物层单元的加电参数 （如电流强度、 时间长短、 变化幅度和频 率） 可以彼此相同也可不同。
所述导电聚合物层的厚度可以是均一的， 也可以是不均一的。 导电聚 合物层的厚度可以在几十纳米至几毫米间， 为便于在体外用倒置显微镜观 察， 将导电聚合物的厚度控制在 500 纳米以下较好， 这样不但可以保证较 高的透光率， 同时可以保证聚合物同基质之间较好的附着性 。
制造所述导电聚合物层的原料为聚苯胺、 聚吡咯、 聚噻吩及其衍生 物、 共聚物或混合物家族中的一种。 导电聚合物的导电性质可以根据需要 在空间和时间上进行调节。 在用微电极进行电生理信号记录时， 可以将导 电聚合物层调节为非导电状态， 也可以保持为导电状态， 其前提是对电生 理信号的测量不产生不良的影响。
所述微电极为若干个， 并组成微电极阵列。 制作微电极的材料为金、 铂或铟锡氧化物 （Π0) 。
所述导电聚合物层和所述微电极可以是相连的， 也可以是不相连的， 当不相连时， 所述导电聚合物层和所述微电极之间具有 1微米至 50微米的 距离。 各单元导电聚合物与微电极之间的距离可以是一致的， 也可以是不 一致的。
为了防止培养细胞逃逸， 所述基质上导电聚合物以外且非细胞体生长 及微电极测量的部分镀有隔离材料层。 隔离材料层由对细胞无毒性作用的 非导体材料 （如聚酰亚胺等生物兼容性较好的材料） 制成， 厚度一般为 5 一 100微米。
本发明的另一个目的是提供较简单地制造上述剌激动物细胞并记录其 生理信号的装置的方法。
一种生产刺激动物细胞并记录其生理信号的装置的方法， 是以蒸镀或 溅射的方式将良导体微电极及其引线沉积在基质上， 然后在基质上通过
PVD, CVD,电聚合或大分子自组装等方法沉积至少一个单元的具有设计需要 图案的导电聚合物层； 导电聚合物层下沉积一部分金属引线与加电电极连 接。
本发明的又一目的是提供使用剌激动物细胞并记录其生理信号的装置 的方法。
使用刺激动物细胞并记录其生理信号的装置的方法， 是将导电聚合物 连续或间歇通电， 电刺激可以是直流也可以是交流， 电流强度为 pA 至 mA 级， 对于交流电刺激， 其剌激频率为 l-106Hz ; 通过微电极， 以电流、 电势 或阻抗电信号的方式测量被测细胞中产生的生理信号。
本发明刺激动物细胞并记录其生理信号的装置， 其形状可以是二维的 方形、 圆形及各种不规则形中的任何一种， 也可以是三维的中空圆柱体、 球体、 正方体、 长方体、 锥形体及各种不规则立体中的任何一种。 下面结合附图及具体实施例对本发明做进一步说明。
附图说明
图 1为本发明装置的局部结构示意图。
图 2为本发明装置的外观图。
图 3为微电极阵列的局部放大图。
图 4为用于体外研究神经网络的单个培养单元剖面图。 图 5为用于植入体内的本发明装置立体图。
实施发明的最佳方式
实施例 1、 制造本发明装置
在厚度为 350 微米的四方形玻璃基质的一侧表面上， 用常规的光刻方 法用金加工一层厚度为 300 纳米， 具有设计需要图案的测量微电极， 加电 电极 （与聚合物形成图案部分相连） 和引线。
给加电电极通电， 用电化学聚合的方法在与加电电极相连的金表面聚 合一层厚 100纳米的聚苯胺导电聚合物， 形成所需的图案。
通过旋涂的方法在表面形成一层隔离层， 通过光刻的方法形成所需结 构。
经过上述步骤， 即可以得到本发明剌激动物细胞并记录其生理信号的 装置。
在该实施例中， 选择基质的范围很宽， 只要是不良导体均可， 基质的 厚度及形状可根据实际需要确定， 为了有效利用空间， 可以在基质的多个 面上进行上述制作。 电极层还可以用铂， IT0 等电的良导体制成， 厚度在 30 -400 纳米之间较好。 导电聚合物层材料的选择余地也很大， 根据需要 而定， 可以是聚苯胺、 聚吡咯、 聚噻吩及其衍生物、 共聚物或混合物中的 一种。 电极和导电聚合物层的加工， 以及阁案的形成还可以采用其他的镀 膜及光刻的方法。
实施例 2、 本发明的结构
如图 1、 图 2、 图 3所示， 本发明刺激动物细胞并记录其生理信号的装 置包括厚度为 400微米的四方形硅片基质 4; 设于基质 4上 100纳米厚的 聚吡咯导电聚合物层 3， 导电聚合物层 3 上有两个电极引出端 （图中未表 示） ； 设于基质 4上 60纳米厚的 16个铂微电极 2， 每个微电极 2都设有 输出端 （图中未表示） ； 导电聚合物层 3和微电极 2之间具有 25微米的距 离； 基质 4上导电聚合物层 3 以外且非细胞体生长及微电极测量的部分设 有聚酰亚胺隔离材料层 1， 该层厚度为 10微米， 形成宽度在 3— 7微米之 间的沟槽， 以防细胞逃逸。
如图 2 所示， 基层 4 上结合有用生物兼容性较好的材料 （如聚酰亚 胺） 制成的小培养腔 5, 用于盛纳培养液， 培养腔 5 的高度通常大于 100 毫米， 其面积根据装置中微电极阵列中所含电极的个数和分布而定。
在该实施例中， 为了使导电聚合物层在培养液或体液长期浸泡的环境 中不至于脱落， 在基质与导电聚合物层之间还可以设有中间层 （图中未显 示）
在该实施例中， 根据实际需要决定装置的形状、 大小及在基质的具体 那个面或那几个面上设置导电聚合物层和微电极。 如图 5 所示， 为便于植 入体内进行相应的研究， 在基质 4 的两侧表面均设有导电聚合物层 3、 微 电极 2， 两面都可以放置神经细胞， 以增大利用率和细胞植入密度。 如图 5 所示， 植入式的本发明的装置的表面设有覆盖层 6， 它的作用是防止细胞 在插入体内过程中损伤， 也为了防止细胞在体内逃逸， 材料为生物兼容性 好、 稳定性好的非导体材料为佳 （如聚酰亚胺） 。
实施例 3、 利用本发明的装置进行体外神经网络的研究
如图 4所示， 将神经细胞 7置于本发明的装置上， 将导电聚合物 3间 歇通电， 电刺激可为直流电或交流电， 剌激频率为 1- 10¾z; 通过微电极 2 测量被测细胞中产生的生理信号。
通过对神经细胞的间歇通电 （恒压 lOOmV) ，培养数天后， 对比通电组与 对照组细胞的生长情况， 如胞体尺寸， 突起长度等指标。 另外， 通过给与细 胞电、 化学等信号的刺激， 利用微电极测量细胞的胞外膜电位的变化， 研究 细胞对各种刺激的响应。
实施例 4、 利用本发明的装置植入体内对神经网络的研究
将如图 5所示本发明的植入式装置植入体内， 首先通过装置中培养的神 经细胞网络连接损伤的神经纤维， 通过该装置测量局部神经组织中神经元 在体内对外界刺激的响应 （包括电、 药物等刺激） ； 同时， 可以通过该装 置对局部神经组织进行电刺激， 刺激损伤神经的修复与再生。
工业应用
本发明巧妙地将导电聚合物和微电子加工技术领域的最新发展应用于 神经科学， 通过基于导电聚合物的电刺激达到有效控制神经突起生长方向 和生长速度， 可以更有效地在体外记录神经网络电生理信号， 使更深入了 解神经网络的信息传递和反应机理成为可能。 在体内可以使植入的神经细 胞更有效地与主体神经系统整合， 进而修复损伤的神经组织， 具有医学上 重要的应用价值。 同时该装置还可望应用于治疗尿失禁、 视网膜损伤等神 经方面的疾病。
本发明的装置不仅适用于对取自各种动物神经组织不同部位的细胞进 行研究， 也适用于对非神经细胞分化而得的细胞进行研究。

Claims

权利要求书 .
I、 刺激动物细胞并记录其生理信号的装置， 其特征在于它包括非良导 体的基质， 在所述基质的至少一面上设有具加电电极的至少一个单元导电 聚合物层和至少一个良导体微电极。
2、 根据权利要求 1所述的刺激动物细胞并记录其生理信号的装置， 其 特征在于： 在所述基质与导电聚合物层之间设有中间层。
3、 根据权利要求 2所述的剌激动物细胞并记录其生理信号的装置， 其 特征在于： 所述中间层为金或铂。
4、 根据权利要求 1所述的剌激动物细胞并记录其生理信号的装置， 其 特征在于： 所述基质为二维平面。
5、 根据权利要求 1所述的刺激动物细胞并记录其生理信号的装置， 其 特征在于： 所述基质上设有用于细胞定位的凹坑。
6、 根据权利要求 1或 2或 3或 4或 5所述的剌激动物细胞并记录其生 理信号的装置， 其特征在于： 所述导电聚合物层呈节点断开的网格状。
7、 根据权利要求 1或 2或 3或 4或 5所述的刺激动物细胞并记录其生 理信号的装置， 其特征在于： 所述导电聚合物层为若干个单元， 各单元彼 此相连共用一对加电电极。
8、 根据权利要求 1或 2或 3或 4或 5所述的刺激动物细胞并记录其生 理信号的装置， 其特征在于： 所述导电聚合物层为若干个单元， 各单元彼 此不相连， 各自拥有一对加电电极。
9、 根据权利要求 1所述的刺激动物细胞并记录其生理信号的装置， 其 特征在于： 所述导电聚合物层为聚苯胺、 聚吡咯、 聚噻吩及其衍生物、 共 聚物或混合物家族中的一种。
10、 根据权利要求 1 所述的剌激动物细胞并记录其生理信号的装置， 其特征在于： 所述微电极为若干个， 并组成微电极阵列。
II、 根据权利要求 1 所述的刺激动物细胞并记录其生理信号的装置， 其特征在于： 所述制作微电极的材料为金、 铂或铟锡氧化物。
12、 根据权利要求 1 所述的刺激动物细胞并记录其生理信号的装置， 其特征在于： 所述导电聚合物层和所述微电极相连。
13、 根据权利要求 1 所述的刺激动物细胞并记录其生理信号的装置， 其特征在于： 所述导电聚合物层和所述微电极之间具有 1微米至 50微米的 距离。
14、 根据权利要求 1 所述的刺激动物细胞并记录其生理信号的装置， 其特征在于： 所述基质上导电聚合物以外且非细胞体生长及微电极测量的 部分镀有隔离材料层。
15、 一种生产刺激动物细胞并记录其生理信号的装置的方法， 是以蒸 镀或溅射的方式将良导体微电极及其引线沉积在基质上， 然后在基质上通 过 PVD，CVD，电聚合或大分子自组装等方法沉积至少一个单元的具有设计需 要图案的导电聚合物层； 导电聚合物层下沉积一部分金属引线与加电电极 连接。
16、 根据权利要求 15所述的方法， 其特征在于： 在所示装置的表层上 加工一层隔离层， 光刻形成指定结构。
17、 根据权利要求 15所述的方法， 其特征在于： 在镀敷所述导电聚合 物层之前， 用光刻的方法在基质上加工一层具有设计需要图案的中间层。
18、 一种使用刺激动物细胞并记录其生理信号的装置的方法， 是将导 电聚合物连续或间歇通电， 电刺激可以是直流也可以是交流， 电流强度为 pA至 mA级， 对于交流电刺激， 其剌激频率为 l-10¾z ; 通过微电极， 用电 流、 电势或阻抗电信号的方式测量被测细胞中产生的生理信号。
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