WO2023246212A1 - 用于检测心肌细胞电信号的微针阵列电极及其制备方法 - Google Patents

用于检测心肌细胞电信号的微针阵列电极及其制备方法 Download PDF

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WO2023246212A1
WO2023246212A1 PCT/CN2023/083939 CN2023083939W WO2023246212A1 WO 2023246212 A1 WO2023246212 A1 WO 2023246212A1 CN 2023083939 W CN2023083939 W CN 2023083939W WO 2023246212 A1 WO2023246212 A1 WO 2023246212A1
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layer
base
electrode
microneedle array
microneedle
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PCT/CN2023/083939
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English (en)
French (fr)
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朱楚洪
柯明
徐文晖
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中国人民解放军陆军军医大学
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Publication of WO2023246212A1 publication Critical patent/WO2023246212A1/zh

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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
    • 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
    • G01N27/02—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating impedance
    • 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
    • G01N27/02—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating impedance
    • G01N27/04—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating impedance by investigating resistance

Definitions

  • the present invention relates to the field of biomedical technology, and in particular to a microneedle array electrode for detecting electrical signals of myocardial cells and a preparation method thereof.
  • the potential of cardiomyocytes includes the resting potential and the action potential during excitement.
  • the extramembrane potential is positive and the intramembrane potential is negative;
  • the potential of the cardiomyocytes undergoes reverse polarization, that is, the extramembrane potential occurs.
  • the potential temporarily becomes negative, and the intramembrane potential temporarily becomes positive; then the cardiomyocytes return to their original polarization state, that is, repolarization or repolarization.
  • myocardial action potential the electrophysiological state of myocardial cells can be accurately grasped. Detecting indicators such as intra/external signal conduction speed, transmembrane voltage and electrical impedance of cardiomyocytes can effectively evaluate the function of cardiomyocytes.
  • microneedle array electrodes have been widely used in biological and medical fields.
  • the size of microneedle electrodes is generally in the micron range.
  • the microneedle electrode passes through or contacts the object to be measured. Because the size of the microneedle is relatively small, it will not cause damage to the object to be measured.
  • the array-type microneedle electrode can contact multiple objects to be measured at the same time, which can High-throughput detection of the tested object.
  • microneedle electrodes cannot monitor cell status in real time and cannot detect intracellular signaling, single cell membrane surface voltage, and electrical impedance.
  • the present invention provides a microneedle array electrode for detecting electrical signals of cardiomyocytes and a preparation method thereof, which can detect cell status in real time, and can detect intracellular signal conduction, single cell surface voltage and electrical impedance.
  • the main technical solutions adopted by the present invention include:
  • Embodiments of the present invention provide a microneedle array electrode for detecting electrical signals of cardiomyocytes.
  • the microneedle array electrode is used to be placed on a PCB board.
  • the microneedle array electrode includes a plurality of microneedle electrode units arranged in an array; the microneedle electrode unit includes a base layer, a metal layer, an insulating layer and a PDMS porous film layer in order; the base layer
  • the bottom layer includes a base and two pairs of microneedle electrode pairs arranged on the base.
  • the base is provided with soldering pads.
  • Each pair of microneedle electrode pairs includes two needle bodies.
  • the needle bodies are made of transparent material.
  • the metal layer is sprayed on the base layer to An auxiliary metal wire is formed on the base, and a connecting electrode is formed on the top of the needle body.
  • the connecting electrode is connected to one end of the auxiliary metal wire, and the other end of the auxiliary metal wire is connected to the pad.
  • a single cardiomyocyte can cover the tops of two needle bodies at the same time. Connect the electrode; the insulating layer is set on the base layer and exposes the top of the needle body and the pad, and the PDMS porous film layer covers the insulating layer.
  • the needle body has a rotary structure, and the distance between the central axes of the needle body in each pair of microneedle electrodes is 30-50 ⁇ m.
  • the material of the base and the needle body in the base layer is high-transmittance glass.
  • the needle body is a cylinder, and the diameter of the cylinder is 7-10 ⁇ m.
  • the insulation layer is made of polyimide.
  • the soldering pad is connected to the port on the parallel cable on the PCB board through a wire; a plurality of reference electrodes are provided on the PCB board, and the reference electrodes are connected to the ports on the series cable on the PCB board.
  • the invention also provides a method for preparing a microneedle array electrode for detecting electrical signals of cardiomyocytes, which includes the following steps;
  • S2 Clean the base with acetone, pure water, absolute ethanol, and pure water in sequence.
  • the cleaning time should be no less than 5 minutes;
  • S3 Spin-coat a layer of photoresist on one side of the base, then use a mask to form a base layer with needles through etching, and remove excess photoresist;
  • S5 Form a conductive layer on the surface of the dried base layer through magnetron sputtering metal layer.
  • the thickness of the conductive layer is 150nm, so that the top of the needle body has a connecting electrode and the base has an auxiliary metal line;
  • S6 Spin-coat insulating material on the conductive layer except for the top of the needle body and the position of the pad on the base so that the thickness of the insulating material does not exceed 2 ⁇ m, and an insulating layer is formed after baking;
  • S7 Cover the PDMS porous film layer on the insulating layer to form a microneedle array electrode.
  • step S7 includes the following steps;
  • step S8 it also includes step S8;
  • the present invention provides a microneedle array electrode for detecting myocardial cell electrical signals. Since it is provided with multiple microneedle electrode units, and the microneedle electrode unit includes a base layer, a metal layer, an insulating layer and a PDMS porous film layer, And because the needle body in the basal layer is made of transparent material and has good light transmittance, it is convenient for observation under a microscope and high-temperature sterilization, and is more suitable for cell culture. Since the insulating layer is provided to cover the auxiliary metal lines in the metal layer on the base layer, current can be avoided between the auxiliary metal lines on the base, thereby preventing interference with potential detection.
  • Microneedle array electrodes can accurately detect single cell surface voltage and electrical impedance.
  • the invention provides a method for preparing a microneedle array electrode for detecting electrical signals of myocardial cells.
  • the prepared microneedle array electrode is equipped with multiple microneedle electrode units, and the microneedle electrode
  • the pole unit includes a basal layer, a metal layer, an insulating layer and a PDMS porous film layer. Since the needle body in the basal layer is made of transparent material, it has good light transmittance and is convenient for observation under a microscope and high-temperature sterilization, making it more suitable for cell culture. Since the insulating layer is provided to cover the auxiliary metal lines in the metal layer on the base layer, current can be avoided between the auxiliary metal lines on the base, thereby preventing interference with potential detection.
  • Microneedle array electrodes can accurately detect single cell surface voltage and electrical impedance.
  • Figure 1 is a schematic structural diagram of the microneedle array electrode of the present invention.
  • Figure 2 is an enlarged view of part A in Figure 1;
  • Figure 3 is a schematic structural diagram of the microneedle array electrode and PCB board of the present invention.
  • Figure 4 is a schematic structural diagram of the PDMS porous membrane.
  • Microneedle array electrode 11: Microneedle electrode unit; 111: Needle body; 112: Connection electrode; 113: Auxiliary metal wire; 114: Welding pad;
  • an embodiment of the present invention provides a microneedle array electrode 1 for detecting electrical signals of myocardial cells.
  • the microneedle array electrode 1 is used to be arranged on a PCB board 2.
  • the column electrode 1 includes a plurality of microneedle electrode units 11 arranged in an array.
  • the microneedle electrode unit 11 includes a base layer, a metal layer, an insulating layer and a PDMS porous film layer in sequence.
  • the base layer includes a base and two pairs of microneedle electrode pairs arranged on the base.
  • the base is provided with pads.
  • each pair of microneedle electrode pairs includes two needle bodies 111.
  • the needle bodies 111 are made of transparent material.
  • the metal layer is sprayed on the base layer to form an auxiliary metal line 113 on the base, and the top of the needle body 111 forms a connecting electrode 112.
  • the connecting electrode 112 is connected to one end of the auxiliary metal wire 113, and the other end of the auxiliary metal wire 113 is connected to the pad 114.
  • a single cardiomyocyte can cover the connecting electrodes 112 at the top of the two needles 111 at the same time.
  • the insulating layer is arranged on the base layer and The top of the needle 111 (that is, the connecting electrode 112 is exposed) and the pad 114 are exposed, and the PDMS porous film layer covers the insulating layer.
  • the arrangement of the auxiliary metal lines 113 is designed according to actual requirements.
  • PDMS is polydimethylsiloxane.
  • This embodiment provides a microneedle array electrode 1 for detecting electrical signals of myocardial cells because it is provided with multiple microneedle electrode units 11, and the microneedle electrode unit 11 includes a base layer, a metal layer, an insulating layer and PDMS.
  • the insulating layer is provided to cover the auxiliary metal lines in the metal layer on the base layer, current can be avoided between the auxiliary metal lines on the base, thereby avoiding interference with potential detection.
  • a single cardiomyocyte can cover the two connecting electrodes 112 in the electrode pair, by detecting the electrical signal between the two connecting electrodes 112, the electrical signal conduction speed in the cell can be calculated, so it is used to detect the electrical signal of the cardiomyocyte.
  • the microneedle array electrode 1 of the signal can accurately detect the surface voltage and electrical impedance of a single cell.
  • the needle body 111 has a rotary structure.
  • the needle body 111 is a cylinder, and the diameter of the cylinder is 7-10 ⁇ m.
  • the needle body 111 can also be a vertebral body.
  • the distance between the central axes of the needle bodies 111 in each pair of microneedle electrodes is 30-50 ⁇ m.
  • the needle body 111 and the base are both made of highly transparent glass, and the base is made of glass. Since the needle body 111 is made of highly transparent glass, which is harder than stainless steel or plastic and has good light transmittance, it is convenient for observation under a microscope and high-temperature sterilization, and is more suitable for cell culture.
  • PDMS has good biocompatibility and is conducive to the two-dimensional adherent growth of induced cardiomyocytes.
  • the PDMS porous membrane layer is replaceable, preventing extracellular secretions from affecting cell growth.
  • the material of the insulation layer is polyimide.
  • the auxiliary metal wires 113 of the microneedle electrode unit 11 correspond to the pads 114 one by one.
  • the pads 114 are connected to the ports on the parallel cable 21 on the PCB board through wires.
  • multiple parameters are provided on the PCB board 2.
  • the comparison electrode 22 and the reference electrode 22 are connected to the ports on the series cable 23 on the PCB board 2, and the spare pins on the PCB board 2 are connected to the peripheral signal acquisition circuit respectively.
  • the interface on the series cable 23 can be directly connected to a computer or other equipment to read the signal measured by the connection electrode 112 and the electrical signal measured by the reference electrode 22 .
  • the cardiomyocytes are two-dimensionally adherent and planted on the porous membrane, and the cardiomyocytes are spread on a pair of microneedle electrodes, and the connecting electrode 112 on one of the needles 111 is given a
  • the current value is measured from the signal connected to the electrode 112 on the other needle body 111, and the conduction rate, transmembrane voltage and electrical impedance of the signal within (between) myocardial cells are calculated.
  • the microneedle array electrode 1 has multiple microneedle electrode units 11, when the cells are adherently planted and reach a certain density, at least one cardiomyocyte can completely cover a pair of microneedle electrodes, and the electrical activity of a single cardiomyocyte can be detected. Signal.
  • the microneedle array since the microneedle array includes multiple microneedle electrode units 11, the signal conduction rate between two cardiomyocytes on multiple pairs of microneedle electrodes can also be measured, and the (between) cells in multiple cardiomyocytes can be detected simultaneously with high throughput. ) signaling.
  • the detected electrical signal can be compared with the reference signal, and the signal of the cardiomyocyte can be accurately obtained, thereby improving the accuracy of the detection.
  • the diameter of the needle body 111 is several micrometers, when combined with the cell surface, the damage to the cells is less.
  • the size of the microneedle array electrode 1 is a rectangular structure of 2cm ⁇ 2cm, including 60 microneedle electrode units 11.
  • Each microneedle electrode unit 11 is provided with two pairs of microneedle electrodes.
  • Each pair of microneedle electrodes includes two needles 111.
  • the needles 111 are cylinders with a diameter of 7 ⁇ m and a height of 21 ⁇ m.
  • the distance between the central axes of the cylinders is 35 ⁇ m.
  • the material of the cylinder is high-transmittance glass, which facilitates observation of cell growth status.
  • the thickness of the base is 500 ⁇ m.
  • the porous membrane is made of PDMS and has a conical structure.
  • PDMS has good biocompatibility and high transmittance, and can be used for cell growth and observation.
  • the porous membrane has a porous structure with a pore diameter of 8 ⁇ m. Two holes form a pair. The center distance of a pair of holes is 35 ⁇ m. The thickness of the porous membrane does not exceed 20 ⁇ m, which corresponds to the needles 111 on the microneedle electrode pair.
  • the invention also provides a method for preparing a microneedle array electrode 1 for detecting electrical signals of cardiomyocytes, which includes the following steps;
  • S2 Clean the base with acetone, pure water, absolute ethanol, and pure water in sequence.
  • the cleaning time should be no less than 5 minutes;
  • S5 Use magnetron sputtering to form a conductive layer on the surface of the dried base layer.
  • the metal layer is made of gold and the thickness of the conductive layer is 150 nm.
  • the top of the needle body 111 has a connecting electrode 112 on the base. With auxiliary metal wire 113;
  • step S7 includes the following steps;
  • This embodiment provides a method for preparing a microneedle array electrode 1 for detecting electrical signals of cardiomyocytes.
  • the prepared microneedle array electrode 1 is provided with a plurality of microneedle electrode units 11, and the microneedle electrode units 11 It includes a basal layer, a metal layer, an insulating layer and a PDMS porous film layer, and because the needle body 111 in the basal layer is made of transparent material, it has good light transmittance and is convenient for observation under a microscope and high-temperature sterilization, and is more suitable for cell culture. Since the insulating layer is provided to cover the auxiliary metal lines in the metal layer on the base layer, current can be avoided between the auxiliary metal lines on the base, thereby preventing interference with potential detection.
  • a single cardiomyocyte can cover the two connecting electrodes 112 in the electrode pair, by detecting the electrical signal between the two connecting electrodes 112, the electrical signal conduction speed in the cell can be calculated, so it is used to detect the electrical signal of the cardiomyocyte.
  • the microneedle array electrode 1 of the signal can accurately detect the surface voltage and electrical impedance of a single cell.
  • connection In the present invention, unless otherwise clearly stated and limited, the terms “installation”, “connection”, “connection”, “fixing” and other terms should be understood in a broad sense. For example, it can be a fixed connection or a detachable connection. , or integrated; it can be a mechanical connection or an electrical connection; it can be a direct connection or an indirect connection through an intermediate medium; it can be an internal connection between two elements or an interaction between two elements.
  • connection connection
  • fixing and other terms should be understood in a broad sense. For example, it can be a fixed connection or a detachable connection. , or integrated; it can be a mechanical connection or an electrical connection; it can be a direct connection or an indirect connection through an intermediate medium; it can be an internal connection between two elements or an interaction between two elements.
  • a first feature is “on” or “below” a second feature, which may mean that the first and second features are in direct contact, or the first and second features are in direct contact through an intermediary. indirect contact.
  • the first feature is “on” or “above” the second feature and “above” may mean that the first feature is directly above or diagonally above the second feature, or simply means that the first feature is higher in level than the second feature.
  • the first feature being “below”, “below” and “under” the second feature may mean that the first feature is directly below or diagonally below the second feature, or simply means that the first feature is lower in level than the second feature.
  • the terms “one embodiment”, “some embodiments”, “embodiments”, “examples”, “specific examples” or “some examples”, etc. refer to the description in conjunction with the embodiment or example.
  • a specific feature, structure, material, or characteristic described is included in at least one embodiment or example of the invention.
  • the schematic expressions of the above terms are not necessarily directed to the same embodiment or example.
  • the specific features, structures, materials or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.
  • those skilled in the art may combine and combine different embodiments or examples and features of different embodiments or examples described in this specification unless they are inconsistent with each other.

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Abstract

用于检测心肌细胞电信号的微针阵列电极（1）及其制备方法。该用于检测心肌细胞电信号的微针阵列电极（1），由于设有多个微针电极单元（11），且微针电极单元（11）包括基底层、金属层、绝缘层和PDMS多孔膜层，且由于基底层中针体为透明材质，透光性好，便于显微镜下观测和高温灭菌，更适合细胞培养。由于设有绝缘层覆盖基底层上的金属层中的辅助金属线，能够避免底座上的辅助金属线之间形成电流，避免对电位检测形成干扰。由于单一心肌细胞能够覆盖在电极对中的两个连接电极，通过检测两个连接电极之间的电信号，能够计算出细胞内的电信号传导速，进而能够精确的检测到单一细胞表面电压和电阻抗。

Description

用于检测心肌细胞电信号的微针阵列电极及其制备方法 技术领域
本发明涉及生物医学技术领域，尤其涉及一种用于检测心肌细胞电信号的微针阵列电极及其制备方法。
背景技术
心肌细胞的电位包括静息电位和兴奋时的动作电位，当静息时，膜外电位为正，膜内电位为负；当心肌细胞兴奋时，心肌细胞的电位发生反极化，即膜外电位暂时变负，膜内电位暂时变正；接着心肌细胞恢复原来的极化状态，即再极化或复极化。通过检测心肌动作电位可以精确掌握心肌细胞的电生理状态。检测心肌细胞内/外信号传导速度、跨膜电压和电阻抗等指标能有效评价心肌细胞的功能。
目前，微针阵列电极已经广泛应用于生物及医学领域。微针电极的尺寸一般为微米级。在实际使用中，微针电极穿过或者接触被测对象，因微针的尺寸比较微小，不会对被测对象产生损伤，且阵列式的微针电极能够同时接触多个被测对象，能够高通量的对被测对象进行检测。
然而，现有的微针电极无法实时监测细胞状态，并且无法检测细胞内的信号传导、单细胞膜表面电压和电阻抗。
发明内容
(一)要解决的技术问题
鉴于现有技术的上述缺点、不足，本发明提供一种用于检测心肌细胞电信号的微针阵列电极及其制备方法，能够实时检测细胞状态，且能够检测细胞内的信号传导、单细胞表面电压和电阻抗。
(二)技术方案
为了达到上述目的，本发明采用的主要技术方案包括：
本发明实施例提供一种用于检测心肌细胞电信号的微针阵列电极， 微针阵列电极用于设置在PCB板上，微针阵列电极包括呈阵列排布的多个微针电极单元；微针电极单元依次包括基底层、金属层、绝缘层和PDMS多孔膜层；基底层包括底座及设置在底座上的两对微针电极对，底座上设有焊盘，每对微针电极对均包括两个针体，针体为透明材质，金属层喷涂在基底层上以使底座上形成辅助金属线、使针体的顶端形成连接电极，连接电极与辅助金属线的一端连通，辅助金属线的另一端与焊盘连接，单一心肌细胞能够同时覆盖两个针体顶端的连接电极；绝缘层设置在基底层上且露出针体的顶端及焊盘，PDMS多孔膜层覆盖在绝缘层上。
优选地，针体为回转体结构，每对微针电极对内的针体的中心轴线的间距为30-50μm。
优选地，基底层中底座和针体的材质均为高透光玻璃。
优选地，针体为圆柱体，圆柱体的直径为7-10μm。
优选地，绝缘层的材质为聚酰亚胺。
优选地，焊盘通过导线与PCB板上的并联排线上的端口连接；PCB板上设有多个参比电极，参比电极与PCB板上的串联排线上的端口连接。
本发明还提供了一种用于检测心肌细胞电信号的微针阵列电极的制备方法，包括如下步骤；
S1：选择厚度为500μm、4英寸的高透光玻璃片作为基底层的底座；
S2：依次用丙酮、纯水、无水乙醇、纯水对底座进行清洗，清洗时间不少于5分钟；
S3：在底座的一面上旋涂一层光刻胶，再利用掩模通过刻蚀形成具有针体的基底层，去除多余的光刻胶；
S4：将具有针体的基底层依次用无水乙醇和纯水进行清洗，清洗时间不少于5分钟，清洗完毕后，晾晒1小时，得到干燥的基底层；
S5：将经过干燥后的基底层的表面通过磁控溅射金属层形成导电层，导电层的厚度为150nm，使针体的顶端具有连接电极，底座上具有辅助金属线；
S6：在导电层上除针体的顶端和底座上焊盘的位置外，均旋涂绝缘材料，以使绝缘材料的厚度不超过2μm，烘烤后形成绝缘层；
S7：在绝缘层的上覆盖PDMS多孔膜层，形成微针阵列电极。
优选地，步骤S7包括如下步骤；
S71：配制PDMS预聚物溶液，在真空箱中去除气泡，抽取时的真空度为-15kPa，抽取的时间不少于30分钟；
S72：在绝缘层上旋涂一层PDMS预聚物，以使PDMS预聚物的厚度不超过20μm，将具有圆锥形结构的阳模倒扣覆盖在PDMS预聚物上，在85℃的温度下固化2小时；
S73：取下阳模，用刀切割下多余的PDMS膜，在显微镜下对准覆盖在基底层上，露出针体的顶端和底座的焊盘，制得微针阵列电极。
优选地，还包括步骤S8；
S8：将微针阵列电极上的焊盘通过导线与PCB板上的并联排线的端口一一对应连接。
(三)有益效果
本发明的有益效果是：
本发明的提供的一种用于检测心肌细胞电信号的微针阵列电极，由于设有多个微针电极单元，且微针电极单元包括基底层、金属层、绝缘层和PDMS多孔膜层，且由于基底层中针体为透明材质，透光性好，便于显微镜下观测和高温灭菌，更适合细胞培养。由于设有绝缘层覆盖基底层上金属层中的辅助金属线，能够避免底座上的辅助金属线之间形成电流，进而避免对电位检测形成干扰。且由于单一心肌细胞能够覆盖在电极对中的两个连接电极，通过检测两个连接电极之间的电信号，能够计算出细胞内的电信号传导速，故该用于检测心肌细胞电信号的微针阵列电极能够精确的检测到单一细胞表面电压和电阻抗。
本发明提供的一种用于检测心肌细胞电信号的微针阵列电极的制备方法，制备出的微针阵列电极，由于设有多个微针电极单元，且微针电 极单元包括基底层、金属层、绝缘层和PDMS多孔膜层，且由于基底层中针体为透明材质，透光性好，便于显微镜下观测和高温灭菌，更适合细胞培养。由于设有绝缘层覆盖基底层上金属层中的辅助金属线，能够避免底座上的辅助金属线之间形成电流，进而避免对电位检测形成干扰。且由于单一心肌细胞能够覆盖在电极对中的两个连接电极，通过检测两个连接电极之间的电信号，能够计算出细胞内的电信号传导速，故该用于检测心肌细胞电信号的微针阵列电极能够精确的检测到单一细胞表面电压和电阻抗。
附图说明
图1为本发明微针阵列电极的结构示意图；
图2为图1中的A部分的放大图；
图3为本发明微针阵列电极以及PCB板的结构示意图；
图4为PDMS多孔膜的结构示意图。
【附图标记说明】
1：微针阵列电极；11：微针电极单元；111：针体；112：连接电极；113：辅助金属线；114：焊盘；
2：PCB板；21：并联排线；22：参比电极；23：串联排线。
具体实施方式
为了更好的理解上述技术方案，下面将参照附图更详细地描述本发明的示例性实施例。虽然附图中显示了本发明的示例性实施例，然而应当理解，可以以各种形式实现本发明而不应被这里阐述的实施例所限制。相反，提供这些实施例是为了能够更清楚、透彻地理解本发明，并且能够将本发明的范围完整的传达给本领域的技术人员。
实施例一
如图1和图3所示，本发明实施例提供一种用于检测心肌细胞电信号的微针阵列电极1，微针阵列电极1用于设置在PCB板2上，微针阵 列电极1包括呈阵列排布的多个微针电极单元11。
如图2所示，微针电极单元11依次包括基底层、金属层、绝缘层和PDMS多孔膜层，基底层包括底座及设置在底座上的两对微针电极对，底座上设有焊盘，每对微针电极对均包括两个针体111，针体111为透明材质，金属层喷涂在基底层上以使底座上形成辅助金属线113、使针体111的顶端形成连接电极112，连接电极112与辅助金属线113的一端连通，辅助金属线113的另一端与焊盘114连接，单一心肌细胞能够同时覆盖两个针体111顶端的连接电极112，绝缘层设置在基底层上且露出针体111的顶端(即露出连接电极112)和焊盘114，PDMS多孔膜层覆盖在绝缘层上。应当说明的是，辅助金属线113的排布方式根据实际需求设计。应当说明的是，PDMS为聚二甲基硅氧烷。
本实施例的提供的一种用于检测心肌细胞电信号的微针阵列电极1，由于设有多个微针电极单元11，且微针电极单元11包括基底层、金属层、绝缘层和PDMS多孔膜层，且由于基底层中针体111为透明材质，透光性好，便于显微镜下观测和高温灭菌，更适合细胞培养。由于设有绝缘层覆盖基底层上的金属层中的辅助金属线，能够避免底座上的辅助金属线之间形成电流，进而避免对电位检测形成干扰。且由于单一心肌细胞能够覆盖在电极对中的两个连接电极112，通过检测两个连接电极112之间的电信号，能够计算出细胞内的电信号传导速，故该用于检测心肌细胞电信号的微针阵列电极1能够精确的检测到单一细胞表面电压和电阻抗。
在本实施例中，针体111为回转体结构，具体为针体111为圆柱体，圆柱体的直径为7-10μm。当然，针体111也可为椎体。每对微针电极对内的针体111的中心轴线的间距为30-50μm。针体111和底座的材质均为高透光玻璃，底座的材质为玻璃。由于针体111为高透光玻璃，比不锈钢或者塑料硬度大，透光性好，便于显微镜下观测和高温灭菌，更适合细胞培养。
其中，PDMS具有较好的生物相容性，有利于诱导的心肌细胞二维贴壁生长。此外，PDMS多孔膜层可替换，避免了细胞外分泌物影响细胞的生长。绝缘层的材质为聚酰亚胺。
其中，微针电极单元11的辅助金属线113与焊盘114一一对应，焊盘114通过导线与PCB板上的并联排线21上的端口连接，同时在PCB板2上设有多个参比电极22，参比电极22与PCB板2上的串联排线23上的端口连接，PCB板2上空余引线脚分别与外围信号采集电路连接。当然串联排线23上的接口可直接与计算机等设备连接读取连接电极112测量的信号和参比电极22测得的电信号。
当使用微针阵列电极1检测心肌细胞电信号时，将心肌细胞二维贴壁种植在多孔膜上，心肌细胞铺展在一对微针电极上，给其中一个针体111上的连接电极112一个电流值，测量另一个针体111上的连接电极112的信号，通过计算得到信号在心肌细胞内(间)的传导速率，跨膜电压和电阻抗。且由于该微针阵列电极1具有多个微针电极单元11，当细胞贴壁种植达到一定密度后，至少一个心肌细胞能够完整覆盖于一对微针电极上，可检测到单个心肌细胞的电信号。同时，由于该微针阵列包括多个微针电极单元11，也可测量多对微针电极上两个心肌细胞间的信号传导速率，并且可同时高通量检测到多个心肌细胞内(间)的信号传导。由于PCB板2上设有参比电极22，可将检测的电信号与参比信号进行比较，可以准确获取心肌细胞的信号，从而提高检测的准确性。此外，由于针体111的直径为几个微米当与细胞表面结合时，对细胞的损伤较小。
实施例二
在本实施例中，微针阵列电极1的尺寸为2cm×2cm的矩形结构，包括60个微针电极单元11，当然可根据实际需求设置多个微针电极单元11。每个微针电极单元11上设有两对微针电极对，每对微针电极对包括两个针体111，针体111为直径是7μm的圆柱体，其高度为21μm，两 个圆柱体中心轴线的间距为35μm。其中，圆柱体的材质为高透光玻璃，便于观测细胞生长状态。底座的厚度为500μm。
如图4所示，多孔膜的材质为PDMS制成，为圆锥形结构，PDMS具有很好的生物相容性、高透光度，可用于细胞的生长和观测。其中，多孔膜具有多孔结构，孔径8μm，两个孔为一对，一对孔的中心间距为35μm，多孔膜的厚度不超过20μm，与微针电极对上的针体111一一对应。
实施例三
本发明还提供了一种用于检测心肌细胞电信号的微针阵列电极1的制备方法，包括如下步骤；
S1：选择厚度为500μm、4英寸的高透光玻璃片作为基底层的底座；
S2：依次用丙酮、纯水、无水乙醇、纯水对底座进行清洗，清洗时间不少于5分钟；
S3：在底座的一面上旋涂一层光刻胶，再利用掩模通过刻蚀形成具有针体111的基底层，去除多余的光刻胶；
S4：将具有针体111的基底层依次用无水乙醇和纯水进行清洗，清洗时间不少于5分钟，清洗完毕后，晾晒1小时，得到干燥的基底层；
S5：将经过干燥后的基底层的表面通过磁控溅射金属层形成导电层，其中金属层的材质为金，导电层的厚度为150nm，使针体111的顶端具有连接电极112，底座上具有辅助金属线113；
S6：在导电层上除针体111的顶端和底座上焊盘114的位置外，均旋涂绝缘材料，以使绝缘材料的厚度不超过2μm，烘烤后形成绝缘层；
S7：通过旋涂法在绝缘层的上覆盖PDMS多孔膜层，形成微针阵列电极1。
其中，步骤S7包括如下步骤；
S71：配制PDMS预聚物溶液，在真空箱中去除气泡，抽取时的真空度为-15kPa，抽取的时间不少于30分钟；
S72：在绝缘层上旋涂一层PDMS预聚物，以使PDMS预聚物的厚度不超过20μm，将具有圆锥形结构的阳模倒扣覆盖在PDMS预聚物上，在85℃的温度下固化2小时；
S73：取下阳模，用刀切割下多余的PDMS膜，在显微镜下对准覆盖在基底层上，露出针体111的顶端和底座的焊盘114，制得微针阵列电极1。
S8：将微针阵列电极1上的焊盘114通过焊接导线与PCB板上的并联排线21的端口一一对应连接。
本实施例提供的一种用于检测心肌细胞电信号的微针阵列电极1的制备方法，制备出的微针阵列电极1，由于设有多个微针电极单元11，且微针电极单元11包括基底层、金属层、绝缘层和PDMS多孔膜层，且由于基底层中针体111为透明材质，透光性好，便于显微镜下观测和高温灭菌，更适合细胞培养。由于设有绝缘层覆盖基底层上金属层中的辅助金属线，能够避免底座上的辅助金属线之间形成电流，进而避免对电位检测形成干扰。且由于单一心肌细胞能够覆盖在电极对中的两个连接电极112，通过检测两个连接电极112之间的电信号，能够计算出细胞内的电信号传导速，故该用于检测心肌细胞电信号的微针阵列电极1能够精确的检测到单一细胞表面电压和电阻抗。
在本发明中，除非另有明确的规定和限定，术语“安装”、“相连”、“连接”、“固定”等术语应做广义理解，例如，可以是固定连接，也可以是可拆卸连接，或成一体；可以是机械连接，也可以是电连接；可以是直接相连，也可以通过中间媒介间接相连；可以是两个元件内部的连通或两个元件的相互作用关系。对于本领域的普通技术人员而言，可以根据具体情况理解上述术语在本发明中的具体含义。
在本发明中，除非另有明确的规定和限定，第一特征在第二特征“上”或“下”，可以是第一和第二特征直接接触，或第一和第二特征通过中间媒介间接接触。而且，第一特征在第二特征“之上”、“上方” 和“上面”，可以是第一特征在第二特征正上方或斜上方，或仅仅表示第一特征水平高度高于第二特征。第一特征在第二特征“之下”、“下方”和“下面”，可以是第一特征在第二特征正下方或斜下方，或仅仅表示第一特征水平高度低于第二特征。
在本说明书的描述中，术语“一个实施例”、“一些实施例”、“实施例”、“示例”、“具体示例”或“一些示例”等的描述，是指结合该实施例或示例描述的具体特征、结构、材料或者特点包含于本发明的至少一个实施例或示例中。在本说明书中，对上述术语的示意性表述不必须针对的是相同的实施例或示例。而且，描述的具体特征、结构、材料或者特点可以在任一个或多个实施例或示例中以合适的方式结合。此外，在不相互矛盾的情况下，本领域的技术人员可以将本说明书中描述的不同实施例或示例以及不同实施例或示例的特征进行结合和组合。
尽管上面已经示出和描述了本发明的实施例，可以理解的是，上述实施例是示例性的，不能理解为对本发明的限制，本领域的普通技术人员在本发明的范围内可以对上述实施例进行改动、修改、替换和变型。

Claims (9)

  1. 用于检测心肌细胞电信号的微针阵列电极，其特征在于，所述微针阵列电极用于设置在PCB板上，所述微针阵列电极包括呈阵列排布的多个微针电极单元；
    所述微针电极单元依次包括基底层、金属层、绝缘层和PDMS多孔膜层；
    所述基底层包括底座及设置在所述底座上的两对微针电极对，所述底座上设有焊盘，每对微针电极对均包括两个针体，所述针体为透明材质，所述金属层喷涂在所述基底层上以使所述底座上形成辅助金属线、使所述针体的顶端形成连接电极，所述连接电极与所述辅助金属线的一端连通，所述辅助金属线的另一端与所述焊盘连接，单一所述心肌细胞能够同时覆盖两个所述针体顶端的所述连接电极；
    所述绝缘层设置在所述基底层上且露出所述针体的顶端及所述焊盘，所述PDMS多孔膜层覆盖在所述绝缘层上。
  2. 如权利要求1所述的用于检测心肌细胞电信号的微针阵列电极，其特征在于：
    所述针体为回转体结构，每对微针电极对内的所述针体的中心轴线的间距为30-50μm。
  3. 如权利要求1所述的用于检测心肌细胞电信号的微针阵列电极，其特征在于：
    所述基底层中所述底座和所述针体的材质均为高透光玻璃。
  4. 如权利要求1所述的用于检测心肌细胞电信号的微针阵列电极，其特征在于：
    所述针体为圆柱体，所述圆柱体的直径为7-10μm。
  5. 如权利要求1所述的用于检测心肌细胞电信号的微针阵列电极，其特征在于：
    所述绝缘层的材质为聚酰亚胺。
  6. 如权利要求1所述的用于检测心肌细胞电信号的微针阵列电极， 其特征在于：
    所述焊盘通过导线与所述PCB板上的并联排线上的端口连接；
    所述PCB板上设有多个参比电极，所述参比电极与所述PCB板上的串联排线上的端口连接。
  7. 用于检测心肌细胞电信号的微针阵列电极的制备方法，其特征在于：包括如下步骤；
    S1：选择厚度为500μm、4英寸的高透光玻璃片作为基底层的底座；
    S2：依次用丙酮、纯水、无水乙醇、纯水对所述底座进行清洗，清洗时间不少于5分钟；
    S3：在所述底座的一面上旋涂一层光刻胶，再利用掩模通过刻蚀形成具有针体的基底层，去除多余的光刻胶；
    S4：将具有针体的基底层依次用无水乙醇和纯水进行清洗，清洗时间不少于5分钟，清洗完毕后，晾晒1小时，得到干燥的所述基底层；
    S5：将经过干燥后的所述基底层的表面通过磁控溅射金属层形成导电层，所述导电层的厚度为150nm，使针体的顶端具有连接电极，所述底座上具有辅助金属线；
    S6：在导电层上除所述针体的顶端和所述底座上焊盘的位置外，均旋涂绝缘材料，以使所述绝缘材料的厚度不超过2μm，烘烤后形成绝缘层；
    S7：在所述绝缘层的上覆盖PDMS多孔膜层，形成微针阵列电极。
  8. 如权利要求7所述的用于检测心肌细胞电信号的微针阵列电极的制备方法，其特征在于：所述步骤S7包括如下步骤；
    S71：配制PDMS预聚物溶液，在真空箱中去除气泡，抽取时的真空度为-15kPa，抽取的时间不少于30分钟；
    S72：在所述绝缘层上旋涂一层PDMS预聚物，以使所述PDMS预聚物的厚度不超过20μm，将具有圆锥形结构的阳模倒扣覆盖在所述PDMS预聚物上，在85℃的温度下固化2小时；
    S73：取下阳模，用刀切割下多余的PDMS膜，在显微镜下对准覆盖在基底层上，露出所述针体的顶端和所述底座的焊盘，制得微针阵列电极。
  9. 如权利要求7所述的用于检测心肌细胞电信号的微针阵列电极的制备方法，其特征在于：还包括步骤S8；
    S8：将所述微针阵列电极上的焊盘通过导线与PCB板上的并联排线的端口一一对应连接。
PCT/CN2023/083939 2022-06-22 2023-03-26 用于检测心肌细胞电信号的微针阵列电极及其制备方法 WO2023246212A1 (zh)

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