WO2013170641A1 - 单细胞阵列微芯片及其制造、电测量和电穿孔方法 - Google Patents

单细胞阵列微芯片及其制造、电测量和电穿孔方法 Download PDF

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WO2013170641A1
WO2013170641A1 PCT/CN2013/070613 CN2013070613W WO2013170641A1 WO 2013170641 A1 WO2013170641 A1 WO 2013170641A1 CN 2013070613 W CN2013070613 W CN 2013070613W WO 2013170641 A1 WO2013170641 A1 WO 2013170641A1
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electrodes
microelectrode
cells
electrical measurement
positioning
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French (fr)
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朱荣
郭霄亮
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清华大学
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Priority to US14/328,425 priority Critical patent/US9695412B2/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N13/00—Treatment of microorganisms or enzymes with electrical or wave energy, e.g. magnetism, sonic waves
    • 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
    • G01N33/483—Physical analysis of biological material
    • G01N33/4833—Physical analysis of biological material of solid biological material, e.g. tissue samples, cell cultures
    • G01N33/4836—Physical analysis of biological material of solid biological material, e.g. tissue samples, cell cultures using multielectrode arrays
    • 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
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00277—Apparatus
    • B01J2219/00497—Features relating to the solid phase supports
    • B01J2219/00527—Sheets
    • B01J2219/00533—Sheets essentially rectangular
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00583—Features relative to the processes being carried out
    • B01J2219/00603—Making arrays on substantially continuous surfaces
    • B01J2219/00653—Making arrays on substantially continuous surfaces the compounds being bound to electrodes embedded in or on the solid supports
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00718—Type of compounds synthesised
    • B01J2219/0072—Organic compounds
    • B01J2219/0074—Biological products
    • B01J2219/00743—Cells

Definitions

  • the invention relates to the field of biological detection technology, in particular to a single cell array microchip and a manufacturing method thereof, an electrical measurement method and an electroporation method.
  • Cells are the basic unit of life activity, and cell measurement and analysis have drawn great attention in the scientific and medical fields.
  • Cellular measurements generally include biomechanical measurements, electrical measurements, motion behaviors, and physicochemical properties.
  • the measurement methods used are: microscopic observation, electrochemical measurement, optical measurement, etc., where microscopic observation and optical measurement often require cells.
  • Chemical or fluorescent labeling is complicated, has a single function, and changes the original characteristics of the cells to some extent.
  • Electrochemical measurement does not require labeling, no damage to cells, continuous and real-time measurement of cells in a natural culture state, and has higher sensitivity than conventional chemical methods, measurement can be fully automated, and no manual intervention is required.
  • DEP dielectrophoresis
  • the technical problem to be solved by the present invention is to provide a single cell array microchip and a method for manufacturing, electrical measurement and electroporation thereof, which can effectively improve the electrical measurement efficiency and precision of a single cell, and can improve the electroporation efficiency of a single cell. And controllability, reducing cell death.
  • the present invention provides a single cell array microchip comprising a substrate, an array of a plurality of positioning microelectrodes, an array of a plurality of electrical measuring microelectrode pairs, and a sample cell microcavity, wherein each phase
  • the adjacent four positioning microelectrodes constitute a positioning unit arranged in a diamond shape
  • Each of the plurality of positioning microelectrodes of the array type is connected by a transverse electrode connection, or two of the plurality of positioning microelectrodes of the array are arranged in a diamond shape.
  • the opposite positioning microelectrode pairs are respectively connected to the positioning microelectrodes in the adjacent positioning unit through the transverse electrode connection and the longitudinal electrode connection;
  • the center of the positioning unit is distributed with an electrical measurement micro-electrode pair, and the two electrodes of the electrical measurement micro-electrode pair are respectively connected to the electrodes of the adjacent electrical measurement micro-electrode pairs through the lateral electrode connection and the longitudinal electrode connection .
  • the lateral electrode wiring and the longitudinal electrode wiring are respectively arranged in two wiring layers, and the two wiring layers are separated by an insulating layer.
  • the substrate is an insulating material or a silicon material coated with an insulating layer.
  • the sample cell microcavity is a polymer material or a glass material.
  • a method for manufacturing the aforementioned single cell array microchip comprising the steps of:
  • a method for electrically measuring cells using the aforementioned single cell array microchip comprising the steps of:
  • S3 traversing the array formed by the plurality of electrical measurement microelectrode pairs by using horizontal and vertical scanning, respectively applying voltage to the two electrodes of each of the electrical measurement microelectrode pairs, and measuring the current signal between the two electrodes;
  • S4 determining, according to the measured current signal between the two electrodes in each of the electrical measurement microelectrode pairs, whether the cells in the electrode of the electrical measurement microelectrode are positioned;
  • S5 Apply voltage to the two electrodes of the electrical measurement microelectrode pair where the cells are located at the electrode, measure the current signal between the two electrodes, and complete electrical measurement of the cells.
  • the step S4 further comprises: pre-calibrating the current range of the electrode between the two electrodes in the microelectrode pair and the cell is not positioned, according to the measured The current range corresponding to the current between the two electrodes of the microelectrode pair is electrically determined to determine whether the cell is positioned.
  • a method of electroporating cells using the aforementioned single cell array microchip comprising the steps of:
  • S33 traversing the array formed by the plurality of electrical measurement microelectrode pairs by using horizontal and vertical scanning, respectively applying voltage to the two electrodes of each of the electrical measurement microelectrode pairs, and measuring the current signal between the two electrodes;
  • S44 determining, according to the measured current signal between the two electrodes of the pair of microelectrodes, whether cells in the electrode of the pair of electrodes are positioned;
  • S55 Applying a pulse voltage signal to the two electrodes of the electrical measurement microelectrode pairs in which the cells are located at the electrodes, respectively, to achieve electroporation of cells positioned at the electrodes of the electrical measurement microelectrode pairs.
  • the step S44 further comprises: pre-measuring the range of currents between the two electrodes in the microelectrode pair by the experiment and the cells are positioned and the cells are not positioned, according to the measured The current range corresponding to the current between the two electrodes of the microelectrode pair is electrically determined to determine whether the cell is positioned.
  • the invention combines cell localization with cell electrical measurement and electroporation, adopts label-free, non-invasive micro-manipulation, localization and parameter measurement and analysis technology, and can realize in-situ online measurement and analysis of single cell array and multi-mode;
  • the position of the cells is fixed, which can effectively improve the electrical detection accuracy of the cells, and can improve the electroporation efficiency of the cells and reduce the cell death rate.
  • the invention can also introduce automatic control technology into cell micro-manipulation and electrical measurement, and automatically control cell positioning through electrical measurement feedback, and finally realize rapid, accurate and multi-mode cell automated measurement and analysis.
  • 1 is a schematic view showing the electrode structure of the single cell array microchip of the present invention.
  • FIG. 2 is a schematic view showing the structure of an electrode connection of a single cell array microchip according to Embodiment 1 of the present invention
  • FIG. 3 is a schematic view showing the structure of an electrode connection of a single cell array microchip according to Embodiment 2 of the present invention.
  • 4a-4f are flow charts showing a process for fabricating a single cell array microchip according to Embodiment 3 of the present invention.
  • Embodiment 1 As shown in FIG. 1-2, the present invention provides a single cell array microchip comprising a substrate 1, an array of a plurality of positioning microelectrodes 2, an array of a plurality of electrical measurement microelectrode pairs 3 and a sample cell microcavity 4, wherein each adjacent four positioning microelectrodes 2 constitute a positioning unit arranged in a diamond shape, the center of the positioning unit is distributed with an electrical measuring microelectrode pair 3; each row of positioning microelectrodes 2 Connected by a lateral electrode connection 5, the two electrodes of the electrical measurement microelectrode pair 3 are connected to the electrodes of the adjacent electrical measurement microelectrode pair 3 via a transverse electrode connection 5 and a longitudinal electrode connection 6, respectively.
  • the lateral electrode line 5 and the longitudinal electrode line 6 are respectively disposed in two wiring layers, and the two wiring layers are separated by an insulating layer.
  • the substrate 1 is an insulating material such as glass or a silicon material overlying an insulating layer.
  • the sample cell microcavity 4 is a PDMS polymer material or a glass material.
  • the insulating layer is a silicon nitride, silicon dioxide or polymer material.
  • the sample cell microcavity 4 can be fabricated by using a mold.
  • the lateral electrode connection of the electrical measurement microelectrode pair 3 is electrically and electrically outputted through the pad 14, the longitudinal electrode connection of the microelectrode pair 3 is electrically input and electrically outputted through the pad 15; the lateral electrode of the microelectrode 2 is positioned
  • the wires are electrically input through the pads 16.
  • Embodiment 2 As shown in FIG. 3, the present embodiment is different from Embodiment 1 in that two opposite positioning microelectrodes 2 in each positioning unit arranged in a diamond shape are respectively passed through a lateral electrode connection line 5 and The longitudinal electrode wiring 6 is connected to the positioning microelectrode 2 in the adjacent positioning unit.
  • Embodiment 3 A method for manufacturing the aforementioned single cell array microchip, comprising the following steps:
  • the embodiment provides a specific manufacturing process of a single cell array microchip, which uses a glass piece as a substrate, and then sequentially performs the following process steps on the substrate in the same direction:
  • Example 4 A method of electrically measuring cells using the aforementioned single cell array microchip, comprising the steps of:
  • S3 traversing the array formed by the plurality of electrical measurement microelectrode pairs 3 by means of horizontal and vertical scanning, respectively applying voltages to the two electrodes of each of the electrical measurement microelectrode pairs 3, and measuring the current signals between the two electrodes;
  • S4 determining, according to the measured current signal between the two electrodes of each of the microelectrode pairs 3, whether the cells in the electrode of the electrical measurement microelectrode pair 3 are positioned;
  • the current range of the two electrodes in the electrode between the two electrodes in the cell is located and the cells are not positioned, and the current between the two electrodes of the microelectrode pair 3 is measured according to the measured electrical quantity.
  • the corresponding current range determines whether the cell is located.
  • the cells are positioned.
  • the cells are not positioned.
  • S5 Apply voltage to the two electrodes of the electrical measurement microelectrode pair 3 at which the cells are located at the electrode, measure the current signal between the two electrodes, and complete electrical measurement of the cells.
  • the voltage applied to the measuring electrode can be a direct current, alternating current or pulsed signal.
  • Example 5 A method of electroporating cells using the aforementioned single cell array microchip, comprising the steps of:
  • S33 traversing the array formed by the plurality of electrical measurement microelectrode pairs 3 by using horizontal and vertical scanning, applying a voltage to each of the two electrodes of the electrical measurement microelectrode pair 3, and measuring a current signal between the two electrodes;
  • S44 determining, according to the measured current signal between the two electrodes of the microelectrode pair 3, whether the cells in the electrode of the electrical measurement microelectrode pair 3 are positioned;
  • the current range of the two electrodes in the electrode between the two electrodes in the cell is located and the cells are not positioned, and the current between the two electrodes of the microelectrode pair 3 is measured according to the measured electrical quantity.
  • the corresponding current range determines whether the cell is located.
  • the cells are positioned.
  • the cells are not positioned.
  • S55 Applying a pulse voltage signal to the two electrodes of the electrical measurement microelectrode pair 3 at which the cells are positioned at the electrodes, respectively, to achieve electroporation of cells positioned at the electrodes of the electrical measurement microelectrode pair 3.
  • the invention combines cell localization with cell electrical measurement and electroporation, adopts label-free, non-invasive micro-manipulation, localization and parameter measurement and analysis technology, and can realize in-situ online measurement and analysis of single cell array and multi-mode;
  • the position of the cells is fixed, which can effectively improve the electrical detection accuracy of the cells, and can improve the electroporation efficiency of the cells and reduce the cell death rate.
  • the invention can also introduce automatic control technology into cell micro-manipulation and electrical measurement, and automatically control cell positioning through electrical measurement feedback, and finally realize rapid, accurate and multi-mode cell automated measurement and analysis.

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Abstract

一种单细胞阵列微芯片及其制造方法以及利用该单细胞阵列微芯片对细胞进行电测量和电穿孔的方法，该单细胞阵列微芯片包括基底（1）、阵列式的多个定位微电极（2）、阵列式的多个电测量微电极对（3）和样品池微腔体（4）。该电测量和电穿孔的方法是将细胞定位与细胞电测量和电穿孔相结合，采用无标记、无损伤的微操作、定位和参数测量分析技术，实现单细胞阵列、多模式的原位在线测量和分析；由于定位后的细胞位置固定，可有效提高细胞的电学检测精度，提升细胞的电穿孔效率，降低细胞死亡率。

Description

单细胞阵列微芯片及其制造、电测量和电穿孔方法
技术领域
本发明涉及生物检测技术领域，尤其涉及一种单细胞阵列微芯片及其制造、电测量和电穿孔方法。
背景技术
细胞是生命活动的基本单元，细胞测量和分析已引起科学界和医疗领域的极大关注。细胞测量一般包括生物力学测量、电学测量、运动行为和物理化学特性测量等，采用的测量方法主要有：显微观察、电化学测量、光学测量等，其中显微观察和光学测量常常需要对细胞进行化学或者荧光标记，其操作复杂、功能单一，并在一定程度上改变了细胞原有的特质。而电化学测量则不需要标记，对细胞无损伤，能够对细胞在自然培养状态下连续、实时测量，具有比常规化学方法更高的灵敏度、测量可以全自动进行、无需人工干预等诸多优势。
电化学测量技术中，为实现单细胞原位精细测量，细胞定位必不可少，在众多微操作技术中，介电泳（dielectrophoresis，DEP）被广泛用于各种细胞、病毒、DNA等研究，成为生命科学领域研究细胞和生物分子不可或缺的有效工具。
现有的有关细胞电测量技术的器件通常不对细胞进行定位，由于细胞分布的随机性，其电测量的效率较低、精度差；且现有的细胞电穿孔技术通常不对细胞进行定位、测量，由于细胞分布的随机性，其电穿孔的效率低、精度差、细胞死亡率高、可控性不好。
发明内容
（一）要解决的技术问题
本发明要解决的技术问题是：提供一种单细胞阵列微芯片及其制造、电测量和电穿孔方法，其能够有效提高单细胞的电测量效率、精度，并可提升单细胞的电穿孔效率和可控性、降低细胞死亡率。
（二）技术方案
为解决上述问题，本发明提供了一种单细胞阵列微芯片，包括基底、阵列式的多个定位微电极、阵列式的多个电测量微电极对和样品池微腔体，其中，每相邻的四个定位微电极构成一个排列呈菱形的定位单元；
所述阵列式的多个定位微电极中的每一行定位微电极通过横向电极连线连接，或者，所述阵列式的多个定位微电极中，每个排列呈菱形的定位单元中的两对相对的定位微电极对分别通过横向电极连线和纵向电极连线与相邻定位单元中的定位微电极连接；
所述定位单元的中心位置分布有一电测量微电极对，所述电测量微电极对中的两个电极分别通过横向电极连线和纵向电极连线与相邻电测量微电极对中的电极连接。
前述的单细胞阵列微芯片中，所述横向电极连线和纵向电极连线分别布置在两层连线层中，两连线层之间由绝缘层隔开。
前述的单细胞阵列微芯片中，所述基底为绝缘材料或上覆有绝缘层的硅材料。
前述的单细胞阵列微芯片中，所述样品池微腔体为聚合物材料或者玻璃材料。
一种前述的单细胞阵列微芯片的制造方法，包括以下步骤：
A：在绝缘的基底上沉积第一层金属层并刻蚀，使所述第一层金属层上形成纵向电极连线；
B：沉积第一层绝缘层并刻蚀，使所述第一层绝缘层上露出电极窗口；
C：沉积第二层金属层并刻蚀，使所述第二层金属层上形成横向电极连线；
D：沉积第二层绝缘层并刻蚀，使所述第二层绝缘层上露出电极窗口；
E：沉积第三层金属层并刻蚀，使所述第三层金属层上形成定位微电极和电测量微电极对；
F：黏附样品池微腔体。
一种利用前述单细胞阵列微芯片对细胞进行电测量的方法，包括以下步骤：
S1：将细胞溶液放入样品池微腔体中；
S2：向定位微电极施加交流信号，细胞在负的介电力的作用下向电场中场强较低的位置运动；
S3：采用横、纵向扫描的方式，遍历由多个电测量微电极对构成的阵列，分别对每个电测量微电极对中的两电极施加电压，测量两电极间的电流信号；
S4：根据测得的每个电测量微电极对中的两电极间的电流信号，判断电测量微电极对中的电极处是否有细胞被定位；
S5：分别对电极处定位有细胞的电测量微电极对中的两电极施加电压，测量两电极间的电流信号，完成对细胞的电测量。
前述的对细胞进行电测量的方法中，所述步骤S4进一步包括：提前通过实验标定电测量微电极对中两电极间的在细胞被定位和细胞未被定位时的电流范围，根据测得的电测量微电极对中两电极间的电流所对应的电流范围判断细胞是否被定位。
一种利用前述单细胞阵列微芯片对细胞进行电穿孔的方法，包括以下步骤：
S11：将细胞溶液放入样品池微腔体中；
S22：向定位微电极施加交流信号，细胞在负的介电力的作用下向电场中场强较低的位置运动；
S33：采用横、纵向扫描的方式，遍历由多个电测量微电极对构成的阵列，分别对每个电测量微电极对中的两电极施加电压，测量两电极间的电流信号；
S44：根据测得的电测量微电极对中两电极间的电流信号，判断电测量微电极对中的电极处是否有细胞被定位；
S55：分别对电极处定位有细胞的电测量微电极对中的两电极施加脉冲电压信号，实现对被定位在电测量微电极对的电极处的细胞的电穿孔。
前述的对细胞进行电穿孔的方法中，所述步骤S44进一步包括：提前通过实验标定电测量微电极对中两电极间的在细胞被定位和细胞未被定位时的电流范围，根据测得的电测量微电极对中两电极间的电流所对应的电流范围判断细胞是否被定位。
（三）有益效果
本发明将细胞定位与细胞电测量和电穿孔相结合，采用无标记、无损伤的微操纵、定位和参数测量分析技术，可实现单细胞阵列、多模式的原位在线测量和分析；由于定位后的细胞位置固定，可有效提高细胞的电学检测精度，并可提升细胞的电穿孔效率、降低细胞死亡率。本发明还可将自动化控制技术引入细胞微操纵和电测量中，通过电测量反馈进行细胞定位的自动控制，最终可实现快速、精确和多模式的细胞自动化测量和分析。
附图说明
图1为本发明所述单细胞阵列微芯片的电极结构示意图；
图2为本发明实施例1中单细胞阵列微芯片的电极连线结构示意图；
图3为本发明实施例2中单细胞阵列微芯片的电极连线结构示意图；
图4a-4f为本发明实施例3中所述单细胞阵列微芯片的制作工艺的流程图。
具体实施方式
下面结合附图和实施例，对本发明的具体实施方式作进一步详细描述。以下实施例用于说明本发明，但不用来限制本发明的范围。
实施例1：如图1-2所示，本发明提供了一种单细胞阵列微芯片，包括基底1、阵列式的多个定位微电极2、阵列式的多个电测量微电极对3和样品池微腔体4，其中，每相邻的四个定位微电极2构成一个排列呈菱形的定位单元，所述定位单元的中心位置分布有一电测量微电极对3；每一行定位微电极2通过横向电极连线5连接，所述电测量微电极对3中的两个电极分别通过横向电极连线5和纵向电极连线6与相邻电测量微电极对3中的电极连接。
所述横向电极连线5和纵向电极连线6分别布置在两层连线层中，两连线层之间由绝缘层隔开。
所述基底1为绝缘材料（如玻璃）或上覆有绝缘层的硅材料。所述样品池微腔体4为PDMS聚合物材料或玻璃材料。所述绝缘层为氮化硅、二氧化硅或聚合物材料。所述样品池微腔体4可采用模具制作而成。电测量微电极对3的横向电极连线通过焊盘14电输入和电输出，电测量微电极对3的纵向电极连线通过焊盘15电输入和电输出；定位微电极2的横向电极连线通过焊盘16电输入。
实施例2：如图3所示，本实施例与实施例1不同的是，所述排列呈菱形的每个定位单元中的两个相对的定位微电极2，分别通过横向电极连线5和纵向电极连线6与相邻定位单元中的定位微电极2连接。
实施例3：一种前述的单细胞阵列微芯片的制造方法，包括以下步骤：
A：在绝缘的基底1上沉积第一层金属层并刻蚀，使所述第一层金属层上形成纵向电极连线6；
B：沉积第一层绝缘层并刻蚀，使所述第一层绝缘层上露出电极窗口；
C：沉积第二层金属层并刻蚀，使所述第二层金属层上形成横向电极连线5；
D：沉积第二层绝缘层并刻蚀，使所述第二层绝缘层上露出电极窗口；
E：沉积第三层金属层并刻蚀，使所述第三层金属层上形成定位微电极2和电测量微电极对3；
F：黏附样品池微腔体4。
如图4a-图4f所示，本实施例提供了一种单细胞阵列微芯片的具体制作工艺，其采用玻璃片作为基底，然后沿同一方向在所述基底上顺序进行如下工艺步骤：
步骤（a）：在玻璃基底1上溅射一层钛/金（Ti/Au）薄膜形成第一层金属层7，通过光刻图形化该金属层形成电测量微电极对的纵向电极连线6；
步骤（b）：沉积一层SiO2薄膜8，刻蚀绝缘层露出电极（或焊盘）窗口9；
步骤（c）：溅射第二层金属层钛/金（Ti/Au）薄膜10，通过光刻图形化该金属层形成横向电极连线5；
步骤（d）：沉积第二层SiO2薄膜11，刻蚀绝缘层露出电极（或焊盘）窗口12；
步骤（e）：溅射第三层金属层钛/金（Ti/Au）薄膜13，通过光刻图形化该金属层形成定位微电极2、电测量微电极对3和焊盘14、15、16、17；
步骤（f）：将样品池微腔体4黏附在芯片上，使得阵列式定位微电极2和电测量微电极对3位于样品池微腔体4内，焊盘14、15、16、17位于样品池微腔体4外。
实施例4：一种利用前述单细胞阵列微芯片对细胞进行电测量的方法，包括以下步骤：
S1：将细胞溶液放入样品池微腔体4中；
S2：向定位微电极2施加交流信号，在相对电极上施加相同的正弦信号，在相邻电极上施加相位相差180°的正弦信号；细胞在负的介电力的作用下向电场中场强较低的位置运动；
S3：采用横、纵向扫描的方式，遍历由多个电测量微电极对3构成的阵列，分别对每个电测量微电极对3中的两电极施加电压，测量两电极间的电流信号；
S4：根据测得的每个电测量微电极对3中的两电极间的电流信号，判断电测量微电极对3中的电极处是否有细胞被定位；
本步骤中，需提前通过实验标定电测量微电极对3中两电极间的在细胞被定位和细胞未被定位时的电流范围，根据测得的电测量微电极对3中两电极间的电流所对应的电流范围判断细胞是否被定位。
若测得的电测量微电极对3中两电极间的电流落在电测量微电极对3中两电极间的在细胞被定位时的电流范围内，则有细胞被定位。
若测得的电测量微电极对3中两电极间的电流落在电测量微电极对3中两电极间的在细胞未被定位时的电流范围内，则细胞未被定位。
S5：分别对电极处定位有细胞的电测量微电极对3中的两电极施加电压，测量两电极间的电流信号，完成对细胞的电测量。对测量电极施加的电压可以是直流、交流或脉冲信号。
实施例5：一种利用前述单细胞阵列微芯片对细胞进行电穿孔的方法，包括以下步骤：
S11：将细胞溶液放入样品池微腔体4中；
S22：向定位微电极2施加交流信号，在相对电极上施加相同的正弦信号，在相邻电极上施加相位相差180°的正弦信号，细胞在负的介电力的作用下向电场中场强较低的位置运动；
S33：采用横、纵向扫描的方式，遍历由多个电测量微电极对3构成的阵列，对每个电测量微电极对3中的两电极施加电压，测量两电极间的电流信号；
S44：根据测得的电测量微电极对3中两电极间的电流信号，判断电测量微电极对3中的电极处是否有细胞被定位；
本步骤中，需提前通过实验标定电测量微电极对3中两电极间的在细胞被定位和细胞未被定位时的电流范围，根据测得的电测量微电极对3中两电极间的电流所对应的电流范围判断细胞是否被定位。
若测得的电测量微电极对3中两电极间的电流落在电测量微电极对3中两电极间的在细胞被定位时的电流范围内，则有细胞被定位。
若测得的电测量微电极对3中两电极间的电流落在电测量微电极对3中两电极间的在细胞未被定位时的电流范围内，则细胞未被定位。
S55：分别对电极处定位有细胞的电测量微电极对3中的两电极施加脉冲电压信号，实现对被定位在电测量微电极对3的电极处的细胞的电穿孔。
以上实施方式仅用于说明本发明，而并非对本发明的限制，有关技术领域的普通技术人员，在不脱离本发明的精神和范围的情况下，还可以做出各种变化和变型，因此所有等同的技术方案也属于本发明的范畴，本发明的专利保护范围应由权利要求限定。
工业实用性
本发明将细胞定位与细胞电测量和电穿孔相结合，采用无标记、无损伤的微操纵、定位和参数测量分析技术，可实现单细胞阵列、多模式的原位在线测量和分析；由于定位后的细胞位置固定，可有效提高细胞的电学检测精度，并可提升细胞的电穿孔效率、降低细胞死亡率。本发明还可将自动化控制技术引入细胞微操纵和电测量中，通过电测量反馈进行细胞定位的自动控制，最终可实现快速、精确和多模式的细胞自动化测量和分析。

Claims (1)

  1. 权 利 要 求 书
    1、一种单细胞阵列微芯片，其特征在于，包括基底（1）、阵列式的多个定位微电极（2）、阵列式的多个电测量微电极对（3）和样品池微腔体（4），其中，每相邻的四个定位微电极（2）构成一个排列呈菱形的定位单元；
    所述阵列式的多个定位微电极（2）中的每一行定位微电极（2）通过横向电极连线（5）连接，或者，所述阵列式的多个定位微电极（2）中，每个排列呈菱形的定位单元中的两个相对的定位微电极（2）分别通过横向电极连线（5）和纵向电极连线（6）与相邻定位单元中的定位微电极（2）连接；
    所述定位单元的中心位置分布有一电测量微电极对（3），所述电测量微电极对（3）中的两个电极分别通过横向电极连线（5）和纵向电极连线（6）与相邻电测量微电极对（3）中的电极连接。
    2、如权利要求1所述的单细胞阵列微芯片，其特征在于，所述横向电极连线（5）和纵向电极连线（6）分别布置在两层连线层中，两连线层之间由绝缘层隔开。
    3、如权利要求1所述的单细胞阵列微芯片，其特征在于，所述基底（1）为绝缘材料或上覆有绝缘层的硅材料。
    4、如权利要求1所述的单细胞阵列微芯片，其特征在于，所述样品池微腔体（4）为聚合物材料或玻璃材料。
    5、一种权利要求1-4中任一项所述的单细胞阵列微芯片的制造方法，其特征在于，包括以下步骤：
    A：在绝缘的基底（1）上沉积第一层金属层并刻蚀，使所述第一层金属层上形成纵向电极连线（6）；
    B：沉积第一层绝缘层并刻蚀，使所述第一层绝缘层上露出电极窗口；
    C：沉积第二层金属层并刻蚀，使所述第二层金属层上形成横向电极连线（5）；
    D：沉积第二层绝缘层并刻蚀，使所述第二层绝缘层上露出电极窗口；
    E：沉积第三层金属层并刻蚀，使所述第三层金属层上形成定位微电极（2）和电测量微电极对（3）；
    F：黏附样品池微腔体（4）。
    6、一种利用权利要求1-4中任一项所述单细胞阵列微芯片对细胞进行电测量的方法，其特征在于，包括以下步骤：
    S1：将细胞溶液放入样品池微腔体（4）中；
    S2：向定位微电极（2）施加交流信号，细胞在负的介电力的作用下向电场中场强较低的位置运动；
    S3：采用横、纵向扫描的方式，遍历由多个电测量微电极对（3）构成的阵列，分别对每个电测量微电极对（3）中的两电极施加电压，测量两电极间的电流信号；
    S4：根据测得的每个电测量微电极对（3）中的两电极间的电流信号，判断电测量微电极对（3）中的电极处是否有细胞被定位；
    S5：分别对电极处定位有细胞的电测量微电极对（3）中的两电极施加电压，测量两电极间的电流信号，完成对细胞的电测量。
    7、如权利要求6所述的对细胞进行电测量的方法，其特征在于，所述步骤S4进一步包括：提前通过实验标定电测量微电极对（3）中两电极间的在细胞被定位和细胞未被定位时的电流范围，根据测得的电测量微电极对（3）中两电极间的电流所对应的电流范围判断细胞是否被定位。
    8、一种利用权利要求1-4中任一项所述单细胞阵列微芯片对细胞进行电穿孔的方法，其特征在于，包括以下步骤：
    S11：将细胞溶液放入样品池微腔体（4）中；
    S22：向定位微电极（2）施加交流信号，细胞在负的介电力的作用下向电场中场强较低的位置运动；
    S33：采用横、纵向扫描的方式，遍历由多个电测量微电极对（3）构成的阵列，分别对每个电测量微电极对（3）中的两电极施加电压，测量两电极间的电流信号；
    S44：根据测得的电测量微电极对（3）中两电极间的电流信号，判断电测量微电极对（3）中的电极处是否有细胞被定位；
    S55：分别对电极处定位有细胞的电测量微电极对（3）中的两电极施加脉冲电压信号，实现对被定位在电测量微电极对（3）的电极处的细胞的电穿孔。
    9、如权利要求8所述的对细胞进行电穿孔的方法，其特征在于，所述步骤S44进一步包括：提前通过实验标定电测量微电极对（3）中两电极间的在细胞被定位和细胞未被定位时的电流范围，根据测得的电测量微电极对（3）中两电极间的电流所对应的电流范围判断细胞是否被定位。
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