CN103630579A - Cell impedance analysis chip and apparatus - Google Patents

Cell impedance analysis chip and apparatus Download PDF

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Publication number
CN103630579A
CN103630579A CN201310061079.4A CN201310061079A CN103630579A CN 103630579 A CN103630579 A CN 103630579A CN 201310061079 A CN201310061079 A CN 201310061079A CN 103630579 A CN103630579 A CN 103630579A
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impedance analysis
impedance
measuring
cell
area
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赵湛
谭静
方震
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Institute of Electronics of CAS
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Institute of Electronics of CAS
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Abstract

The invention provides a cell impedance analysis chip and an apparatus. The chip comprises: an insulative substrate whose center position is defined as an impedance analysis area, a minisize metal electrode array formed on the insulative substrate, an insulative protection layer and a measure trap; the minisize metal electrode array comprises N minisize metal electrodes which are insulative mutually and uniformly distributed at the periphery of the impedance analysis area in a radiation shape, and the sensitive part at the inner side of each minisize metal electrode stretches into the impedance analysis area, wherein N is 4 or more; the insulative protection layer covers other areas except the impedance analysis area, the inner sensitive parts of the minisize metal electrodes and outer side lead wire parts; the measure trap is formed on the periphery insulative protection layer of the impedance analysis area; and an accommodation area for accommodating a to-be-analyzed sample is formed on the impedance analysis area, and the inside radius of the accommodation area is equal to or slightly greater than the radius of the impedance analysis area. The provided cell impedance analysis chip and the apparatus are capable of performing impedance measuring and analysis on a single cell.

Description

Chip and instrument for cell impedance analysis
Technical Field
The invention belongs to the technical field of micro sensor chips, and particularly relates to a cell impedance analysis chip and a cell impedance analysis instrument capable of detecting and analyzing biological cell impedance.
Background
With the social concern about environment, health and the like, people have higher and higher requirements on biomedical detection. The study of cell level can obtain more accurate and comprehensive information reflecting biological physiological state and process, and can make people better understand some special cell functions in cell population, and more deeply know cell individual difference, cell-cell interaction and information transmission, and physiological influence stimulated by neurotransmitter, medicine or poison. Analysis at the cellular level is of great and profound significance.
Due to technical limitations, conventional biological analysis methods generally use a large number of cells as research objects, and reflect physiological information of the cells with average results. Due to the distribution of various chemical components in biological tissues and the high degree of heterogeneity of the cells themselves, extensive cellular analysis often leads to inaccurate or even erroneous conclusions. It is therefore important to directly analyze the properties at the cellular and sub-cellular level. According to different impedance characteristics of cells under different physiological states, the micro multi-electrode impedance analysis chip is manufactured by using the MEMS process, and the multi-electrode data acquisition enriches the sampling information and improves the analysis precision and resolution.
Reference 1 (chinese patent, patent application No. 98813315) proposes a cell impedance analysis chip. FIG. 1A is a schematic diagram of a cell impedance analysis chip according to the prior art. FIG. 1B is an enlarged view of the central impedance analysis region of the cellular impedance analysis chip shown in FIG. 1A. Referring to fig. 1A and 1B, the cell impedance analyzing chip includes an insulating substrate, a reference electrode, a conductive wiring connected to the reference electrode, a measuring micro-electrode array provided on the insulating substrate, a conductive pattern for the measuring micro-electrode wiring, an electrical contact connected to an end of the conductive pattern, an insulating film covering a surface of the conductive pattern and the reference electrode wiring, and a wall surrounding a region including the measuring micro-electrodes on a surface of the insulating film, the cell potential measuring electrode for measuring electrophysiological activities while culturing cells or cell tissues in the region surrounded by the wall, wherein the reference electrodes having a smaller impedance than the measuring micro-electrodes are respectively arranged at a plurality of positions in the region surrounded by the wall and outside the measuring micro-electrode array, the measuring regions are insulated from each other, and the electrical contact for the reference electrode is connected to an end of the conductive pattern connected to the reference electrode, and wherein the measurement microelectrodes are insulated from each other, the reference electrodes from each other, and the measurement microelectrodes and the reference electrodes from each other.
In the process of implementing the present invention, the inventors found that the cell impedance analysis chip described above has the following technical defects: (1) the electrode structure is arranged in a planar array and is only suitable for analyzing nerve cells with larger sizes; (2) it can not scan and image the impedance distribution of the cell, and can carry out dynamic measurement and analysis on the life activity in the cell.
Disclosure of Invention
Technical problem to be solved
To solve one or more of the above problems, the present invention provides a chip and an apparatus for cell impedance analysis.
(II) technical scheme
According to an aspect of the present invention, there is provided a cell impedance analysis chip including: an insulating substrate, the central position of which is defined as an impedance analysis area; the miniature metal electrode array is formed on the insulating substrate and comprises N miniature metal electrodes which are insulated with each other, the N miniature metal electrodes are uniformly distributed on the circumference of the impedance analysis area in a radial shape, a sensitive part at the inner side of each miniature metal electrode extends into the impedance analysis area, wherein N is more than or equal to 4; the insulating protective layer covers other areas except the impedance analysis area and the inner sensitive part and the outer lead part on the miniature metal electrode; and the measuring trap is formed on the peripheral insulating protection layer of the impedance analysis area, an accommodating area for accommodating a sample to be analyzed is formed above the impedance analysis area, and the inner radius of the accommodating area is equal to or slightly larger than that of the impedance analysis area.
According to another aspect of the present invention, there is also provided a cell impedance analyzing apparatus including the above cell impedance analyzing chip. The instrument further comprises: the device comprises a driving current module, a voltage measuring module and an impedance image reconstruction module; the drive current module is provided with two electrodes, and the two electrodes are connected to two miniature metal electrodes in the N miniature metal electrodes according to a preset current drive module and used for inputting drive current to a sample in the measurement trap; the voltage measuring module is provided with two electrodes which are connected to the other two micro metal electrodes except the two micro metal electrodes according to a preset voltage measuring mode and used for measuring the voltage generated by the sample in the measuring well under the excitation of the driving current on the two micro metal electrodes; and the impedance image reconstruction module is used for reconstructing an impedance image of the sample in the impedance analysis area according to the voltage of the miniature metal electrode obtained by combining the plurality of current driving modes and the voltage measuring mode.
(III) advantageous effects
According to the technical scheme, the cell impedance analysis chip and the cell impedance analysis instrument have the following beneficial effects:
(1) the microelectrode which is distributed in a ring shape is arranged on the periphery of the measuring trap which is slightly larger than the size of the cell, so that the impedance measurement analysis can be carried out on the single cell. Specifically, the method comprises the following steps: according to the physical phenomenon that different physiological states of cells have different electrical impedance characteristics, a safe and constant voltage or current is applied to a sample, and the current or voltage on the surface of the sample is measured through a measuring electrode to obtain the internal electrical impedance distribution. Impedance measurement analysis can be carried out on small-sized single cells, particularly spherical or similar cells, and the electrodes are arranged around the cells, so that the interference on cell analysis and test is small;
(2) the cells can be imaged by impedance scanning, and the anatomy and structure of the moving tissue of the cells are reflected by the electrical characteristics. The method adopts a current driving voltage measurement driving measurement mode, the current driving mode comprises an adjacent driving mode, a cross driving mode and a relative driving mode, the adopted voltage measurement mode is used for measuring the voltages of other adjacent electrodes except for an exciting electrode, data are collected by adopting a proper current driving mode and a proper voltage measurement mode, and impedance image reconstruction is carried out on a sample in an impedance analysis area, so that biological information of cell activity is reflected visually and comprehensively through images.
(3) The chip extracts electrical characteristic information related to physiological and pathological states of the micro-nano scale cell sample, can reflect the anatomical structure of the micro-nano scale cell sample, and can also give a functional image result. The apparatus has the characteristics of miniaturized structure, sensitization measurement, non-invasive sample, information diversification and the like, can realize impedance measurement and analysis of cell and subcellular levels, provides a powerful basis for researching physiological changes of cells, and has important significance for prediction, diagnosis and treatment of diseases.
Drawings
FIG. 1A is a schematic diagram of a cell impedance analysis chip according to the prior art;
FIG. 1B is an enlarged view of the central impedance analysis region of the cellular impedance analysis chip shown in FIG. 1A
FIG. 2A is a schematic top view of a cell impedance analysis chip according to an embodiment of the present invention;
FIG. 2B is a cross-sectional view of the cellular impedance analysis chip shown in FIG. 2A taken along the A-A direction;
FIG. 3 is a schematic structural diagram of a cell impedance analysis apparatus according to an embodiment of the present invention.
[ description of main reference symbols of the invention ]
1-an insulating substrate; 2-micro metal electrode array
3-insulating protective layer; 4-a measurement well;
5-sample cover.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is described in further detail below with reference to specific embodiments and the accompanying drawings. It should be noted that in the drawings or description, the same drawing reference numerals are used for similar or identical parts. Implementations not depicted or described in the drawings are of a form known to those of ordinary skill in the art. Additionally, while exemplifications of parameters including particular values may be provided herein, it is to be understood that the parameters need not be exactly equal to the respective values, but may be approximated to the respective values within acceptable error margins or design constraints.
The invention provides a chip and an instrument for cell impedance analysis, which are fused with a cell impedance characteristic principle and an electrical impedance imaging technology, so as to realize impedance analysis of biological cells.
In one exemplary embodiment of the present invention, a cell impedance analysis chip is provided. FIG. 2A is a schematic top view of a cell impedance analysis chip according to an embodiment of the present invention. FIG. 2B is a cross-sectional view of the cellular impedance analysis chip shown in FIG. 2A taken along the A-A direction. Referring to fig. 2A and 2B, the cell impedance analysis chip of the present embodiment includes: an insulating substrate 1 whose central position is defined as an impedance analysis region; the miniature metal electrode array 2 comprises N miniature metal electrodes which are insulated with each other, the N miniature metal electrodes are uniformly distributed on the circumference of the impedance analysis area in a radial shape, a sensitive part at the inner side of each miniature metal electrode extends into the impedance analysis area, wherein N is more than or equal to 4; the insulating protective layer 3 covers the region of the miniature metal electrode except the inner sensitive part and the outer lead part; the measuring trap 4 is positioned at the periphery of the impedance analysis area, the height of the measuring trap is higher than that of the impedance analysis area, so that an accommodating area for accommodating a sample to be analyzed is formed above the impedance analysis area, and the inner radius of the accommodating area is equal to or slightly larger than that of the impedance analysis area; and the sample cover 5 is fixed on the insulating protective layer outside the measuring trap, and the height of the sample cover is higher than that of the measuring trap so as to isolate the analysis blocking area from the external environment.
The technical details of each component of the cell impedance analysis chip according to the embodiment of the present invention are described in detail below.
In this example, the insulating substrate is 1.5X 1.5cm2But the invention is not limited thereto. One skilled in the art can also select other types and sizes of insulating substrates, such as silicon wafers with silicon nitride deposited on the surface or ITO glass, as desired.
A circular impedance analysis region is provided in the central region of the glass sheet, the circular impedance analysis region having dimensions of 50 μm for the convenience of measuring the impedance characteristics of cells or subcells. It will be clear to the skilled person that the dimensions of the circular impedance analysis region depend on the size of the cells in the sample, typically on the micrometer scale, and can be specifically determined according to the measurement requirements.
In the cell impedance analysis chip shown in fig. 2 and 3, 16 micro metal electrodes are distributed on the upper surface of the glass plate around the impedance analysis region. The size and the shape of the micro metal electrodes are the same, the electrode spacing is the same, and the line width of the top ends of the electrodes is the same as the line width of the electrode spacing. Of course, the number of micro metal electrodes is not limited to 16, and may be (but is not limited to) 8, 16 or 32.
Each miniature metal electrode can be used as an excitation electrode input signal and can also be used as a response electrode output signal. The material of the micro metal electrode array can be (but is not limited to) gold or platinum, which is fabricated by using sputtering and lift-off process.
The top end of the inner side sensitive part of the miniature metal electrode is in a linear or arc shape, is uniformly distributed on the central circular impedance analysis area, has the length of about 2-10 mu m extending into the impedance analysis area, and is used for adding measuring current to a sample in the impedance analysis area or collecting a voltage signal generated by the impedance of the sample to be analyzed; the outer lead part is rectangular and can be connected with an external circuit through a lead wire and used for receiving measuring current or transmitting a voltage signal collected by the sensitive part outwards.
In the present embodiment, the insulating protection layer 3 is a silicon nitride layer, and the thickness thereof is 8000A. When the silicon nitride layer is prepared, firstly, a silicon nitride insulating layer is manufactured above the whole substrate including the micro metal electrode array positioned on the substrate by utilizing a Plasma Enhanced Chemical Vapor Deposition (PECVD) process, then, the silicon nitride layer of the impedance analysis area and the lead part at the outer side of the micro metal electrode is removed by utilizing an ion etching process, wherein the radius of the silicon nitride in the central area is slightly larger than that of the impedance analysis area and is about 55 mu m. It is particularly emphasized that the sensitive part of the micro-metal electrode extending into the impedance analysis area is exposed and not covered by the silicon nitride layer. In addition, the material of the insulating protection layer 3 may also be an organic polymer having insulating properties, such as: polyimide, polydimethylsiloxane, or a photoresist having an insulating property, and the like.
Referring to fig. 2 and 3, a measurement well is formed by a polymer right above the central circular impedance analysis region for accommodating a sample to be measured. The electrodes are exposed within the measurement well and are in contact with the sample. The measuring well is cylindrical and is made of SU-8, and has a height of 30 μm, an inner radius of 60 μm and an outer radius of 600 μm.
Of course, it should be clear to one skilled in the art that the dimensions of the measurement well, the inside radius of the well, and the outside radius of the well can all be flexibly adjusted as desired. The material for preparing the polymer trap can be polyimide, Polydimethylsiloxane (PDMS) or other biocompatible polymers besides SU-8 photoresist.
The cell impedance analysis signal further comprises a sample cover 5 for storing a buffer. The inner diameter of the inner cylindrical groove of the sample cover is larger than the outer diameter of the measuring trap, and the sample cover is fixed on the insulating protective layer outside the measuring trap. In this embodiment, the material of the sample cover is polyimide or PDMS, and the shape thereof may be circular, square or rectangular. The radius of the cylindrical groove is 2mm, and the wall thickness is 2 mm.
In performing the measurement, the sample cover is pierced by the needle for injecting the sample, the sample is injected into the measurement well, and then the needle is pulled out. The buffer does not flow out of the pin hole due to the surface tension of the buffer. Through this sample lid, reduce the sample evaporation, reduce the influence of environment to the experiment. For the chip holding the sample cover, it is disposable. When no sample cap is included, it can be reused, but such use is not recommended.
Thus, the introduction of the cell impedance analysis chip is completed. According to the description of the present embodiment, those skilled in the art should be able to clearly understand the cell impedance analysis chip of the present invention in combination with their own expertise.
In another exemplary embodiment of the present invention, a cell impedance analysis apparatus is also provided. As shown in fig. 3, the apparatus comprises: the cell impedance analysis chip, the driving current module, the measuring voltage module and the impedance image reconstruction module are used for analyzing the cell impedance. The drive current module is provided with two electrodes, and the two electrodes are connected to two miniature metal electrodes in the N miniature metal electrodes according to a preset current drive module and used for inputting drive current to the sample in the measurement trap. The voltage measuring module is provided with two electrodes which are connected to the other two micro metal electrodes except the two micro metal electrodes according to a preset voltage measuring mode and used for measuring the voltage generated by the sample in the measuring well under the excitation of the driving current on the two micro metal electrodes. And the impedance image reconstruction module is used for reconstructing an impedance image of the sample in the impedance analysis area according to the voltage of the miniature metal electrode obtained by combining the plurality of current driving modes and the voltage measuring mode. The current driving mode has an adjacent, crossed and opposite driving mode, and the voltage measuring mode is used for measuring the voltage of other adjacent electrodes except the excitation electrode.
Thus, the present embodiment of the cellular impedance analyzer is described. According to the description of the present embodiment, those skilled in the art should be able to clearly understand the cell impedance analysis apparatus of the present invention in combination with their own expertise.
Furthermore, it should be noted that the above definitions of the elements are not limited to the specific structures or shapes mentioned in the embodiments, and those skilled in the art may easily substitute them, for example: the corresponding sample trap structure and electrode arrangement mode can be designed for the cells with specific shapes.
In summary, according to the physical phenomenon that different physiological states of cells have different electrical impedance characteristics, a safe and constant voltage or current is applied to the sample, and the current or voltage on the surface of the sample is measured by the measuring electrodes to reconstruct an internal electrical impedance distribution image, so that biological information of the sample can be intuitively and comprehensively reflected through the image.
The above-mentioned embodiments are intended to illustrate the objects, technical solutions and advantages of the present invention in further detail, and it should be understood that the above-mentioned embodiments are only exemplary embodiments of the present invention, and are not intended to limit the present invention, and any modifications, equivalents, improvements and the like made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims (10)

1. A cell impedance analysis chip, comprising:
an insulating substrate, the central position of which is defined as an impedance analysis area;
the miniature metal electrode array is formed on the insulating substrate and comprises N miniature metal electrodes which are insulated with each other, the N miniature metal electrodes are uniformly distributed on the circumference of the impedance analysis area in a radial shape, a sensitive part at the inner side of each miniature metal electrode extends into the impedance analysis area, and N is more than or equal to 4;
the insulating protective layer covers other areas except the impedance analysis area and the inner sensitive part and the outer lead part on the miniature metal electrode; and
and the measuring trap is formed on the peripheral insulating protection layer of the impedance analysis area, an accommodating area for accommodating a sample to be analyzed is formed above the impedance analysis area, and the inner radius of the accommodating area is equal to or slightly larger than that of the impedance analysis area.
2. The cell impedance analysis chip of claim 1, further comprising:
and the sample cover is fixed on the insulating protective layer outside the measuring trap and is higher than the measuring trap so as to isolate the sample accommodating area to be analyzed from the external environment.
3. The cell impedance analysis chip according to claim 2, wherein the material of the sample cover is polyimide, PDMS, which is hollow and stores buffer solution inside.
4. The cell impedance analysis chip according to claim 1, wherein the size and shape of each of the N micro metal electrodes are the same, the electrode pitch between the micro electrodes is the same, and the electrode tip line width is the same as the electrode pitch line width.
5. The cell impedance analysis chip according to claim 4, wherein the top of the inner sensitive portion of the micro metal electrode is shaped as a line or arc, and is uniformly and symmetrically distributed on the central circular impedance analysis area.
6. The cell impedance analysis chip of claim 1, wherein the impedance analysis region is circular and has a radius of 50 μm;
the length of the miniature metal electrode extending into the impedance analysis area is about 2-10 μm;
the insulating protective layer forms a circular ring at the periphery of the impedance analysis area, and the radius of the circular ring is 55 mu m; the inner radius of the measuring trap is 60 μm, and the outer radius of the measuring trap is 600 μm.
7. The cell impedance analysis chip according to any one of claims 1 to 6, wherein the insulating substrate is a glass sheet, ITO glass, or a silicon wafer with silicon nitride deposited on the surface.
8. The cell impedance analysis chip according to any one of claims 1 to 6, wherein the material of the insulating protective layer is silicon nitride, polyimide, or polydimethylsiloxane;
the measuring trap is made of SU-8 photoresist, polyimide and polydimethylsiloxane.
9. A cell impedance analyzing apparatus comprising the cell impedance analyzing chip according to any one of claims 1 to 8, further comprising: the device comprises a driving current module, a voltage measuring module and an impedance image reconstruction module; wherein,
the driving current module is provided with two electrodes, and the two electrodes are connected to two miniature metal electrodes in the N miniature metal electrodes according to a preset current driving module and used for inputting driving current to the sample in the measuring well;
the voltage measuring module is provided with two electrodes which are connected to the other two micro metal electrodes except the two micro metal electrodes according to a preset voltage measuring mode and used for measuring the voltage generated by the sample in the measuring trap on the two micro metal electrodes under the excitation of the driving current;
and the impedance image reconstruction module is used for reconstructing an impedance image of the sample in the impedance analysis area according to the voltage of the miniature metal electrode obtained by combining the plurality of current driving modes and the voltage measuring mode.
10. The cellular impedance analysis instrument of claim 9, wherein the current driving mode has an adjacent, crossed and opposite driving mode, and the voltage measuring mode is to measure voltages of adjacent electrodes except for the excitation electrode.
CN201310061079.4A 2013-02-27 2013-02-27 Cell impedance analysis chip and apparatus Pending CN103630579A (en)

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CN108037159A (en) * 2017-11-02 2018-05-15 江苏大学 A kind of cytoactive detection method and device based on impedance spectrum integration feature
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CN108037159A (en) * 2017-11-02 2018-05-15 江苏大学 A kind of cytoactive detection method and device based on impedance spectrum integration feature
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WO2024145528A1 (en) * 2022-12-29 2024-07-04 The Board Of Trustees Of The Leland Stanford Junior University Devices, systems and methods for electrophysical recordings of suspension cultures

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Application publication date: 20140312