CN103630579A - Cell impedance analysis chip and apparatus - Google Patents

Abstract

The invention provides a chip and an instrument for cell impedance analysis. The chip includes: an insulating substrate, the central position of which is defined as an impedance analysis area; a miniature metal electrode array is formed on the insulating substrate, including N miniature metal electrodes insulated from each other, and the N miniature metal electrodes are uniformly distributed in a radial shape. On the circumference of the impedance analysis area, the inner sensitive part of each miniature metal electrode extends into the impedance analysis area, where N≥4; the insulating protective layer covers the inner sensitive part and the outer lead part of the impedance analysis area and the miniature metal electrode other regions; and the measurement well, which is formed on the surrounding insulating protective layer of the impedance analysis region, forms an accommodating area above the impedance analysis area to accommodate the sample to be analyzed, and the inner radius of the accommodating area is equal to or slightly Greater than the radius of the impedance analysis area. The cell impedance analysis chip and instrument provided by the invention can perform impedance measurement and analysis on single cells.

Description

Chip and Instrument for Cell Impedance Analysis

technical field

The invention belongs to the technical field of micro sensor chips, and in particular relates to a cell impedance analysis chip and an instrument capable of detecting and analyzing biological cell impedance.

Background technique

As society pays more attention to the environment and health, people have higher and higher requirements for biomedical testing. Research at the cell level can obtain more accurate and comprehensive information reflecting biological physiological states and processes, and can also enable people to better understand some special cell functions in cell populations, and gain a deeper understanding of individual differences in cells, cell Deeper information such as interaction and information transmission between neurotransmitters, physiological effects of drug or toxic stimulation, etc. Analysis at the cellular level has great and far-reaching significance.

Due to technical limitations, conventional biological analysis methods generally use a large number of cells as the research object, and use the average result to reflect the physiological information of the cells. Due to the highly heterogeneous distribution of various chemical components in biological tissues and the cells themselves, inaccurate or even wrong conclusions are often drawn from the analysis of a large number of cells. It is therefore important to directly analyze properties at the cellular and subcellular level. According to the different impedance characteristics of cells in different physiological states, a micro multi-electrode impedance analysis chip is produced by using MEMS technology. Multi-electrode data collection enriches the sampling information and improves the analysis accuracy and resolution.

Reference 1 (Chinese patent, patent application number: 98813315) proposes a cell impedance analysis chip. FIG. 1A is a schematic diagram of a prior art cell impedance analysis chip. FIG. 1B is an enlarged view of the central impedance analysis area of the cell impedance analysis chip shown in FIG. 1A . Please refer to Figure 1A and Figure 1B, the cell impedance analysis chip includes an insulating substrate, a reference electrode, a conductive wiring connected to the reference electrode, a measurement microelectrode array arranged on the insulating substrate, and a conductive pattern for the measurement microelectrode wiring , an electrical contact connected to an end of the conductive pattern, an insulating film covering the surface of the conductive pattern and a reference electrode wiring, and a wall surrounding an area containing a measuring microelectrode on the surface of the insulating film, the cell Potentiometric measuring electrodes are used to measure electrophysiological activity during the cultivation of cells or cell tissues in the area enclosed by the wall, wherein a reference electrode having a lower impedance than the measuring microelectrode is respectively arranged in the area enclosed by the wall At a plurality of locations, and outside of said array of measuring microelectrodes, insulated from each other in said measuring region, the electrical contacts for the reference electrodes are connected to the ends of the conductive patterns connected to the reference electrodes, and wherein each measuring microelectrode The electrodes, the reference electrodes, and the measuring microelectrodes and reference electrodes are insulated from each other.

In the process of realizing the present invention, the inventors found that the above-mentioned cell impedance analysis chip has the following technical defects: (1) it is an electrode structure arranged in a planar array, which is only suitable for the analysis of nerve cells with larger sizes; (2) It cannot scan and image the impedance distribution of cells, and perform dynamic measurement and analysis of life activities in cells.

Contents of the invention

(1) Technical problems to be solved

In order to solve one or more of the above problems, the present invention provides a chip and an instrument for cell impedance analysis.

(2) Technical solution

According to one aspect of the present invention, a cell impedance analysis chip is provided. The cell impedance analysis chip includes: an insulating substrate, the central position of which is defined as an impedance analysis area; a miniature metal electrode array is formed on the insulating substrate, including insulation from each other. N miniature metal electrodes, the N miniature metal electrodes are evenly distributed on the circumference of the impedance analysis area in a radial shape, and the sensitive part inside each miniature metal electrode extends into the impedance analysis area, where N≥4; the insulating protective layer , covering other areas except the impedance analysis area and the inner sensitive part and the outer lead part on the micro metal electrode; An accommodating area for the sample to be analyzed is placed, and the inner radius of the accommodating area is equal to or slightly larger than the radius of the impedance analysis area.

According to another aspect of the present invention, a cell impedance analysis instrument including the above-mentioned cell impedance analysis chip is also provided. The instrument also includes: a drive current module, a measurement voltage module, and an impedance image reconstruction module; wherein, the drive current module has 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. On the metal electrode, it is used to input the driving current to the sample in the measurement well; the measurement voltage module has two electrodes, and the two electrodes are connected to the other two miniature metal electrodes in addition to the above two miniature metal electrodes according to the preset voltage measurement mode. On the electrode, it is used to measure the voltage generated by the two miniature metal electrodes under the excitation of the above-mentioned drive current by the sample in the measurement well; The voltage of the micro metal electrode obtained by the combination of the impedance analysis area is used to reconstruct the impedance image of the sample.

(3) Beneficial effects

It can be seen from the above technical scheme that the cell impedance analysis chip and instrument of the present invention have the following beneficial effects:

(1) Microelectrodes distributed in a ring are used and arranged around the measurement well slightly larger than the size of the cells, so that the impedance measurement and analysis of single cells can be performed. Specifically: According to the physical phenomenon that different physiological states of cells have different electrical impedance characteristics, by applying a safe and constant voltage or current to the sample, and measuring the current or voltage on the surface of the sample through the measuring electrode, the internal electrical impedance distribution is obtained. . Impedance measurement analysis can be performed on small-sized single cells, especially spherical or similar-shaped cells, and the electrodes are arranged around the cells, which has little interference with cell analysis tests;

(2) Impedance scanning imaging can be performed on cells, and electrical characteristics can be used to reflect the anatomy and structure of cell activities. The drive measurement method of current drive voltage measurement is adopted. The current drive mode has adjacent, cross, and relative drive modes. The voltage measurement mode used is to measure the voltage of other adjacent electrodes except the excitation electrode. By adopting an appropriate current drive mode and voltage measurement mode to collect data, and reconstruct the impedance image of the sample in the impedance analysis area, so as to intuitively and comprehensively reflect the biological information of cell activities through the image.

(3) The electrical characteristic information related to the physiological and pathological state of the micro-nanoscale cell sample extracted by the chip can not only reflect its anatomical structure, but also give functional image results. The instrument has the characteristics of miniaturized structure, sensitive measurement, non-invasive sample, and diversified information. It can realize impedance measurement and analysis at the cell and subcellular level, provide a strong basis for studying the physiological changes of cells, and predict and diagnose diseases. , Treatment is of great significance.

Description of drawings

1A is a schematic diagram of a cell impedance analysis chip in the prior art;

Figure 1B is an enlarged view of the central impedance analysis area of the cell impedance analysis chip shown in Figure 1A

2A is a schematic top view of a cell impedance analysis chip according to an embodiment of the present invention;

2B is a cross-sectional view of the cell impedance analysis chip shown in FIG. 2A along the direction A-A;

Fig. 3 is a schematic structural diagram of a cell impedance analysis instrument according to an embodiment of the present invention.

[Description of the main component symbols of the present invention]

1-insulating substrate; 2-miniature metal electrode array

3-insulation protection layer; 4-measurement well;

5-Sample cover.

Detailed ways

In order to make the object, technical solution and advantages of the present invention clearer, the present invention will be described in further detail below in conjunction with specific embodiments and with reference to the accompanying drawings. It should be noted that, in the drawings or descriptions of the specification, similar or identical parts all use the same figure numbers. Implementations not shown or described in the accompanying drawings are forms known to those of ordinary skill in the art. Additionally, while illustrations of parameters including particular values may be provided herein, it should be understood that the parameters need not be exactly equal to the corresponding values, but rather may approximate the corresponding values within acceptable error margins or design constraints.

The invention integrates the principle of cell impedance characteristics and electrical impedance imaging technology, and provides a chip and an instrument for cell impedance analysis, so as to realize the impedance analysis of biological cells.

In an 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 cell impedance analysis chip shown in FIG. 2A along the direction A-A. Please refer to Fig. 2A and Fig. 2B, the cell impedance analysis chip of this embodiment comprises: an insulating substrate 1, whose central position is defined as an impedance analysis area; a miniature metal electrode array 2, including N miniature metal electrodes insulated from each other, the N The miniature metal electrodes are radially evenly distributed on the circumference of the impedance analysis area, and the sensitive part inside each miniature metal electrode extends into the impedance analysis area, wherein N≥4; the insulating protective layer 3 covers the impedance analysis area. Areas other than the inner sensitive part and the outer lead part on the miniature metal electrode; the measurement well 4 is located around the impedance analysis area, and its height is higher than the height of the impedance analysis area, so that An accommodating area for accommodating samples to be analyzed is formed above, and the inner radius of the accommodating area is equal to or slightly larger than the radius of the impedance analysis area; the sample cover 5 is fixed on the insulating protective layer outside the measuring well, and its height is higher than The height of the measurement well isolates the barrier analysis region from the external environment.

The technical details of each component of the cell impedance analysis chip according to the embodiment of the present invention will be described in detail below.

In this embodiment, the insulating substrate is a glass sheet of 1.5×1.5 cm ² , but the present invention is not limited thereto. Those skilled in the art can also choose other types and sizes of insulating substrates as required, such as silicon wafers or ITO glass with silicon nitride deposited on the surface.

A circular impedance analysis area is set in the central area of the glass slide. In order to facilitate the measurement of the impedance characteristics of cells or sub-cells, the scale of the circular impedance analysis area is 50 μm. It should be clear to those skilled in the art that the size of the circular impedance analysis region depends on the size of the cells in the sample, and is generally in the micron size, which can be specifically determined according to measurement requirements.

In the cell impedance analysis chip shown in Fig. 2 and Fig. 3, 16 miniature metal electrodes are distributed on the upper surface of the glass slide around the impedance analysis area. Wherein, the size and shape of the miniature metal electrodes are the same, and the distance between the electrodes is the same, and the line width of the top of the electrode is the same as the line width of the distance between the electrodes. Of course, the number of micro metal electrodes is not limited to 16, it can be (but not limited to) 8, 16 or 32.

Each miniature metal electrode can be used as an excitation electrode to input a signal, and can also be used as a response electrode to output a signal. The material of the micro metal electrode array can be (but not limited to) gold or platinum, which is fabricated by sputtering and lift-off process.

The top shape of the inner sensitive part of the miniature metal electrode is linear or arc-shaped, evenly distributed on the central circular impedance analysis area, and its length extending into the impedance analysis area is about 2-10 μm, which is used for samples in the impedance analysis area Add measuring current or collect the voltage signal generated by the impedance of the sample to be analyzed; the outer lead part is rectangular and can be connected to the external circuit through the lead wire to receive the measuring current or transmit the voltage signal collected by the sensitive part to the outside.

In this embodiment, the insulating protection layer 3 is a silicon nitride layer with a thickness of 8000 Å. When preparing the silicon nitride layer, first use the plasma enhanced chemical vapor deposition (PECVD) process to make a silicon nitride insulating layer on the entire substrate, including the micro metal electrode array on it, and then use the ion etching process Remove the silicon nitride layer in the impedance analysis area and the outer lead of the micro-metal electrode, and the radius of the silicon nitride in the central area is slightly larger than that of the impedance analysis area, which is about 55 μm. It should be emphasized that the sensitive part of the micro metal electrode protruding into the impedance analysis area is exposed and not covered by the silicon nitride layer. In addition, the material of the insulating protective layer 3 can also be an organic polymer with insulating properties, such as polyimide, polydimethylsiloxane, or photoresist with insulating properties.

Please refer to Figure 2 and Figure 3, right above the central circular impedance analysis area, a measuring well is constructed of polymer to accommodate the sample to be tested. The electrodes are slightly exposed in the measuring well and are in contact with the sample. The measurement well is cylindrical, its material is SU_8, its height is 30 μm, its inner radius is 60 μm, and its outer radius is 600 μm.

Of course, those skilled in the art should understand that the size of the measurement well, the inner radius of the well and the outer radius of the well can be flexibly adjusted as required. In addition to the SU-8 photoresist, the material for preparing the polymer well can also be polyimide, polydimethylsiloxane (PDMS) or other biocompatible polymers.

The cell impedance analysis signal also includes a sample cover 5 for storing buffer. The inner diameter of the inner cylindrical groove of the sample cover is larger than the outer diameter of the measuring well, and the sample cover is fixed on the insulating protection layer outside the measuring well. In this embodiment, the sample cover is made of polyimide or PDMS, and its shape can be round, square or rectangular. Its cylindrical groove has a radius of 2 mm and a wall thickness of 2 mm.

When performing a measurement, the sample is injected into the measuring well by piercing the sample cap with the needle that injects the sample, and then the needle is pulled out. Due to the surface tension of the buffer, the buffer does not flow out through the pinhole. Through the sample cover, the evaporation of the sample is reduced, and the influence of the environment on the experiment is reduced. As for the chip with the fixed sample cover, it is single use. When the sample cap is not included, it can be reused, but not recommended.

So far, the introduction of the cell impedance analysis chip of this embodiment is completed. According to the description of this embodiment, combined with their own professional knowledge, those skilled in the art should be able to have a clear understanding of the cell impedance analysis chip of the present invention.

In another exemplary embodiment of the present invention, a cell impedance analysis instrument is also provided. As shown in Fig. 3, the instrument includes: the above-mentioned cell impedance analysis chip, a driving current module, a measuring voltage module and an impedance image reconstruction module. Wherein, the driving current module has two electrodes, and the two electrodes are connected to two of the N microscopic metal electrodes according to the preset current driving module, and are used to input the driving current to the sample in the measurement well. The measurement voltage module has two electrodes, which are connected to the other two miniature metal electrodes in addition to the above two miniature metal electrodes according to the preset voltage measurement mode, and are used to measure the sample in the measurement well when the above driving Under the excitation of current, the voltage generated by the two tiny metal electrodes. The impedance image reconstruction module is used to reconstruct the impedance image of the sample in the impedance analysis area according to the voltage of the micro metal electrode obtained by combining multiple current drive modes and voltage measurement modes. The current drive mode includes adjacent, cross, and relative drive modes, and the voltage measurement mode adopted is to measure the voltage of other adjacent electrodes except the excitation electrode.

So far, the introduction of the cell impedance analysis instrument of this embodiment is completed. According to the description of this embodiment, combined with their own professional knowledge, those skilled in the art should be able to have a clear understanding of the cell impedance analysis instrument of the present invention.

In addition, it should be noted that the above definition of each element is not limited to the various specific structures or shapes mentioned in the embodiments, and those skilled in the art can easily and well-known replace them, for example: for a specific shape Cells can design the corresponding sample well structure and electrode arrangement.

To sum up, according to the physical phenomenon that different physiological states of cells have different electrical impedance characteristics, the present invention reconstructs the internal resistance by applying a safe and constant voltage or current to the sample, and measuring the current or voltage on the surface of the sample through the measuring electrodes. The electrical impedance distribution image can reflect the biological information of the sample intuitively and comprehensively through the image.

The specific embodiments described above have further described the purpose, technical solutions and beneficial effects of the present invention in detail. It should be understood that the above descriptions are only specific embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention shall be included within 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.

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