An electrical impedance detection electrode chip for adherent single cells and a detection method thereof

Abstract

The present invention discloses an electrical impedance detection electrode chip for single cells attached to a wall and a detection method thereof, comprising a central electrode (1) whose projected area is less than or equal to that of a single cell when suspended; a combined ring electrode surrounding the central electrode (1) arranged on the same substrate; the central electrode and the combined ring electrode constitute a pair of measuring electrodes; the combined ring electrode constitutes a gradually increasing measuring area required by the single cell attached to the wall under the condition of growth. An electrode array composed of a plurality of pairs of central electrodes and the measuring electrodes constitutes a gradually increasing measuring area required by the single cell array attached to the wall under the condition of growth, division and proliferation. As cells divide and proliferate, the cell attachment area gradually expands, and the combination of electrodes changes accordingly, so that the impedance change of a monoclonal cell group formed by a single cell can be detected, and the cell cycle, proliferation and other behaviors can be analyzed from the impedance change process.

Description

Technical Field

[0001] The invention relates to various fields such as biological cell impedance detection, microelectrode design technology, and in particular to a cell electrical impedance detection electrode chip design. Background Art

[0002] Due to the heterogeneity between cells, the results obtained from the average effects of a group of cells in many biological studies cannot provide sufficient useful information. Therefore, single-cell research has become a hot topic in recent years.

[0003] In the diagnosis of many cancers, we can find that the "key cells" in the diseased tissue have strong proliferation and differentiation capabilities, and some of them can also metastasize, which is one of the important reasons for the deterioration of the disease. Therefore, it is of great significance to study the biological characteristics of cell state such as proliferation, differentiation, and migration at the single cell level. Some researchers have proved that the cell electrical impedance technology can be used to monitor and study the cell state at the single cell level (Kurz et al.). Because the technology itself is non-destructive, non-labeled, and can monitor the state for a long time, the use of cell electrical impedance technology for single cell research has unique advantages.

[0004] To achieve single-cell electrical impedance detection, it is generally necessary to precisely operate on individual cells, efficiently and non-destructively capture single living cells on electrodes, and allow cells to adhere and grow on electrodes (i.e., adhere to the wall), so that the cell state can be monitored by detecting the cell electrical impedance. This requires not only efficient capture of cells, but more importantly, the design of the electrode is key to detecting the electrical impedance of single cells attached to the wall. Existing electrode designs can detect short-term behaviors such as cell adhesion, but it is difficult to achieve long-term monitoring.

[0005] How to make the electrode sensitive enough to detect the state changes of attached single cells, and further monitor the biological behaviors of single cells such as growth, division and proliferation, cell cycle, etc. over a long period of time, while meeting the requirements of general micro-processing technology for glass electrodes, and layout a sufficient number of electrodes on a limited single layer to realize array-based single-cell detection, is the focus and difficulty of electrode design, and is the problem solved by the present invention. Summary of the invention

[0006] The purpose of the present invention is to propose an electrical impedance detection electrode chip for adherent single cells and a detection method thereof, which utilizes a combined ring electrode to form a gradually increasing measurement area required as the adherent single cells grow, and the electrode system can be used to construct an electrode array chip to realize electrical impedance detection of adherent cells and their growth process.

[0007] The present invention discloses an electrical impedance detection electrode chip for single cells attached to the wall, comprising a central electrode 1 whose projected area is less than or equal to that of the single cell when suspended; a combined ring electrode surrounding the central electrode 1 arranged on the same substrate; the central electrode and the combined ring electrode forming a pair of measuring electrodes; electrode leads are respectively provided on the central electrode and the combined ring electrode to connect the electrodes to pads at the edge of the chip; the electrode leads are all covered with an insulating layer; the combined ring electrode forms a gradually increasing measuring area required by the single cell attached to the wall during growth, the combined ring electrode at least comprises an inner ring electrode 2 and an outer ring electrode 3, when the single cell is just attached to the wall, the central electrode 1 and the inner ring electrode 2 are selected as a pair of measuring electrodes, and when the area of ​​the single cell increases to completely cover the inner ring electrode 2 and exceeds the range of the inner ring electrode 2 after division, the central electrode 1 and the inner ring electrode 2 are short-circuited as one electrode, and the outer ring electrode 3 is selected as another electrode for impedance detection.

[0008] The present invention discloses an electrode array chip for detecting electrical impedance of single adherent cells, comprising an electrode array composed of a plurality of pairs of central electrodes and combined ring electrodes, which forms a gradually increasing measurement area required by a group of adherent cells during growth, division and proliferation.

[0009] The present invention discloses an electrical impedance detection electrode chip for single cells attached to the wall, comprising a central electrode 1 whose projected area is less than or equal to that of the single cell when suspended; a combined ring electrode arranged on the same substrate and surrounding the central electrode 1; electrode leads are respectively arranged on the central electrode and the combined ring electrode; an insulating layer is covered on the electrode leads; the combined ring electrode constitutes a gradually increasing measurement area required when the single cell attached to the wall grows, divides and proliferates after being captured; the combined ring electrode at least comprises an inner ring electrode 2 and an outer ring electrode 3, when the single cell is just attached to the wall, the central electrode 1 and the inner ring electrode 2 are selected as a pair of electrodes, and when the area of ​​the single cell increases to completely cover the inner ring electrode 2 and exceeds the range of the inner ring electrode 2 after division, the central electrode 1 and the inner ring electrode 2 are short-circuited as one electrode, and the outer ring electrode 3 is selected as another electrode for impedance detection, and also comprises an alternating electric field applied between the central electrode 1 and the combined ring electrode, which is used to attract the suspended single cell to the central electrode 1 and capture the suspended single cell.

[0010] The present invention provides an electrode array chip for electrical impedance detection of single cells attached to a wall, comprising an electrode array composed of a plurality of pairs of central electrodes and measuring electrodes composed of combined ring electrodes. The alternating electric field applied between each central electrode 1 and each combined ring electrode is used to attract suspended single cells to each central electrode 1, thereby capturing a plurality of suspended single cells; thus forming a gradually increasing measurement area required as the attached single cells grow, divide and proliferate after being captured.

[0011] The present invention provides an electrical impedance detection method for adherent single cells, wherein the specific steps of the method are as follows:

[0012] After the suspended single cell adheres to the surface of the central electrode, a pair of electrodes is formed by the central electrode 1 and the combined circle electrode. For the electrical impedance detection of the single cell attached to the wall in the growth state, when the single cell just adheres to the wall, the central electrode 1 and the inner circle electrode 2 are selected as a pair of electrodes. When the area of ​​the single cell increases to completely cover the inner circle electrode 2 and exceeds the range of the inner circle electrode 2 after division, the central electrode 1 and the inner circle electrode 2 are short-circuited as one electrode, and the outer circle electrode 3 is selected as the other electrode for impedance detection.

[0013] The present invention provides an electrical impedance detection method for adherent single cells, wherein the specific steps of the method are as follows:

[0014] After the suspended single cell is captured and attached to the surface of the central electrode, a pair of electrodes consisting of the central electrode 1 and the combined circle electrode are used to perform impedance detection on the single cell attached to the surface after capture and in the growth state. When the single cell has just attached to the surface, the central electrode 1 and the inner circle electrode 2 are selected as a pair of electrodes. When the area of ​​the single cell increases after division to completely cover the inner circle electrode 2 and exceed the range of the inner circle electrode 2, the central electrode 1 and the inner circle electrode 2 are short-circuited as one electrode, and the outer circle electrode 3 is selected as the other electrode for impedance detection.

[0015] The present invention provides an electrical impedance detection method for adherent single cells, wherein the specific steps of the method are as follows:

[0016] After the suspended single cells adhere to the central electrode surface, the electrical impedance of the attached single cells and single cell clones under growth conditions is detected.

[0017] The present invention provides an electrical impedance detection method for adherent single cells, wherein the specific steps of the method are as follows:

[0018] After the suspended single cells are captured and attached to the surface of the central electrode, a pair of electrodes consisting of the central electrode 1 and the combined ring (annular) electrode is used to perform electrical impedance detection on the attached single cells and single cell clones after capture and in the growth state.

[0019] The present invention provides an electrical impedance detection method for adherent single cells, wherein the specific steps of the method are as follows:

[0020] After the suspended single cells adhere to the surface of the central electrode, the electrode array composed of multiple pairs of central electrodes and combined ring electrodes, which are composed of measuring electrodes, simultaneously performs electrical impedance detection on the single cells, single cell arrays or group cells in the growth state.

[0021] The present invention provides an electrical impedance detection method for adherent single cells, wherein the specific steps of the method are as follows:

[0022] After the suspended single cells are captured and attached to the surface of the central electrode, the electrode array composed of multiple pairs of central electrodes and combined ring electrodes is used to simultaneously perform electrical impedance detection on each single cell, single cell array or group of cells after capture, growth, division and proliferation.

[0023] Compared with the prior art, the advantage of the design of the present invention is that when a single cell is captured on the central electrode and grows attached to the wall, the inner circle electrode and the central electrode work together to detect the impedance of the single cell. As the cell divides, the cell attachment area gradually expands. When it approaches the area of ​​the inner circle electrode, the outer circle electrode and the inner circle electrode work together to detect the impedance change of the monoclonal cell formed by the single cell, and the cell cycle, proliferation and other behaviors are analyzed from the change process of the electrical impedance.

[0024] By setting up an extended outer ring electrode, cell impedance detection can be achieved when a cell divides and proliferates into more cells.

[0025] The electrode array can be used to simultaneously detect the electrical impedance of multiple single cells arranged in an array in the test area after they adhere to the wall and grow and divide and proliferate. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 A schematic diagram of an electrode structure for electrical impedance detection of adherent cells;

[0027] Figure 2 A schematic diagram of the insulating layer structure of an electrical impedance detection electrode for adherent cells;

[0028] Figure 3 A cross-sectional view of an electrode chip structure for electrical impedance detection of adherent cells;

[0029] Figure 4 Schematic diagram of the electrode array chip of multiple single cell detection units according to an embodiment of the present invention; the part in the box is Figure 1 The corresponding structure;

[0030] Reference numerals:

[0031] 1. Central electrode, 2. Inner electrode, 3. Outer electrode, 4. Electrode lead; 5. Insulation layer (with Figure 1 Corresponding), 6. Structure without insulating layer, 7. Chip substrate, 8. Pad. DETAILED DESCRIPTION

[0032] The embodiments of the present invention will be described in further detail below with reference to the accompanying drawings.

[0033] The embodiments of the present invention will be described in further detail below with reference to the accompanying drawings.

[0034] As Figure 1 to Figure 2 The electrical impedance detection electrode structure for adherent cells shown is an embodiment. Among them, the small electrode located in the middle is the central electrode 1, and the inner circle electrode 2 and the outer circle electrode 3 are also provided, which surround the central electrode 1 from the periphery, and the areas of the inner circle electrode 2 and the outer circle electrode 3 gradually increase; electrode leads are respectively provided on the central electrode 1, the first peripheral electrode 2 and the second peripheral electrode 3; the suspended cells are close in size to the central electrode 1, so the central electrode 1 cooperates with the electrode 2 or the electrode 3 to capture a single cell on the electrode 1, and the cell is close in size to the electrode 2 after being adhered to the wall, at which time the electrode 1 and the inner circle electrode 2 are used for detection; the adherent cells further divide and proliferate, and the area gradually expands to the area of ​​the outer circle electrode 3, at which time the electrode 1 and the electrode 2 are short-circuited as one electrode and used together with the electrode 3 for detection.

[0035] Specifically, the central electrode 1 is circular in shape with a diameter of 5 to 15 microns; the inner ring electrode 2 is a ring surrounding the central electrode 1, and the inner and outer diameters of the ring are 7 to 50 microns, wherein the inner diameter is larger than the outer diameter of the central electrode 1; the outer ring electrode 3 is a ring surrounding the inner ring electrode 2, and the inner and outer diameters of the ring are 15 to 200 microns, wherein the inner diameter is larger than the outer diameter of the inner ring electrode 2; more extended outer ring electrodes may be arranged around the outer ring electrode 3.

[0036] The central electrode 1, the inner circle electrode 2, and the outer circle electrode 3 are independent and not connected to each other; all electrodes are connected to the edge of the chip through leads and connected to the back-end impedance detection circuit.

[0037] The combination of electrodes is adjusted as needed. As a pair of electrodes required for electrical impedance measurement, different electrodes can be combined for impedance detection. The paired combinations include central electrode 1 and inner circle electrode 2, central electrode 1 and outer circle electrode 3, inner circle electrode 2 and outer circle electrode 3, inner circle electrode 2 and outer circle electrode 3 are short-circuited and then combined with central electrode 1, central electrode 1 and inner circle electrode 2 are short-circuited and then combined with outer circle electrode 3, and central electrode 1 and outer circle electrode 3 are short-circuited and then combined with inner circle electrode 2. If the outer circle is expanded, more combinations will be added accordingly. That is, the short circuit of adjacent electrodes or different electrodes can form a combined electrode with the adjacent electrodes outside or inside.

[0038] like Figure 4 , which is a general view of the electrode array chip of the single cell detection unit according to an embodiment of the present invention.

[0039] The chip preparation method of the present invention is specifically performed as follows:

[0040] Optimize the layout on the glass sheet and design the electrodes, leads and insulation layer layout:

[0041] Electrode lead layout design: Design pads and leads at the edge of the glass electrode; design an electrode array in the center of the glass chip; design the electrode layout, the diameter of the electrode area at the detection site is 20-40μm, in order to ensure the processing accuracy and reduce the electrode's own impedance, the thinnest line width of the electrode is 5μm, and the thinnest line width of the lead is 10μm. Design the insulation layer layout according to the pattern of the electrode, pad, and lead.

[0042] Gold electrodes and insulating layers are processed on the glass chip according to the layout:

[0043] 1. First, a layer of titanium (30nm) and gold (300nm) are sputtered on clean glass;

[0044] 2. Use electrode layout ( Figure 1 ) patterning the metal layer by photolithography, and obtaining the required electrode pattern through steps such as coating, exposure, washing, etching, and stripping;

[0045] 3. Process SiO on the electrode surface through PECVD process 2 / Si 3 N 4 / SiO 2 Thin film, then use insulating layer layout ( Figure 2 ), the thin film layer is patterned by a photolithography process, and a patterned insulating layer is obtained by the same steps of coating, exposure, elution, etching, and degumming, so that the electrode part is exposed and the lead is covered by the insulating layer to improve the detection sensitivity and reduce signal interference;

[0046] Design a PCB interface board that matches the glass chip, and connect the electrode chip to the PCB board through soldering;

[0047] Epoxy AB glue was used to cover the welding points to ensure insulation. After the glue dried, a layer of PDMS mixture was covered on the glue and cured to prevent the toxic effect of the glue on the cells;

[0048] A fence was fixed on the chip PCB board with PDMS to form a culture chamber, and a transparent plastic cover was added;

[0049] After the device was processed, it was soaked in distilled water overnight, rinsed with PBS, and sterilized by ultraviolet light;

[0050] The PCB board leads out the electrode leads and connects them to the impedance spectrum analyzer to detect the impedance between the electrodes on the chip.

[0051] The present invention provides a detection method for an electrical impedance detection electrode structure for adherent cells, and the specific steps of the method are as follows:

[0052] After a single cell is captured on the electrode surface, a pair of electrodes consisting of the central electrode 1 and the inner circle electrode 2 is used for electrical impedance detection to detect the adhesion and growth of the single cell;

[0053] When the impedance of the cells detected by the central electrode 1 and the inner circle electrode 2 has reached the threshold, or the cells are observed to be completely attached to the wall by the microscope, the central electrode 1 and the inner circle electrode 2 are short-circuited through their respective electrode leads to become one electrode, and then together with the outer circle electrode 3, a cell measurement electrode pair is formed for impedance detection, or only the inner circle electrode 2 and the outer circle electrode 3 are used to form a cell measurement electrode pair for impedance detection;

[0054] In the process of cell division, proliferation and reattachment, as the number of cells increases, the impedance formed by cell measurement becomes larger and larger.

[0055] When more cells are detected to proliferate from the division of a cell, an outer circle electrode is further set up, and the central electrode 1, the inner circle electrode 2 and the outer circle electrode 3 are short-circuited to form an electrode, which together with the extended outer circle electrode forms a pair of electrodes for impedance detection. The inner circle electrode 2, the outer circle electrode 3 or the outer circle three electrodes alone can also be used as one electrode to form a cell measurement electrode pair with the outer circle electrode, and so on.

[0056] Embodiment of a method for fixing a single cell to an electrode detection site:

[0057] (1) External chip-assisted method

[0058] A PDMS (polydimethylsiloxane) pipe with a single cell capture function is attached to the chip electrode surface and aligned under a microscope to position the cell capture structure on the electrode (1) surface;

[0059] The adherent cells are digested and suspended in a cell culture medium to form a cell suspension of a certain density;

[0060] The processed PDMS chip is attached to the bottom of a clean culture dish. After plasma cleaning, a small amount of culture medium without cells is dripped into it. Aspirate carefully from the outlet with a syringe to allow the culture medium to fill the channel.

[0061] To capture cells, drip a small amount of cell suspension from the inlet, and aspirate from the outlet with a syringe. When the capture effect meets the requirements under a microscope, stop aspiration and drip cell culture medium again to clean the excess cells in the inlet and pipeline;

[0062] Aspirate the excess liquid at the inlet, add a large amount of cell culture medium, and transfer the culture dish to the incubator for cell culture;

[0063] After the cells adhere to the wall, the PDMS layer is peeled off, more culture medium is added, single cell array culture is performed, and the impedance changes are detected to analyze the changes in the physiological state at the single cell level.

[0064] (2) Electrode dielectric force capture method

[0065] A cell suspension of a certain concentration is dripped onto the electrode, and then an alternating electric field of a certain frequency and voltage is applied between electrodes 1, 2, and 3 to attract single cells to electrode 1;

[0066] Apply a certain flow force to wash away the extra cells that are not attracted;

[0067] The chip was allowed to stand still and the AC electric field was removed to allow the cells to grow on the wall and then electrical impedance measurement was started.

Claims

1. An electrical impedance detection electrode chip for adherent single cells, characterized in that: The invention comprises a central electrode (1) having a diameter of 5 to 15 micrometers and a projection area smaller than or equal to that of a single cell when suspended; a combined ring electrode arranged on the same substrate and surrounding the central electrode (1); the central electrode and the combined ring electrode forming a pair of measuring electrodes; electrode leads connecting electrodes and chip edge pads are respectively arranged on the central electrode and the combined ring electrode; an insulating layer is covered on the electrode leads; the combined ring electrode forms a gradually increasing measuring area required by the single cell attached to the wall during growth; the combined ring electrode at least comprises an inner ring electrode (2) and an outer ring electrode (3); when a single cell is just attached to the wall, the central electrode (1) and the inner ring electrode (2) are selected as a pair of measuring electrodes; when the area of ​​the single cell increases to completely cover the inner ring electrode (2) and exceeds the range of the inner ring electrode (2) after division, the central electrode (1) and the inner ring electrode (2) are short-circuited as one electrode, and the outer ring electrode (3) is selected as the other electrode for impedance detection.

2. The electrical impedance detection electrode chip for adherent single cells according to claim 1, characterized in that: The electrodes are used to form a pair of electrode combinations for impedance detection. The central electrode (1), the inner circle electrode (2), and the outer circle electrode (3) include the following six combinations: the central electrode (1) and the inner circle electrode 2 are combined, the central electrode (1) and the outer circle electrode (3) are combined, the inner circle electrode (2) and the outer circle electrode (3) are combined, the central electrode (1) and the inner circle electrode (2) are short-circuited and then combined with the outer circle electrode (3), the inner circle electrode (2) and the outer circle electrode (3) are short-circuited and then combined with the central electrode (1), and the central electrode (1) and the outer circle electrode (3) are short-circuited and then combined with the inner circle electrode (2).

3. The electrical impedance detection electrode chip for adherent single cells according to claim 1, characterized in that: More extended outer ring electrodes are arranged around the outer ring electrode (3).

4. The electrical impedance detection electrode array chip for adherent single cells implemented by the electrical impedance detection electrode chip for adherent single cells as claimed in any one of claims 1 to 3, characterized in that: An electrode array is provided which comprises a plurality of pairs of central electrodes and measuring electrodes formed by combined ring electrodes. The alternating electric field applied between each central electrode (1) and each combined ring electrode is used to attract suspended single cells to each central electrode (1) and capture multiple suspended single cells; thus forming a gradually increasing measuring area required when the attached single cells grow, divide and proliferate after being captured.

5. A detection method for an electrical impedance detection electrode structure for adherent cells implemented based on the electrical impedance detection electrode chip for adherent single cells as described in any one of claims 1 to 3, characterized in that: The specific steps of this method are as follows: After a single cell is captured on the electrode surface, a pair of electrodes is formed by a central electrode (1) and an inner circle electrode (2). Conduct electrical impedance detection to detect the adhesion and growth of the single cell; When the central electrode (1) and the inner electrode (2) detect that the cell impedance has reached the threshold, or when the microscope observes After it is detected that the cells have completely adhered to the wall, the central electrode (1) and the inner circle electrode (2) are short-circuited through their respective electrode leads to become one electrode, and then together with the outer circle electrode (3) form a cell measurement electrode pair for impedance detection; In the process of cell division, proliferation and reattachment, as the number of cells increases, the cell measurement of the barrier Resistance is growing; When more cells are detected to proliferate from the division of a cell, an outer ring electrode is further provided, and the central electrode (1), the inner ring electrode (2) and the outer ring electrode (3) are short-circuited to form an electrode, which together with the extended outer ring electrode forms a pair of electrodes for impedance detection.

6. A detection method for an electrical impedance detection electrode structure for adherent cells as claimed in claim 5, characterized in that: Also includes: Only the inner circle electrode (2) and the outer circle electrode (3) are used to form a cell measurement electrode pair for impedance detection.

7. A detection method for an electrical impedance detection electrode structure for adherent cells as claimed in claim 5, characterized in that: Also includes: When more cells are detected to have proliferated from the division of a cell, the inner circle electrode (2), the outer circle electrode (3) or the outer circle electrode alone (3) is used as an electrode to form a cell measurement electrode pair with the outer circle electrode, and so on.

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