3D cell spatial impedance detection system and method

Abstract

The invention discloses a 3D cell spatial impedance detection system and method, relates to the technical field of cell detection, and aims to solve the problem of poor accuracy of 3D cell spatial impedance detection in the prior art. The system at least comprises an impedance sensor, an electrochemical working unit and an upper computer, the impedance sensor is connected with the electrochemical working unit, and the upper computer is connected with the electrochemical working unit; the impedance sensor comprises a cell culture pool for 3D cell culture and a plurality of plug-in vertical electrode arrays for detecting impedance data of 3D cells in the cell culture pool; the plurality of plug-in vertical electrode arrays are inserted into the cell culture pool, and the distance between the plurality of plug-in vertical electrode arrays is equal to the unit width of the plurality of plug-in vertical electrode arrays; the electrochemical working unit is used for performing constant-current scanning on the impedance sensor, and the impedance sensor returns impedance data to the electrochemical working unit; therefore, the accuracy of 3D cell spatial impedance detection is improved.

Description

Technical Field

[0001] The present invention relates to the technical field of cell detection, and in particular to a detection system and method for 3D cell spatial impedance. Background Art

[0002] With the in-depth development of life science and medical research, the demand for understanding cell behavior, cell-cell interactions, and cell microenvironment responses is increasing. Especially in the fields of tissue engineering, cancer research, drug screening, and regenerative medicine, accurate monitoring of dynamic processes such as cell growth, migration, differentiation, and apoptosis in a three-dimensional (3D) environment is crucial for revealing life mechanisms, optimizing treatment strategies, and developing new biomaterials. Traditional cell culture and analysis techniques are mostly based on two-dimensional (2D) culture systems. Although they are easy to operate and low in cost, they cannot fully simulate the complex cell microenvironment in the body, limiting their research applications under simulated physiological conditions. Therefore, it is particularly important to develop new technologies that can efficiently and accurately monitor cell activity in 3D cell space.

[0003] Among the numerous cell detection technologies, impedance sensing technology has occupied an important position in cell biology research due to its non-invasive, real-time monitoring and high sensitivity. This technology indirectly reflects information such as the number, morphology, adhesion status and proliferation activity of cells by measuring the conductivity or impedance changes between the cell and electrode interface. However, traditional impedance sensor designs are mainly aimed at 2D cell culture systems, and their electrode configurations and signal processing methods are often not suitable for 3D cell culture environments. There are problems such as large signal interference, low resolution, and difficulty in accurately distinguishing information from cell layers at different depths.

[0004] Currently, the detection electrodes commonly used to detect the 3D cell culture environment are large-area monitoring electrodes or coplanar fingertip electrodes. Among them, the large-area monitoring electrodes are to wrap the cell clusters in a flexible wrapping manner to achieve large-area impedance detection of the cell clusters. The coplanar fingertip electrodes are to culture 3D cell spheres above the coplanar fingertip electrodes to achieve impedance monitoring of the entire 3D cells. Since the detection electrodes are distributed around the cell body, the above methods cannot perform spatial dynamic detection of 3D cell models at different positions on the scaffold, that is, they cannot detect the cell impedance inside the cell clusters, and cannot obtain a more accurate 3D cell spatial impedance spectrum.

[0005] Based on this, it is urgent to design a more advanced method for 3D cell spatial impedance detection to solve the problem of low detection accuracy of 3D cell spatial impedance in the existing technology. Summary of the invention

[0006] The purpose of the present invention is to provide a 3D cell spatial impedance detection system and method. By designing a cell culture pool and multiple plug-in vertical electrode arrays, multiple plug-in vertical electrode array sensors are integrated with the internal space of the 3D culture system to obtain an impedance sensor, thereby ensuring the natural growth state of cells during the monitoring process, and being able to perform spatial dynamic detection of 3D cell models at different positions on the cell scaffold, effectively improving the accuracy of 3D cell spatial impedance detection, and solving the problem of low accuracy of 3D cell spatial impedance detection in the prior art.

[0007] In order to achieve the above object, the present invention provides the following technical solutions:

[0008] In a first aspect, the present invention provides a 3D cell spatial impedance detection system, which may at least include:

[0009] Impedance sensor, electrochemical working unit and host computer;

[0010] The impedance sensor is connected to the electrochemical working unit, and the host computer is connected to the electrochemical working unit;

[0011] The impedance sensor comprises a cell culture pool and a plurality of inserted vertical electrode arrays, wherein the plurality of inserted vertical electrode arrays are inserted into the cell culture pool; the cell culture pool is used for 3D cell culture, and the plurality of inserted vertical electrode arrays are used for detecting impedance data of 3D cells in the cell culture pool; the spacing between the plurality of inserted vertical electrode arrays is equal to the unit width of the plurality of inserted vertical electrode arrays;

[0012] The electrochemical working unit is used to perform constant current scanning for the impedance sensor, and the impedance sensor returns impedance data to the electrochemical working unit.

[0013] Preferably, the system may further include a board-level control unit and a multi-way selection unit;

[0014] The multi-way selection unit is connected to the impedance sensor, the electrochemical working unit and the board-level control unit respectively, and the host computer is connected to the board-level control unit;

[0015] The board-level control unit is used to control the multi-way selection unit to connect to a target impedance detection channel; the target impedance detection channel is a target connection channel between the plurality of the insertable vertical electrode arrays and the electrochemical working unit.

[0016] Preferably, the multi-way selection unit may include multiple sub-multi-way selection units; the membrane electrodes of the multiple vertical electrode arrays in the impedance sensor are led out and hot-pressed bonded with multiple soft FPCs at 220°, and then the multiple soft FPCs are respectively connected to the multiple sub-multi-way selection units.

[0017] Preferably, the insertable vertical electrode array may include an insertable vertical electrode array arranged in an M×N matrix, wherein the matrix units are periodic and regular geometric figures, and M and N are both positive integers greater than or equal to 3.

[0018] Preferably, the side length of the matrix unit may be 10 μm to 10 mm, and the spacing between the matrix units may be 0.1 mm.

[0019] Preferably, the material of the cell culture tank may be medical grade transparent polymethyl methacrylate.

[0020] In a second aspect, the present invention provides a method for detecting 3D cell spatial impedance, which is applied to the 3D cell spatial impedance detection system described in the first aspect; the method may include:

[0021] Obtain the target control instruction of the detection target impedance detection channel sent by the host computer;

[0022] Based on the target control instruction, the corresponding impedance detection channel in the impedance sensor is controlled to perform impedance detection on the 3D cell space; the impedance sensor includes a cell culture pool and a plurality of inserted vertical electrode arrays, and the plurality of inserted vertical electrode arrays are inserted into the cell culture pool; the cell culture pool is used for 3D cell culture, and the plurality of inserted vertical electrode arrays are used to detect the impedance data of the 3D cells in the cell culture pool; the spacing between the plurality of inserted vertical electrode arrays is equal to the unit width of the plurality of inserted vertical electrode arrays;

[0023] The electrochemical working unit is controlled to perform constant current scanning on the target impedance detection channel of the impedance sensor to obtain the spatial impedance spectrum data of the 3D cell returned by the impedance sensor.

[0024] Preferably, the plurality of said inserted vertical electrode arrays may include a first inserted vertical electrode array and a second inserted vertical electrode array;

[0025] Before obtaining the target control instruction for detecting the target impedance detection channel sent by the host computer, the method may include: preparing a sandwich structure impedance sensor based on the cell culture pool, the first plug-in vertical electrode array and the second plug-in vertical electrode array;

[0026] In the sandwich structure impedance sensor, from left to right are the first inserted vertical electrode array, the 3D cell sample and the second inserted vertical electrode array.

[0027] Preferably, the controlling the electrochemical working unit to perform constant current scanning on the target impedance detection channel of the impedance sensor to obtain the spatial impedance spectrum data of the 3D cell returned by the impedance sensor may include:

[0028] Based on the electrochemical working unit, the target impedance detection channel is scanned in constant current scanning mode to obtain the spatial impedance spectrum data of the 3D cell returned by the impedance sensor.

[0029] Preferably, the obtaining of the spatial impedance spectrum data of the 3D cells returned by the impedance sensor may further include: determining whether all impedance detection channels in the impedance sensor have been detected, and if so, terminating the detection; if not, switching to the next target impedance detection channel for impedance detection to obtain the spatial impedance spectrum data of the 3D cells corresponding to the impedance detection channel.

[0030] Compared with the prior art, the present invention provides a 3D cell spatial impedance detection system, which may at least include: an impedance sensor, an electrochemical working unit and a host computer; by connecting the impedance sensor to the electrochemical working unit, and connecting the host computer to the electrochemical working unit; the impedance sensor includes a cell culture pool and a plurality of inserted vertical electrode arrays, the plurality of inserted vertical electrode arrays are inserted into the cell culture pool; the cell culture pool is used for 3D cell culture, and the plurality of inserted vertical electrode arrays are used to detect the impedance data of the 3D cells in the cell culture pool; the spacing between the plurality of inserted vertical electrode arrays is equal to the unit width of the plurality of inserted vertical electrode arrays; the electrochemical working unit is used to perform constant current scanning for the impedance sensor, and the impedance The impedance sensor returns impedance data to the electrochemical working unit; based on this, the 3D cells in the cell culture pool are solidified together with multiple inserted vertical electrode arrays to obtain an impedance sensor, which improves the sensitivity and resolution of the spatial distribution of cells in the 3D cell culture system and can accurately capture the subtle changes of cells in three-dimensional space; and the electrochemical working unit is used to perform constant current scanning for the impedance sensor to obtain the impedance data returned by the impedance sensor, and send it to the host computer for data processing and display, which can more accurately reflect key information such as cell quantity, morphology and proliferation activity; thereby improving the accuracy of 3D cell spatial impedance detection and solving the problem of poor accuracy of 3D cell spatial impedance detection in the prior art. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] The drawings described herein are used to provide a further understanding of the present invention and constitute a part of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention. In the drawings:

[0032] Figure 1A first structural schematic diagram of a 3D cell spatial impedance detection system provided by the present invention;

[0033] Figure 2 A second structural schematic diagram of a 3D cell spatial impedance detection system provided by the present invention;

[0034] Figure 3 A schematic diagram of an inserted vertical electrode array in a 3D cell spatial impedance detection system provided by the present invention;

[0035] Figure 4 A physical schematic diagram of a cell culture pool in a 3D cell spatial impedance detection system provided by the present invention;

[0036] Figure 5 A physical schematic diagram of an impedance sensor in a 3D cell spatial impedance detection system provided by the present invention;

[0037] Figure 6 A schematic diagram of the main process of a 3D cell spatial impedance detection method provided by the present invention;

[0038] Figure 7 A schematic diagram of the impedance data acquisition process of a 3D cell spatial impedance detection method provided by the present invention;

[0039] Figure 8 A schematic diagram of the verification effect of a 3D cell spatial impedance detection system and method provided by the present invention.

[0040] Reference numerals: 110 - impedance sensor, 120 - electrochemical working unit, 130 - host computer, 140 - multi-way selection unit, 150 - board-level control unit. DETAILED DESCRIPTION

[0041] In order to facilitate the clear description of the technical solutions of the embodiments of the present invention, in the embodiments of the present invention, the words "first", "second" and the like are used to distinguish the same items or similar items with substantially the same functions and effects. For example, the first threshold and the second threshold are only used to distinguish different thresholds, and do not limit their order of precedence. Those skilled in the art can understand that the words "first", "second" and the like do not limit the quantity and execution order, and the words "first", "second" and the like do not necessarily limit them to be different.

[0042] It should be noted that, in the present invention, words such as "exemplary" or "for example" are used to indicate examples, illustrations or descriptions. Any embodiment or design described as "exemplary" or "for example" in the present invention should not be interpreted as being more preferred or more advantageous than other embodiments or designs. Specifically, the use of words such as "exemplary" or "for example" is intended to present related concepts in a specific way.

[0043] In the present invention, "at least one" means one or more, and "plurality" means two or more. "And / or" describes the association relationship of associated objects, indicating that three relationships may exist. For example, A and / or B can mean: A exists alone, A and B exist at the same time, and B exists alone, where A and B can be singular or plural. The character " / " generally indicates that the associated objects in the previous time are in an "or" relationship. "At least one of the following items" or similar expressions refers to any combination of these items, including any combination of single items or plural items. For example, at least one of a, b or c can mean: a, b, c, the combination of a and b, the combination of a and c, the combination of b and c, or the combination of a, b and c, where a, b, c can be single or multiple.

[0044] First, some English abbreviations involved in the present invention are explained as follows:

[0045] Serial number Chinese Abbreviations English full name 1 3D 3D three-dimensional 2 Insertable vertical electrode array PVEA Plug-inverticalelectrodearray 3 Polymethyl methacrylate PMMA Polymethyl Methacrylate

[0046] In recent years, in order to develop new technologies that can efficiently and accurately monitor cell activities in 3D cell space, researchers have begun to explore impedance sensing technology suitable for 3D cell culture, striving to achieve high-precision monitoring of cell behavior while maintaining the natural growth state of cells. This includes developing electrode arrays with different structures to enhance sensitivity to cell spatial distribution, using advanced signal processing algorithms to improve data resolution and reduce noise interference; and designing adjustable cell culture microenvironments to adapt to different types of cells and research needs. Despite this, existing technologies still face many challenges. First, the unevenness and complexity of cell distribution in 3D cell culture systems require impedance sensors to have higher spatial resolution and depth perception capabilities to accurately capture subtle changes in cells in three-dimensional space; second, how to effectively integrate sensors with 3D culture systems while maintaining cell activity to avoid physical or chemical interference with cell growth is a major difficulty in current technology.

[0047] At present, the detection electrodes commonly used to detect 3D cell culture environment are large-area monitoring electrodes or coplanar fingertip electrodes, both of which use a flexible wrapping method to wrap the cell clusters to achieve impedance detection of the cell clusters. Since the detection electrodes are distributed around the cell body, it is impossible to perform spatial dynamic detection of 3D cell models at different positions on the scaffold, resulting in the impedance spectrum obtained by the impedance detection method in the existing technology has low accuracy, which cannot better support the research needs of different types of cells and hinders the in-depth development of cell biology research.

[0048] Based on this, the present invention provides a 3D cell spatial impedance detection system and method. By designing an impedance sensor with a three-dimensional electrode structure and combining a signal processing algorithm with an intelligent control system, high-sensitivity and high-resolution monitoring of cell growth, migration and morphological changes in three-dimensional space is achieved; a more accurate 3D cell spatial impedance spectrum can be obtained, providing strong technical support for in-depth research on cell behavior, cell-cell interactions and cell microenvironment responses.

[0049] Next, the technical solution of the present invention is described in detail with reference to the accompanying drawings:

[0050] In the first aspect, the present invention provides a 3D cell spatial impedance detection system; please refer to Figure 1 , Figure 1 This is a first structural schematic diagram of a 3D cell spatial impedance detection system provided by the present invention.

[0051] exist Figure 1 The system may include at least:

[0052] Impedance sensor 110 , electrochemical working unit 120 and host computer 130 ; connect the impedance sensor 110 with the electrochemical working unit 120 , and connect the host computer 130 with the electrochemical working unit 120 .

[0053] Specifically, the impedance sensor 110 may include a cell culture pool and a plurality of inserted vertical electrode arrays, wherein the plurality of inserted vertical electrode arrays are inserted into the cell culture pool, that is, the plurality of inserted vertical electrode arrays and the 3D cells in the cell culture pool may be solidified together to form the impedance sensor; the cell culture pool is used for 3D cell culture, and the plurality of inserted vertical electrode arrays are used to detect the impedance data of the 3D cells in the cell culture pool; the spacing between the plurality of inserted vertical electrode arrays is equal to the unit width of the plurality of inserted vertical electrode arrays. The electrochemical working unit is used to perform constant current scanning for the impedance sensor, and the impedance sensor returns impedance data to the electrochemical working unit.

[0054] Based on this, the present invention provides a 3D cell spatial impedance detection system. Since multiple insertable vertical electrode arrays are inserted into a cell culture pool and solidified together with the 3D cells in the cell culture pool to form the impedance sensor, the natural growth state of the cells during the monitoring process is ensured, and subtle changes in the three-dimensional space inside the 3D cells can be detected, including spatial dynamic detection of 3D cell models at different positions on the bracket; and then the impedance sensor is combined with the electrochemical working unit to perform constant current scanning to obtain the impedance data of the 3D cells at different positions in the cell culture pool returned by the impedance sensor, and then the impedance data can be displayed after data analysis and processing on the host computer; thereby achieving high-sensitivity and high-resolution monitoring of cell growth, migration and morphological changes in three-dimensional space, and obtaining 3D cell spatial impedance with higher accuracy, which will not only promote the in-depth development of cell biology research, but also bring new opportunities for disease model construction, drug screening and personalized medicine.

[0055] In another embodiment, in order to meet different detection requirements, such as separately detecting the cell impedance at different positions of the cell pool, the present invention designs a second structure detection system based on the first structure detection system; please refer to Figure 2 , Figure 2 This is a second structural schematic diagram of a 3D cell spatial impedance detection system provided by the present invention.

[0056] exist Figure 2 The system may further include:

[0057] A board-level control unit 150 and a multi-way selection unit 140; the multi-way selection unit 140 is respectively connected to the impedance sensor 110, the electrochemical working unit 120 and the board-level control unit 150, and the host computer 130 is connected to the board-level control unit 150; wherein the board-level control unit 150 is used to control the multi-way selection unit 140 to connect to the target impedance detection channel; the target impedance detection channel is a target connection channel between multiple inserted vertical electrode arrays and the electrochemical working unit.

[0058] It should be noted that, in a specific application scenario, the board-level control unit 150 and the multi-way selection unit 140 can be integrated into a board-level control circuit, wherein the control unit is controlled by a single-chip microcomputer ESP32, the multi-way selection unit can be a plurality of 8-to-1 multiplexers, and a connection interface of an electrochemical working unit is also provided in the board-level control circuit. The board-level control circuit is mainly controlled by ESP32, and instructions are sent to the multiplexer to realize the connection between the impedance sensor and the electrochemical working unit to complete multi-channel impedance acquisition. Finally, the data is transmitted to the host computer via a USB cable.

[0059] Based on this, it is possible to use detection electrodes at different positions of the insertable vertical electrode array sensor to perform impedance detection on cells at different positions in the cell culture pool according to user needs or experimental needs; that is, automatic path switching and data acquisition processing can be performed through the board-level control circuit to achieve M×N multi-channel spatial impedance detection.

[0060] Preferably, the inserted vertical electrode array may include an inserted vertical electrode array arranged in an M×N matrix; wherein the matrix units are periodic regular geometric shapes, including but not limited to circle, rectangle, triangle and other periodic regular geometric shapes; M and N are both positive integers greater than or equal to 3, for example, M or N can be set to a positive integer such as 3, 4, 6 or 8 according to requirements; the corresponding values ​​of M and N can be equal or unequal.

[0061] As an example, see Figure 3 , Figure 3 A physical schematic diagram of an inserted vertical electrode array in a 3D cell spatial impedance detection system provided by the present invention.

[0062] exist Figure 3 In the embodiment, the inserted vertical electrode array is an inserted vertical electrode array arranged in a 4×4 (square) matrix; of course, the inserted vertical electrode array in the present invention can also be an electrode array in other M×N matrix arrangements. The electrode material of the inserted vertical electrode array is preferably a gold electrode material, and can also be replaced by other metals or conductive materials such as ITO; the spatial impedance resolution detected in the impedance sensor is related to the size of the electrode area, and the electrode area can be made smaller within the process allowable range; the preferred electrode side length can be 10μm to 10mm; when the matrix unit is a square, the side length of the square is preferably 2mm, and the spacing between each matrix unit is preferably 0.1mm.

[0063] Specifically, the design of the inserted vertical electrode array can be performed by using L-edit to design the electrode array drawings, as shown below: Figure 3 The square electrode array shown has a total of 16 electrodes, which are 2 mm squares with a spacing of 0.1 mm and arranged in a 4×4 matrix. The thickness of a single inserted vertical electrode array is 0.5 mm. We can use groups A, B, C, and D to mark four different horizontal positions along the vertical direction of the impedance sensor chip, and group every four electrodes with the same horizontal position, that is, the 16 electrodes obtained are A1, B1, C1, D1, A2, B2, C2, D2, A3, B3, C3, D3, A4, B4, C4, and D4.

[0064] Furthermore, the metal leads can be led out to the same side, wherein the line width spacing of the metal leads can be 50 μm to ensure the stability of signal transmission. Metal patterning can be achieved by photolithography stripping technology, that is, first photolithography patterning on the glass substrate, then evaporation deposition of 20 nm chromium as an adhesion layer, then deposition of 200 nm gold, and finally washing away the photoresist with acetone.

[0065] use Figure 3 The electrodes shown in the figure are used to prepare the impedance sensor. Figure 5 . It can be obtained that the distance between the two inserted vertical electrode arrays is 2mm, and the unit electrode size of each inserted vertical electrode array is 2mm. Therefore, in the cell pool, the aspect ratio of each detection environment of the two inserted vertical electrode arrays is consistent, which can significantly improve the sensitivity and resolution of the spatial distribution of cells in the 3D cell culture system, accurately capture the subtle changes of cells in three-dimensional space, improve the accuracy of 3D cell spatial impedance detection, and overcome the shortcomings of traditional single electrodes in depth perception; and the impedance sensor has a simple manufacturing process and low cost. The PVEA chip can be reused many times. 3D cells are cultured in hydrogels, and the changes in cell impedance under different conditions can be simulated by adding different drugs / toxic substances, which has great application prospects.

[0066] It should be noted that the single inserted vertical electrode array in the present invention is a planar electrode array, and the unit size of the microelectrode array is related to the spatial impedance resolution; each planar electrode array serves as an electrical detection multi-channel, connected to the electrochemical working unit to realize 3D cell spatial impedance detection in the cell culture pool.

[0067] Preferably, the multi-way selection unit 140 may include multiple sub-multi-way selection units; the membrane electrodes of the multiple vertical electrode arrays in the impedance sensor are led out and hot-pressed and bonded with multiple soft FPCs at 220°, and then the multiple soft FPCs are respectively connected to the multiple sub-multi-way selection units; thereby, the impedance sensor can be scanned at a constant current by using the electrochemical working unit through the multiple sub-multi-way selection units.

[0068] It should be noted that in Figure 1 In the process, the membrane electrodes of multiple vertical electrode arrays in the impedance sensor can be led out and bonded with multiple soft FPCs by heat pressing at 220°, and then the multiple soft FPCs are respectively connected to the electrochemical working units, so that the impedance sensor can be scanned at a constant current using the electrochemical working unit.

[0069] For further information, see Figures 4 to 5 , Figure 4 A physical schematic diagram of a cell culture pool in a 3D cell spatial impedance detection system provided by the present invention; Figure 5A physical schematic diagram of an impedance sensor in a 3D cell spatial impedance detection system provided by the present invention; it should be noted that, Figure 4 The cell culture pool shown is a 3D cell culture pool into which two insertable vertical electrode arrays can be inserted. Figure 5 The impedance sensor shown is Figure 4 The basis forms the impedance sensor.

[0070] Figure 4 The cell culture pool structure shown can be designed by solidworks drawing, including a cell culture pool matching PVEA, and a slot structure, the width of the slot structure is preferably 3mm, and the height matches PVEA; thereby ensuring that the spacing between the two inserted vertical electrode arrays PVEA is 2mm. Among them, the material of the cell culture pool is preferably medical grade transparent polymethyl methacrylate PMMA.

[0071] Further, PVEA was inserted into the cell culture pool structure, and a hydrogel containing cells was added to obtain Figure 5 The impedance sensor shown. This improvement enables the impedance sensor to accurately capture subtle changes in cells in three-dimensional space, more accurately reflect key information such as cell quantity, morphology and proliferation activity, and provide more powerful data support for cell biology research.

[0072] As an example, the preparation of an impedance sensor for detecting the 3D cell spatial impedance of A549 cells can be accomplished by the following method:

[0073] A549 cells (Cell Resource Center, Peking Union Medical College) were stored in Dulbecco's modified Eagle's medium (DMEM, 11995065, Thermo) supplemented with 10% heat-inactivated fetal bovine serum (FBS, A5669401, Thermo) in a constant temperature and humidity incubator at 37°C and 5% CO2. After the cell monolayer almost reached subconfluence and was in a good state, 1 mL of trypsin / EDTA solution (25200072, Thermo) was added to dissociate the monolayer cells. 3D cell culture requires a cell scaffold, and we chose Matrigel (354234, Corning) as the cell scaffold. The following operations were performed according to Lee's protocol to construct an in vitro 3D cell model. The cell suspension was diluted to different concentrations and mixed with pre-cooled Matrigel solution in a centrifuge tube at a dilution ratio of 1:1. Finally, 200 μL of mixed matrix solution was obtained, with a cell density of 1×10 5 ,3×10 5 ,5×10 5 ,7×10 5cells / well, and the embedded matrix mixture is transferred to the PMMA chamber to form a sandwich structure of PVEA / sample / PVEA. Cross-linking is performed in a humidified incubator at 37°C and 5% CO2 for 30 minutes, and finally the electrode is embedded in Matrigel; thereby obtaining the impedance sensor of the present invention, which can be used to perform spatial impedance detection on cells in the cell pool.

[0074] It should be noted that the present invention only uses A549 cells as an example of the monitored 3D cells. The method for preparing an impedance sensor of the present invention can also be used to monitor other cells that can be cultured into small balls in hydrogels, such as CaCo-2, Ea.hy 926, etc.

[0075] Based on this, when conducting cell experiments, A549 cells are cultured in a Matrigel hydrogel structure, and connected to the electrochemical working unit Autolab through PVEA electrodes on both sides through a multiplexer controlled by a single-chip microcomputer STM32. The electrochemical working unit is used to perform impedance spectrum scanning, and then the data is connected to the computer for data analysis and processing; real-time monitoring and data analysis of cell activities can be achieved, greatly improving experimental efficiency and data processing capabilities.

[0076] In a second aspect, the present invention provides a method for detecting 3D cell spatial impedance, which is applied to the 3D cell spatial impedance detection system described in the first aspect; please refer to Figure 6 , Figure 6 A schematic diagram of the main process of a 3D cell spatial impedance detection method provided by the present invention; the execution subject of the method is a server or terminal equipped with the detection method provided by the present invention, such as a cell detection platform or a handheld detection device, etc.

[0077] exist Figure 6 The methods may include:

[0078] Step 610: Obtain a target control instruction for detecting a target impedance detection channel sent by a host computer.

[0079] Step 620: Based on the target control instruction, the corresponding impedance detection channel in the impedance sensor is controlled to perform impedance detection on the 3D cell space; the impedance sensor includes a cell culture pool and a plurality of inserted vertical electrode arrays, and the plurality of inserted vertical electrode arrays are inserted into the cell culture pool; the cell culture pool is used for 3D cell culture, and the plurality of inserted vertical electrode arrays are used to detect the impedance data of 3D cells in the cell culture pool; the spacing between the plurality of inserted vertical electrode arrays is equal to the unit width of the plurality of inserted vertical electrode arrays.

[0080] Step 630: Control the electrochemical working unit to perform constant current scanning on the target impedance detection channel of the impedance sensor to obtain the spatial impedance spectrum data of the 3D cell returned by the impedance sensor.

[0081] In step 610 to step 630, the system obtains the target control instruction sent by the host computer that needs to detect the target impedance detection channel in the impedance sensor. If the multi-way selection unit and the board-level control unit are not set in the system, the electrochemical working unit can be directly used to perform impedance detection on the 3D cell space according to the target control instruction for the corresponding impedance detection channel in the impedance sensor; if the multi-way selection unit and the board-level control unit are set, the multi-way selection unit and the board-level control unit can be used to determine the corresponding target impedance detection channel, and connect the target impedance detection channel with the electrochemical working unit, so as to use the electrochemical working unit to perform impedance detection on the 3D cell space for the corresponding impedance detection channel in the impedance sensor; the electrochemical working unit is used to perform constant current scanning on the target impedance detection channel of the impedance sensor to obtain the spatial impedance spectrum data of the 3D cell returned by the impedance sensor, and the data is uploaded to the host computer to complete data processing and display.

[0082] Based on this, the present invention provides a method for detecting 3D cell spatial impedance. Because the impedance sensor includes a cell culture pool and multiple inserted vertical electrode arrays, multiple inserted vertical electrode arrays are inserted into the cell culture pool, and the spacing between the multiple inserted vertical electrode arrays is equal to the unit width of the multiple inserted vertical electrode arrays, the impedance sensor can ensure the natural growth state of cells during the monitoring process; and can connect the electrodes at different positions in the multiple inserted vertical electrode arrays as needed to perform spatial dynamic detection of 3D cell models at different positions on the cell scaffold respectively; effectively improve the accuracy of 3D cell spatial impedance detection.

[0083] Preferably, the plurality of inserted vertical electrode arrays may include a first inserted vertical electrode array and a second inserted vertical electrode array.

[0084] In step 620, before obtaining the target control instruction for detecting the target impedance detection channel sent by the host computer, the following may be included: preparing a sandwich structure impedance sensor based on the cell culture pool, the first plug-in vertical electrode array and the second plug-in vertical electrode array; in the sandwich structure impedance sensor, from left to right are the first plug-in vertical electrode array, the 3D cell sample and the second plug-in vertical electrode array. Specifically, the sandwich structure impedance sensor is shown in Figure 5 The spatial structure shown on the left.

[0085] Preferably, in step 630, controlling the electrochemical working unit to perform a constant current scan on the target impedance detection channel of the impedance sensor to obtain the spatial impedance spectrum data of the 3D cells returned by the impedance sensor may include: based on the electrochemical working unit, using a constant current scanning mode to scan the target impedance detection channel to obtain the spatial impedance spectrum data of the 3D cells returned by the impedance sensor.

[0086] Specifically, the electrochemical working unit can be used to scan the EIS impedance spectrum, and the constant current scanning mode is selected, the current is 10 μA, and the frequency range is 10 3 Hz to 10 5 Hz; and in order to avoid the influence of the electric double layer on the electrode surface on the impedance at low frequency, the impedance data for analysis can be the impedance data at 10 kHz.

[0087] As a specific example: when the impedance sensor has two inserted vertical electrode arrays, the electrochemical working unit can be used to perform constant current scanning on the electrodes at relative positions of the two PVEA electrode arrays in the impedance sensor in turn, thereby obtaining the spatial impedance spectrum of the 3D cells.

[0088] Preferably, after obtaining the spatial impedance spectrum data of 3D cells returned by the impedance sensor, the following may be included: determining whether all impedance detection channels in the impedance sensor have completed detection, if so, terminating the detection; if not, switching to the next target impedance detection channel to perform impedance detection, and obtaining the spatial impedance spectrum data of 3D cells in the corresponding impedance detection channel. Thus, each impedance detection channel of the impedance sensor is used to perform impedance detection on 3D cells, and complete spatial impedance spectrum data of 3D cells in the cell culture pool is obtained.

[0089] As a more specific example, see Figure 7 , Figure 7 A schematic diagram of the impedance data acquisition process of a 3D cell spatial impedance detection method provided by the present invention.

[0090] exist Figure 7 In the impedance data acquisition process, the following steps can be performed:

[0091] Step 710: Initialize the equipment of the 3D cell spatial impedance detection system.

[0092] Step 720: For the impedance sensor, use the electrochemical working unit to perform constant current scanning on the target impedance detection channel to obtain the spatial impedance spectrum data of the 3D cell returned by the impedance sensor.

[0093] Step 730: Send the current data to the host computer, which processes and displays the data.

[0094] Step 740: Determine whether the current channel has been collected; if so, execute step 760; if not, execute step 750.

[0095] Step 750 : Switch the frequency, ie switch the scanning frequency of the constant current, and continue to execute step 720 .

[0096] Step 760: Determine whether all channels have been collected, that is, determine whether all target impedance detection channels have been collected; if so, execute step 780; if not, execute step 770.

[0097] Step 770 : Switch to the next target impedance detection channel and execute step 720 .

[0098] Step 780: End the impedance data collection process.

[0099] Based on this, fast real-time and long-term multi-channel detection can be achieved, with the ability to characterize spatial differences; and through the board-level control circuit (board-level control unit and multi-channel selection unit), automatic switching and data acquisition processing can be realized, realizing M×N multi-channel spatial impedance detection.

[0100] Furthermore, in order to verify the technical effect of the 3D cell spatial impedance detection system and method provided by the present invention, the present invention adopts Figures 3 to 5 The impedance sensor is fabricated based on the physical structure shown in the figure, which is used to detect the spatial impedance of 3D cells in the cell pool and analyze the accuracy of the spatial impedance spectrum. Figure 8 , Figure 8 A schematic diagram of the verification effect of a 3D cell spatial impedance detection system and method provided by the present invention. Figure 8 Part a is a schematic diagram of the concentration gradient from top to bottom in the entire cell / matrix gel structure in the cell culture pool; the curve in part b is the change curve of the normalized impedance at different positions of the impedance sensor (A / B / C / D electrodes) at low concentration (10μM ZnO nanoparticles) over time, the curve in part c is the change curve of the normalized impedance at different positions of the impedance sensor (A / B / C / D electrodes) at medium concentration (200μM ZnO nanoparticles) over time, and the curve in part d is the change curve of the normalized impedance at different positions of the impedance sensor (A / B / C / D electrodes) at high concentration (1000μM ZnO nanoparticles) over time.

[0101] Specifically, the 16 electrodes in the impedance sensor can be divided into four groups, that is, every four electrodes with the same horizontal position are divided into one group, and a total of four groups of detection electrodes are obtained. The average normalized impedance of each group is used as the final value of the corresponding horizontal position to minimize the estimation error.

[0102] For nanoparticle exposure, 3D cells cultured for 4 days were washed three times with culture medium for 5 minutes each time. ZnO nanoparticles (21077, Sigma) were dissolved in PBS and sonicated to prevent nanoparticle aggregation. The particle concentration was then adjusted to the target level using PBS and sonicated again. The nanoparticles were then injected onto the top of the Matrigel using a syringe. Due to the diffusion effect within the entire cell / matrigel structure, a top-down concentration gradient was spontaneously formed, such as Figure 8 As shown in part a.

[0103] Furthermore, in order to detect the dynamic diffusion of ZnO nanoparticles, spatial impedance detection was performed on 3D cells. After adding metal nanoparticles to the Matrigel containing cells, they diffused downward in the vertical direction; and we arranged the electrodes in PVEA from top to bottom, marked as A to D, with four electrodes in each layer. Figure 8 A, B, C, and D shown in part a. After the 3D cell model was established, the culture solution containing ZnO metal nanoparticles was applied to the top of the hydrogel to ensure complete coverage; then, the real-time impedance changes of each of the 16 electrodes were monitored to track the dynamic diffusion process of ZnO nanoparticles at different locations of the electrodes.

[0104] As shown in the results, the normalized impedance measured at the top electrode (A) changes fastest, while the bottom electrode (D) changes slowest, which is consistent with the expected diffusion of metal nanoparticles from top to bottom. At low concentrations (10 μM), the impedance change from A to D is minimal, as shown in Figure 2. Figure 8 The impedance of the bottom electrode (D) even shows a slight increase after 12 hours and then decreases. At high concentrations (1000 μM), the impedance measured from A to D decreases rapidly, as shown in Figure 2. Figure 8 The change curve shown in part d; at medium concentration (200μM), the impedance measured from A to D reflects the change of diffusion over time, and the uppermost layer has the largest change, see Figure 8 The change curve shown in part c; Figure 8 In part d, the normalized impedance decreased significantly by 80% and then the rate of decrease slowed down, which is consistent with the toxicity resistance exhibited by 3D cells.

[0105] Based on this, it can be undoubtedly concluded that: by adopting the method of the present invention, a single-layer cell chip for long-term on-chip culture can be constructed, which realizes rapid real-time and long-term multi-channel detection, has the ability to characterize spatial differences, and has higher accuracy of impedance data; and can realize automatic switching and data acquisition processing through board-level control circuits, realizing M×N multi-channel spatial impedance detection; compared with traditional toxicity monitoring experiments, the present invention takes into account that 3D cells are more in line with the actual situation of the human body, and also simulates the diffusion process of metal nanoparticles, which has a larger application scenario in drug experiments.

[0106] Although the present invention is described herein in conjunction with various embodiments, in the process of implementing the claimed invention, those skilled in the art may understand and implement other variations of the disclosed embodiments by viewing the drawings, the disclosure, and the appended claims. In the claims, the word "comprising" does not exclude other components or steps, and "one" or "an" does not exclude multiple situations. A single processor or other unit may implement several functions listed in the claims. Certain measures are recorded in different dependent claims, but this does not mean that these measures cannot be combined to produce good results.

[0107] Although the present invention has been described in conjunction with specific features and embodiments thereof, it is apparent that various modifications and combinations may be made thereto without departing from the spirit and scope of the present invention. Accordingly, this specification and the accompanying drawings are merely exemplary illustrations of the present invention as defined by the appended claims and are deemed to cover any and all modifications, variations, combinations or equivalents within the scope of the present invention. Obviously, those skilled in the art may make various modifications and variations to the present invention without departing from the spirit and scope of the present invention. Thus, the present invention is intended to include such modifications and variations if they fall within the scope of the claims of the present invention and their equivalents.

Claims

1. A 3D cell spatial impedance detection system, characterized in that: At least: Impedance sensor, electrochemical working unit and host computer; The impedance sensor is connected to the electrochemical working unit, and the host computer is connected to the electrochemical working unit; The impedance sensor comprises a cell culture pool and a plurality of inserted vertical electrode arrays, wherein the plurality of inserted vertical electrode arrays are inserted into the cell culture pool; the cell culture pool is used for 3D cell culture, and the plurality of inserted vertical electrode arrays are used for detecting impedance data of 3D cells in the cell culture pool; the spacing between the plurality of inserted vertical electrode arrays is equal to the unit width of the plurality of inserted vertical electrode arrays; The electrochemical working unit is used to perform constant current scanning for the impedance sensor, and the impedance sensor returns impedance data to the electrochemical working unit.

2. The 3D cell spatial impedance detection system according to claim 1, characterized in that: The system also includes a board-level control unit and a multi-way selection unit; The multi-way selection unit is connected to the impedance sensor, the electrochemical working unit and the board-level control unit respectively, and the host computer is connected to the board-level control unit; The board-level control unit is used to control the multi-way selection unit to connect to a target impedance detection channel; the target impedance detection channel is a target connection channel between the plurality of the insertable vertical electrode arrays and the electrochemical working unit.

3. The 3D cell spatial impedance detection system according to claim 2, characterized in that: The multi-way selection unit includes multiple sub-multi-way selection units; the membrane electrodes of the multiple vertical electrode arrays in the impedance sensor are led out and hot-pressed and bonded with multiple soft FPCs at 220°, and then the multiple soft FPCs are respectively connected to the multiple sub-multi-way selection units.

4. The 3D cell spatial impedance detection system according to claim 1, characterized in that: The inserted vertical electrode array comprises an inserted vertical electrode array arranged in an M×N matrix, wherein the matrix units are periodic and regular geometric figures, and M and N are both positive integers greater than or equal to 3.

5. The 3D cell spatial impedance detection system according to claim 4, characterized in that: The side length of the matrix unit is 10 μm to 10 mm, and the spacing between the matrix units is 0.1 mm.

6. The 3D cell spatial impedance detection system according to claim 1, characterized in that: The material of the cell culture tank is medical grade transparent polymethyl methacrylate.

7. A method for detecting 3D cell spatial impedance, characterized in that: The method is applied to the 3D cell spatial impedance detection system according to any one of claims 1 to 6; Methods include: Obtain the target control instruction of the detection target impedance detection channel sent by the host computer; Based on the target control instruction, the corresponding impedance detection channel in the impedance sensor is controlled to perform impedance detection on the 3D cell space; the impedance sensor includes a cell culture pool and a plurality of inserted vertical electrode arrays, and the plurality of inserted vertical electrode arrays are inserted into the cell culture pool; the cell culture pool is used for 3D cell culture, and the plurality of inserted vertical electrode arrays are used to detect the impedance data of the 3D cells in the cell culture pool; the spacing between the plurality of inserted vertical electrode arrays is equal to the unit width of the plurality of inserted vertical electrode arrays; The electrochemical working unit is controlled to perform constant current scanning on the target impedance detection channel of the impedance sensor to obtain the spatial impedance spectrum data of the 3D cell returned by the impedance sensor.

8. The method for detecting 3D cell spatial impedance according to claim 7, characterized in that: The plurality of said inserted vertical electrode arrays include a first inserted vertical electrode array and a second inserted vertical electrode array; Before obtaining the target control instruction for detecting the target impedance detection channel sent by the host computer, the method includes: Based on the cell culture pool, the first insertable vertical electrode array and the second insertable vertical electrode array, a sandwich structure impedance sensor is prepared; In the sandwich structure impedance sensor, from left to right are the first inserted vertical electrode array, the 3D cell sample and the second inserted vertical electrode array.

9. The method for detecting 3D cell spatial impedance according to claim 7, characterized in that: The controlling electrochemical working unit performs constant current scanning on the target impedance detection channel of the impedance sensor to obtain spatial impedance spectrum data of the 3D cell returned by the impedance sensor, including: Based on the electrochemical working unit, the target impedance detection channel is scanned in constant current scanning mode to obtain the spatial impedance spectrum data of the 3D cell returned by the impedance sensor.

10. The method for detecting 3D cell spatial impedance according to claim 7, characterized in that: The step of obtaining the spatial impedance spectrum data of the 3D cell returned by the impedance sensor further includes: It is determined whether all impedance detection channels in the impedance sensor have completed detection. If so, the detection is terminated; if not, the next target impedance detection channel is switched to perform impedance detection to obtain spatial impedance spectrum data of the 3D cell corresponding to the impedance detection channel.

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