A system and method for real-time dynamic in-situ detection of cell microdomain impedance based on scanning electrochemical microscopy

Abstract

This invention belongs to the field of bioelectrochemical detection and cell interface characterization technology, specifically relating to a real-time dynamic in-situ detection system and method for cell micro-region impedance based on scanning electrochemical microscopy. The system includes: a conductive substrate electrode, a SECM probe, a cell culture module, a multi-channel electrochemical workstation, and a control system and imaging software. By precisely scanning near the cell surface using the SECM probe, impedance response signals are acquired simultaneously with the application of alternating excitation signals, enabling real-time dynamic in-situ detection of impedance in micro-regions on the surface of living cells, achieving sub-micron spatial resolution. This invention integrates impedance detection and electrochemical detection functions, enabling label-free, non-invasive continuous monitoring during cell culture, providing a novel high-resolution, multi-parameter synchronous acquisition analytical platform for cell physiological state research, drug screening, and pathological mechanism analysis.

Description

Technical Field

[0001] This invention belongs to the field of bioelectrochemical detection and cell interface characterization technology, specifically relating to a real-time dynamic in-situ detection system and method for cell micro-region impedance based on scanning electrochemical microscopy. Background Technology

[0002] As the basic unit of life, the morphology, structure, physiological state, and functional changes of cells are closely related to their electrical properties. The cell membrane exhibits selective permeability and capacitance. The intercellular junction structures (such as tight junctions and adhesive junctions) and the adhesion state between cells and the matrix all significantly affect the electrical resistance, capacitance, and complex impedance characteristics of the cellular system. Based on these principles, cell impedance detection technology has gradually developed into an important label-free, non-invasive detection method. Cell impedance detection technology measures the changes in the electrical response of a cellular system under an alternating current field to reflect cell number, morphological changes, cell adhesion, and intercellular junction state, and has become one of the important research directions in cell function research and biosensing. Compared with methods such as fluorescent labeling and immunostaining, electrical detection does not rely on exogenous markers and can avoid interference with the physiological state of cells to a certain extent. Therefore, this type of technology has been widely used in biomedical research, drug screening, toxicity evaluation, and cell barrier function research, especially in the study of epithelial and endothelial cell barrier function.

[0003] Scanning electrochemical microscopy (SECM) acquires localized electrochemical response signals on sample surfaces by precisely scanning microelectrodes near the surface. It offers advantages such as high spatial resolution, strong localization capabilities, and in-situ detection. This technology has been widely applied in materials science and electrochemical research and is increasingly being introduced into biological systems to study localized electrochemical activity and mass transport processes on cell surfaces. However, current SECM techniques primarily focus on detecting electrochemical signals such as redox currents. Their system structure and measurement methods have not been optimized for cell impedance detection, and there is a lack of technical solutions that integrate impedance detection functionality with the SECM scanning structure.

[0004] Currently, the main technologies related to cell impedance detection include transepithelial impedance spectroscopy (TEER / EIS), microelectrode array (MEA), and scanning electrochemical microscopy (SECM). TEER / EIS typically involves placing one or more pairs of fixed electrodes in a cell culture vessel. By applying an AC excitation signal to the electrodes, the voltage and current responses are measured to obtain the overall impedance value of the cell layer. Transepithelial impedance spectroscopy (TEER) is primarily used to evaluate the barrier function of the cell layer, while electrochemical impedance spectroscopy (EIS) can acquire impedance information at different frequencies to distinguish between resistive and capacitive components. TEER has fixed electrode positions; the measurement object is usually the entire cell layer or a large culture area; however, the detection results are regional averages with low spatial resolution, making it unsuitable for micro-area or single-cell detection; it is difficult to locate specific cells or local cell regions; and it is mostly static or phased measurements, unsuitable for dynamic micro-area analysis. Microelectrode array technology integrates multiple microelectrodes on a substrate surface. Changes in cell adhesion behavior on the electrodes cause changes in the electrode interface impedance, thus indirectly reflecting the cell state. Multi-channel parallel detection; small electrode size; can obtain a certain degree of spatial distribution information, but the electrode array position is fixed, making scanning measurement impossible; resolution is limited by the electrode spacing, making it difficult to continuously locate and detect dynamically changing micro-regions during cell culture. Scanning electrochemical microscopy (SECM) obtains information on the electrochemical activity distribution of the sample surface by placing microelectrode probes near the sample surface and scanning in three-dimensional space to detect the electrochemical current signal at the probe in real time. SECM is mainly used to study local electrochemical reactions, mass transport, and surface reaction kinetics. It has micron or even submicron spatial resolution; supports non-contact scanning; and can perform in-situ detection of local areas. However, traditional SECM mainly detects electrochemical current signals; it is not specifically designed for cell impedance; and it lacks a structural scheme that integrates impedance measurement and electrochemical signal synchronization.

[0005] In summary, existing cell impedance detection technologies have limitations such as limited spatial resolution, difficulty in achieving long-term in-situ dynamic monitoring, and inability to perform micro-region quantitative analysis of single-cell or subcellular structures. Summary of the Invention

[0006] To address the aforementioned issues, this invention aims to provide a real-time dynamic in-situ detection system and method for cell micro-region impedance based on scanning electrochemical microscopy (SECM), overcoming the technical problems of limited spatial resolution, difficulty in achieving long-term in-situ dynamic monitoring, and inability to perform quantitative analysis of micro-regions for single cells or subcellular structures in existing cell impedance detection technologies.

[0007] To achieve the above objectives, the present invention adopts the following technical solution: In a first aspect, the present invention provides a real-time dynamic in-situ detection system for cell micro-region impedance based on scanning electrochemical microscopy, comprising: Conductive substrate electrodes are used to support cell culture and provide auxiliary electrical signal detection. The SECM probe is suspended above the conductive substrate electrode. The SECM probe serves as a micro-area scanning working electrode that combines SECM scanning and impedance detection functions. The conductive substrate electrode serves as an auxiliary electrode and a cell culture support substrate. The two work together to achieve scanning impedance measurement of cell micro-areas. The cell culture module consists of a quartz glass ring fixed to the conductive base electrode by PDMS to form a cell culture chamber. The chamber is lined with a hydrogel culture medium substrate attached to the surface of the conductive base electrode, forming an integrated electrode-culture medium structure for in situ culture and long-term observation of live cells. The multi-channel electrochemical workstation is connected to the SECM probe and the conductive substrate electrode respectively through the Amplifier1 / 2 multi-channel interconnect circuit, supporting impedance spectroscopy measurement and real-time data acquisition in AC / DC-SECM mode, and realizing synchronous acquisition of impedance signal and DC current signal; The control system and imaging software are connected to the scanning drive mechanism of the multi-channel electrochemical workstation and the SECM probe. The scanning motion and signal acquisition are triggered synchronously by the same clock signal, enabling simultaneous acquisition, processing, and spatiotemporal imaging of electrochemical scanning and impedance data. Furthermore, the conductive substrate electrode is ITO conductive glass.

[0008] Furthermore, the SECM probe is a microelectrode with a diameter ≤10 μm.

[0009] Furthermore, the hydrogel culture medium substrate is a type I rat tail collagen crosslinked polyacrylamide hydrogel, and the inner surface of the culture chamber is hydrophilically treated with 3-(trimethoxymethane)propyl acrylate.

[0010] Furthermore, the multi-channel electrochemical workstation is configured as a PG 618 USB system, supporting multi-channel Amplifier1 / 2, and can realize synchronous acquisition of impedance and DC current in AC / DC-SECM mode.

[0011] Furthermore, the system supports electrochemical impedance spectroscopy measurements over a wide frequency range of 1 Hz to 100 kHz, as well as matrix scanning imaging at a fixed frequency.

[0012] Secondly, the present invention provides a method for real-time dynamic in-situ detection of cell micro-region impedance based on scanning electrochemical microscopy, implemented using the aforementioned system, comprising the following steps: S1. Prepare a conductive substrate electrode and a SECM probe and construct a dual working electrode system, specifically including: fixing a quartz glass ring onto the conductive substrate electrode with PDMS to form a cell culture chamber, preparing a microelectrode with a diameter ≤10 μm as a SECM probe, and connecting the SECM probe and the conductive substrate electrode to the Amplifier1 / 2 channel of a multi-channel electrochemical workstation, respectively. S2. Assemble the multi-channel electrochemical workstation and configure the synchronous acquisition mode. Correctly connect the Amplifier1 / 2 multi-channel interconnection circuit, configure the lock-in amplifier parameters, and set the AC / DC-SECM program to achieve synchronous acquisition of impedance signal and DC current signal. S3. Constructing a hydrogel cell culture system on a conductive substrate electrode, specifically including: hydrophilic treatment of the conductive substrate electrode, preparing a hydrogel culture medium substrate and spreading it on the surface of the conductive substrate electrode to form an integrated electrode-culture medium structure, and inoculating cell suspension for culture until the cells adhere and grow. S4. Synchronous acquisition and imaging: Using the open-circuit voltage of the conductive substrate electrode as the initial voltage, full-frequency EIS testing is performed in the range of 1 Hz to 100 kHz. Then, the AC / DC-SECM program is used to perform matrix scanning at a fixed frequency. The SECM probe scanning motion and signal acquisition are triggered synchronously by the same clock signal to obtain the spatiotemporal impedance spectrum, admittance spectrum, phase spectrum and DC current spectrum.

[0013] Furthermore, the matrix scanning parameters are set as follows: scanning area 50 μm × 50 μm, number of sampling points 21 × 21, scanning speed 5 μm / s, and distance between the SECM probe and the sample surface 3-6 μm.

[0014] Furthermore, during matrix scanning, a progressive curve method is used for probe positioning: the probe is progressively moved along the Z-axis at a speed of -0.5 μm / s or -0.2 μm / s, and the optimal detection distance between the probe and the cell surface is determined based on the current inflection point.

[0015] Furthermore, the method is used for identifying live and dead cells, monitoring drug stimulation response, dynamically assessing cell barrier function, and analyzing cell pathological status.

[0016] Compared with the prior art, the present invention has the following beneficial effects: (1) Realize in-situ scanning of micro-areas and active control of detection area: This invention overcomes the limitations of traditional cell impedance detection techniques, which rely on fixed electrode positions and unadjustable detection areas. By constructing an electrode system comprising a SECM probe and a conductive substrate electrode, and utilizing the three-dimensional scanning function of the SECM probe, active localization and scanning measurement of specific micro-regions on the cell surface are achieved. This system allows for flexible selection of the detection area (such as single cells, cell edges, or intercellular junctions) according to research needs, meeting the requirements for non-destructive, in-situ, and pinpoint measurement of specific micro-regions during cell culture. It solves the technical challenge of existing technologies being unable to accurately locate and track local changes.

[0017] (2) Possesses high spatial resolution at the subcellular level Thanks to the design of microelectrode probes with diameters below 10 μm, this invention significantly improves the spatial resolution of impedance detection. Unlike traditional macro-electrodes or microelectrode arrays, which are limited by electrode spacing, this system can precisely acquire the distribution of micro-regional electrical properties of individual cells or even subcellular structures through precise scanning of the cell surface by microprobes. This high-resolution imaging capability makes it possible to reveal microscopic physiological mechanisms such as differences in local cell membrane structure and uneven distribution of ion channels.

[0018] (3) Supports long-term dynamic real-time monitoring and in-situ culture This invention integrates a hydrogel-based electrode-culture medium integrated cell culture module. By constructing a biocompatible hydrogel substrate (such as collagen-crosslinked polyacrylamide) on a conductive substrate, it not only simulates the in vivo extracellular matrix environment, which is beneficial for cell adhesion, growth, and long-term survival, but also avoids the physical damage or toxic interference caused by direct contact with cells by traditional metal electrodes. Combined with the system's non-contact scanning detection mode, it enables in-situ, continuous, and dynamic impedance monitoring of live cells during long-term culture, fully capturing the temporal information of cell state evolution (such as drug response and barrier function remodeling).

[0019] (4) Realize spatiotemporal synchronous acquisition and multidimensional imaging of multimodal signals This invention achieves strictly synchronous acquisition of SECM probe scanning motion, impedance signals (real, imaginary, phase, and admittance), and DC current signals through the combined use of Amplifier 1 / 2 in a multi-channel electrochemical workstation and a common clock signal triggering mechanism. This method not only generates high-resolution impedance maps but also simultaneously acquires admittance maps, phase maps, and DC current maps, constructing a multi-parameter spatiotemporal imaging platform for the cellular microenvironment. This multimodal data fusion provides comprehensive and reliable data support for in-depth analysis of the electrophysiological characteristics, material transport processes, and coupling mechanisms of cellular microregions, effectively overcoming the shortcomings of existing technologies such as the separation of impedance and electrochemical detection and spatiotemporal data mismatch.

[0020] (5) Provide label-free, non-invasive, and precise analysis methods Based on the above system and method, this invention achieves high-precision detection of dead and live cells, drug-induced responses, cell barrier function assessment, and pathological state analysis without fluorescent labeling or exogenous tracers, and without damaging cell structure or interfering with normal physiological states. This not only improves the authenticity and reliability of the detection data, but also provides a powerful new analytical tool for new drug screening, toxicological evaluation, and basic cell biology research. Attached Figure Description

[0021] Figure 1 : A physical image of a 10μm diameter platinum microelectrode (left) and an ITO substrate electrode (right).

[0022] Figure 2 : A schematic diagram of the connection of a multi-channel electrochemical workstation that supports impedance spectroscopy measurement and real-time data acquisition.

[0023] Figure 3 Comparison of EIS impedance at all frequencies between living and dead cells.

[0024] Figure 4 : AC-SECM-Matrix impedance imaging results of live and dead cells at different frequencies Figure 5 Image of a single live cell AC / DC-SECM-Matrix at 10 kHz (including admittance, phase and DC current spectra). Detailed Implementation

[0025] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments, but this should not be construed as limiting the invention. Unless otherwise specified, the technical means used in the following embodiments are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following embodiments are commercially available unless otherwise specified.

[0026] Currently, cell impedance detection primarily employs fixed electrodes within a culture vessel. An AC excitation signal is applied between the electrodes, and the voltage and current responses are measured to obtain impedance parameters. This technique typically involves measurements after cells have adhered and formed a certain coverage area, and the resulting impedance values ​​reflect the overall or average electrical properties of the cell within the electrode-covered area. While this method has advantages in evaluating the overall cell growth status or cell layer integrity, its measurements are essentially spatially averaged and fail to reflect localized electrical differences within the cell. In real biological systems, cells are not uniformly distributed ideal structures. Even within the same culture system, significant differences in electrical properties can exist between different cells, different regions of the same cell, and between the cell-matrix interface. For example, the cell periphery differs from the central region in membrane structure and adhesion; the intercellular junctions differ from the surface of a single cell in impedance characteristics; and localized areas of the cell often change first after being stimulated by mechanical, drug, or infectious agents. However, existing fixed-electrode impedance detection techniques, due to their fixed detection area, cannot precisely locate and track these localized changes, thus limiting their application in refined cell function studies.

[0027] With the development of cell biology and biomedical engineering, research focus has gradually shifted from static endpoint analysis to continuous monitoring of dynamic cellular behavior. For example, in cell barrier function studies, the formation, remodeling, and disruption of intercellular connections are often a continuous process, and relying solely on single or intermittent impedance measurements is insufficient to accurately reflect the evolution of cell states. However, most existing impedance detection devices are designed for intermittent or intermittent measurements, making it difficult to achieve real-time, continuous, and dynamic impedance monitoring during cell culture. Furthermore, some detection methods require human intervention in the culture system during measurement, which can easily affect the physiological state of cells.

[0028] To improve spatial resolution, some technologies attempt to achieve localized detection by reducing electrode size or employing microelectrode array structures. However, these approaches typically still rely on fixed electrode arrangements, with the detection location predetermined by the electrode array, lacking flexibility, and resolution is limited by electrode spacing and manufacturing processes. Furthermore, detection methods involving direct contact between fixed electrodes and cells may affect cell growth during long-term monitoring, hindering truly non-destructive testing. Therefore, achieving non-contact or weak-contact detection while maintaining high spatial resolution has become a key technical challenge for existing technologies.

[0029] In summary, existing cell impedance detection technologies generally suffer from the following shortcomings in practical applications: ① The detection area is fixed, lacking the ability to locate and scan cell microregions; ② Spatial resolution is limited, making it difficult to reflect electrical differences in local cell regions; ③ It is difficult to achieve in-situ, real-time, and dynamic continuous monitoring during cell culture; ④ Impedance detection and electrochemical detection are usually separate systems, lacking integrated design. Therefore, a new technical solution is urgently needed to achieve in-situ, high-resolution, real-time dynamic detection of cell microregion impedance without interfering with the normal physiological state of cells, and to be combined with electrochemical detection functions to meet the needs of refined cell research.

[0030] Example 1: Construction and basic detection of a real-time dynamic in-situ detection system for cell micro-region impedance based on scanning electrochemical microscopy (SECM).

[0031] 1. Electrode and probe preparation ITO conductive glass was used as the substrate electrode (the glass size of the electrochemical workstation device is 24×24 mm, and the glass thickness is 1.1 mm to meet the requirements of observation by inverted optical microscope), and it was cleaned with ethanol before use. A quartz glass ring (outer diameter: 18 mm, inner diameter: 15 mm, length: 5 mm) was fixed to the ITO surface using polydimethylsiloxane (PDMS) adhesive to form a cell culture chamber. Fabrication of a 10 μm diameter platinum disk microelectrode: A 10 μm diameter platinum wire was cut into 1 cm lengths and fixed to a copper wire with conductive silver paste. After drying, the wire was inserted into a glass capillary tube, and the electrode was drawn using a PC-10 tube puller. The drawn electrode was then further heated and sealed at the tip using a vacuum pump (Vi 240sv) under vacuum conditions. Epoxy resin was applied to the opening of the probe to fix the capillary end and the copper wire joint. The tip was then vertically polished using a needle grinder to obtain a probe with an electrode diameter of 10 μm and an RG ratio (the ratio of the diameter of the electrode tip, including the glass shell, to the diameter of the conductive platinum wire at the center of the electrode) of 2.

[0032] Figure 1 The illustration shows a physical image of a substrate electrode with good conductivity and a microelectrode probe with both SECM scanning and impedance detection functions, prepared according to embodiments of the present disclosure.

[0033] 2. Workstation Configuration The PG 618 USB electrochemical workstation was used, connected to the Amplifier1 / 2 dual-channel amplifier; Configure the lock-in amplifier (phase shift=0, Attenuation=1); A three-electrode system was constructed: a platinum disk microelectrode (working electrode), an ITO substrate (auxiliary electrode), an Ag / AgCl (reference electrode), and a Pt wire (counter electrode).

[0034] Figure 2 A schematic diagram illustrates a multi-channel electrochemical workstation assembled according to an embodiment of the present disclosure to support impedance spectroscopy measurements and real-time data acquisition.

[0035] 3. Construction of hydrogel culture system Surface hydrophilic treatment: The cell culture chamber was cleaned with anhydrous methanol, and 2% 3-(trimethoxymalkyl)propyl acrylate solution was added to the pre-cleaned cell culture chamber ITO substrate electrode for hydrophilic treatment for 5 minutes. Then, 0.5% glutaraldehyde solution was added and allowed to stand for 30 minutes before being rinsed with pure water and dried to make the ITO glass surface hydrophilic. Hydrogel preparation: PBS, 40% acrylamide solution (solution A), 2% methylenebisacrylamide solution (solution B), 10% ammonium persulfate (APS) solution, and N,N,N',N'-tetramethylethylenediamine (TEMED) were mixed according to the proportions in Table 1. 30 μL of the polymerization solution was dropped onto the hydrophilically treated ITO substrate electrode, and a glass slide was used to cover it to form a sandwich structure. After the PA glue had polymerized for a period of time, the glass slide was removed to prepare a hydrogel with a hardness of 1 kPa. The hydrogel was soaked and washed three times with PBS for 5 minutes each time to completely remove the hydrophobic substances on the surface of the PA glue. Table 1 Hydrogel composition Surface protein modification: Under light-protected conditions, Sulfo-SANPAH solution was added dropwise to the PA gel surface until completely covered, and incubated for 5 minutes. Then, it was activated under 365 nm UV irradiation for 10 minutes. The gel was washed three times with 10 mmol / L HEPES buffer for 5 minutes each time, and then incubated overnight at 4°C with 50 μg / mL type I collagen solution to form a culture medium substrate with good biocompatibility suitable for cell growth.

[0036] 4. Simultaneous detection and imaging A cell suspension of a certain number of cells was seeded into a cell culture chamber and cultured in complete culture medium. For SECM assays, a 0.5 mM ferrocene formate solution was prepared using PBS buffer. Before the test, the culture medium was aspirated, and the cells were immersed in 0.5 mM ferrocene formate solution. The ITO substrate electrode and platinum microelectrode prepared in step 1 were used as working electrodes, the Ag / AgCl electrode as the reference electrode, and the platinum wire as the counter electrode. Using the open-circuit voltage of the ITO substrate electrode OCP as the initial voltage and the output frequency from 1 Hz to 100 kHz, EIS tests were performed on cells grown in the ITO substrate electrode culture chamber at all frequencies to obtain cell impedance values ​​at different frequencies. The AC / DC-SECM program was selected, a three-electrode system was connected, the potential was biased at 0.5 V, and an asymptotic curve was plotted using ferrocene formate as the redox medium to determine the probe height and matrix scanning area. Using the OCP open-circuit voltage as the initial voltage, a scan was performed in a fixed area region to obtain multi-parameter spectra at a certain frequency: impedance spectrum, admittance spectrum, phase spectrum, and DC current spectrum.

[0037] Example 2: Cell impedance detection under different cell states Using the system constructed in Example 1, impedance detection was performed on normally growing live MDA-MB-231 cells and dead MDA-MB-231 cells immobilized with 4% paraformaldehyde. Specifically, the detection included: 1. Assemble the measurement system: The SECM assay used a 0.5 mM ferrocene formate solution prepared with PBS buffer. Before the assay, the culture medium was aspirated, and the cells were immersed in 0.5 mM ferrocene formate solution. The reference electrode and counter electrode were also immersed in the solution. The ITO substrate electrode and platinum microelectrode prepared in step 1 were used as working electrodes, the Ag / AgCl electrode as the reference electrode, and the platinum wire as the counter electrode.

[0038] 2. Positioning probe height: First, place the probe below the liquid surface and above the ITO substrate electrode. Then, bias the potential at 0.5 V, using ferrocene formate as the redox medium. Use the probe to perform an asymptotic curve along the cell surface to position the probe on the cell surface. Then, raise the probe to a distance of 3-5 μm from the cell surface. For the asymptotic method: Under low magnification (4x), locate the bright spot (the needle tip). Use a joystick to move the needle to the center of the field of view (observing the liquid surface to prevent needle collision). Adjust to high magnification (10x and 20x) and manually adjust the Z-axis focus to bring the cell and needle close to approximately the same focal length. The obvious dark shadow indicates the electrode. Select 2D Scan mode and begin a asymptotic motion along the Z-axis onto the substrate or cell. Select a stop level of 1% and an asymptotic speed of -0.5 μm / s. Manually stop the asymptotic motion at the current inflection point and measure the required elevation distance.

[0039] 3. Impedance curve measurement and matrix scanning imaging of cellular microregions (1) Curve determination The OCP open-circuit voltage of the electrode was obtained. In the Lockin window, phase shift=0 and Attenuation=1 were set. In the Protocol Editor, the EIS program was selected, and a new protocol named EIS-A1 / A2 was created. The initial voltage E-initial was manually set to the OCP open-circuit voltage value, and the frequency was 1 Hz-20 kHz (or a wider range of 0.01 Hz to 100 kHz). The Current Range channel setting was set to 5 nA initially; this can be left unset, as the program will automatically find the range. After positioning the probe on a specific area of ​​the cell surface, the EIS button was pressed to start the measurement. The current will automatically begin searching, and the current channel was selected as I. mon1 Obtain impedance data at various frequencies: the real part of the impedance, the imaginary part of the impedance, the frequency change value, the phase angle, etc.

[0040] Plot a Nyquist diagram using the real and imaginary parts of the impedance from the impedance curve measurement results, as shown below. Figure 3 As shown, the measurement results indicate that in the high-frequency region, the system impedance is determined by the solution resistance (R0). s The dominant characteristic is directly reflected in the intercept of the horizontal axis; both living and dead cell regions exhibit low solution resistance. In the mid-frequency region, the system is subject to charge transfer impedance (R0). ct The larger the radius of the semicircle, the greater the charge transfer resistance and the slower the reaction rate. The charge transfer resistance is greater in the dead cell region than in the live cell region. In the low-frequency region, the reaction of the system is controlled by diffusion (such as Warburg impedance), which corresponds to a slow mass transfer process. The Nyquist plot of the live cell region shows an obvious mass transfer diffusion process in the low-frequency region.

[0041] (2) Matrix scanning Position the probe on the cell surface, raising it approximately 3-5 μm. Adjust the probe to the appropriate position and perform equidistant scanning. Use 2D-Scan mode, selecting current channel 2 and voltage channel 2. Scanning current changes along the X and Y axes in 2D Scan mode will obtain the cell location information. Set the X / Y region scan size, such as 50 μm × 50 μm, the number of samples to 21, and the speed to 5 μm / s, scanning within a fixed area. Set to a three-electrode system (3Electrode Mode), the scanning program to Matrix Scan, the protocol to AC / DC-SECM, and in the lockin window, set phase shift=0 and Attenuation=1. Select the AC / DC-SECM program using F9, and set the parameters in the program: initial voltage as OCP open circuit voltage, and scan frequency under AC voltage (1 Hz / 100 Hz / 10 kHz). This will produce the following at the selected frequency: Impedance spectrum, Admittance spectrum, Phase spectrum, and DC current graph.

[0042] After matrix scanning, spatial imaging of cellular micro-region impedance can be obtained, and the results are as follows: Figure 4 As shown, Figure 4 The single-cell impedance matrix spectra revealed the following: First, frequency affects the detection resolution, with higher resolution observed at 10 kHz. At the same frequency, cell state influences the impedance detection results, with dead cells exhibiting higher impedance characteristics. Furthermore, impedance values ​​at different locations on the cell surface differ between living and dead cells. At 10 kHz, the impedance values ​​measured at high resolution for living cells ranged from 600 kΩ to 800 kΩ, while those for dead cells ranged from 900 kΩ to 1100 kΩ. These results indicate a significant correlation between cell viability and its surface impedance characteristics. Additionally, the spatiotemporal synchronous acquisition of cell impedance, admittance, phase, and current signals can be achieved, such as… Figure 5 As shown in the figure. The detection results indicate that the method of the present invention can perform micro-region quantitative analysis of single-cell or subcellular structures.

[0043] It should be noted that when numerical ranges are mentioned in the claims of this invention, it should be understood that the two endpoints of each numerical range and any value between the two endpoints can be selected. To avoid redundancy, the present invention describes preferred embodiments.

[0044] Although preferred embodiments of the invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including both the preferred embodiments and all changes and modifications falling within the scope of the invention.

Claims

1. A real-time dynamic in-situ detection system for cell micro-region impedance based on scanning electrochemical microscopy, characterized in that, include: Conductive substrate electrodes are used to support cell culture and provide auxiliary electrical signal detection. SECM probe, suspended above the conductive substrate electrode, serves as a micro-area scanning working electrode that combines SECM scanning and impedance detection functions, while the conductive substrate electrode serves as an auxiliary electrode. The cell culture module consists of a quartz glass ring fixed to the conductive base electrode by PDMS to form a cell culture chamber. The chamber is lined with a hydrogel culture medium substrate attached to the surface of the conductive base electrode, forming an integrated electrode-culture medium structure for in situ culture and long-term observation of live cells. The multi-channel electrochemical workstation is connected to the SECM probe and the conductive substrate electrode respectively through the Amplifier1 / 2 multi-channel interconnect circuit, supporting impedance spectroscopy measurement and real-time data acquisition in AC / DC-SECM mode, and realizing synchronous acquisition of impedance signal and DC current signal; The control system and imaging software are connected to the scanning drive mechanism of the multi-channel electrochemical workstation and the SECM probe. The scanning motion and signal acquisition are triggered synchronously by the same clock signal, so as to realize the synchronous acquisition, processing and spatiotemporal imaging of electrochemical scanning and impedance data.

2. The detection system according to claim 1, characterized in that, The conductive substrate electrode is ITO conductive glass.

3. The detection system according to claim 1, characterized in that, The SECM probe is a microelectrode with a diameter ≤10 μm.

4. The detection system according to claim 1, characterized in that, The hydrogel culture medium substrate is a type I rat tail collagen crosslinked polyacrylamide hydrogel, and the inner surface of the culture chamber is hydrophilically treated with 3-(trimethoxymethane)propyl acrylate.

5. The detection system according to claim 1, characterized in that, The multi-channel electrochemical workstation is configured as a PG618 USB system, which supports the use of multiple channels of Amplifier1 / 2, and can realize synchronous acquisition of impedance and DC current in AC / DC-SECM mode.

6. The detection system according to claim 1, characterized in that, The system supports electrochemical impedance spectroscopy measurements over a wide frequency range from 1 Hz to 100 kHz, as well as matrix scanning imaging at a fixed frequency.

7. A method for real-time dynamic in-situ detection of cellular micro-region impedance based on scanning electrochemical microscopy, implemented using the system described in any one of claims 1-6, characterized in that, Includes the following steps: S1. Prepare a conductive substrate electrode and a SECM probe and construct a dual working electrode system, specifically including: fixing a quartz glass ring onto the conductive substrate electrode with PDMS to form a cell culture chamber, preparing a microelectrode with a diameter ≤10 μm as a SECM probe, and connecting the SECM probe and the conductive substrate electrode to the Amplifier1 / 2 channel of a multi-channel electrochemical workstation, respectively. S2. Assemble the multi-channel electrochemical workstation and configure the synchronous acquisition mode. Correctly connect the Amplifier1 / 2 multi-channel interconnection circuit, configure the lock-in amplifier parameters, and set the AC / DC-SECM program to achieve synchronous acquisition of impedance signal and DC current signal. S3. Constructing a hydrogel cell culture system on a conductive substrate electrode, specifically including: hydrophilic treatment of the conductive substrate electrode, preparing a hydrogel culture medium substrate and spreading it on the surface of the conductive substrate electrode to form an integrated electrode-culture medium structure, and inoculating cell suspension for culture until the cells adhere and grow. S4. Synchronous acquisition and imaging: Using the open-circuit voltage of the conductive substrate electrode as the initial voltage, full-frequency EIS testing is performed in the range of 1 Hz to 100 kHz. Then, the AC / DC-SECM program is used to perform matrix scanning at a fixed frequency. The SECM probe scanning motion and signal acquisition are triggered synchronously by the same clock signal to obtain the spatiotemporal impedance spectrum, admittance spectrum, phase spectrum and DC current spectrum.

8. The detection method according to claim 7, characterized in that, The matrix scanning parameters are set as follows: scanning area 50 μm × 50 μm, number of sampling points 21 × 21, scanning speed 5 μm / s, and distance between SECM probe and sample surface 3-6 μm.

9. The detection method according to claim 8, characterized in that, During matrix scanning, the probe is positioned using a progressive curve method: the probe is progressively moved along the Z-axis at a speed of -0.5 μm / s or -0.2 μm / s, and the optimal detection distance between the probe and the cell surface is determined based on the current inflection point.

10. The detection method according to claim 7, characterized in that, The method is used for identifying live and dead cells, monitoring drug stimulation response, dynamically assessing cell barrier function, and analyzing cell pathological status.

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