Method and apparatus for super-resolution electrochemical imaging of single live cell surface antigens

Abstract

The application provides a single living cell surface antigen super-resolution electrochemical imaging device and method, and a novel single cell surface antigen electrochemical analysis system is constructed. The system has high stability and spatial resolution, and the XY direction spatial resolution reaches 30 nm, and the Z direction spatial resolution is less than 5 nm. By using the system, the application of SICM in biological imaging is realized, a method for high spatial resolution imaging and characterization of single cell surface antigens based on electrochemical signals is developed, and the foundation for synchronous characterization of multiple antigens is laid. The current research results provide detailed spatial information of CEA antigen and related potential organization mechanism on the cell membrane, which will help better understand the relationship between molecular aggregation and its function, and the overall structure of the cell membrane. This has important significance for single cell biology.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of electrochemical imaging, in particular to a method and device for super-resolution electrochemical imaging of surface antigens of single living cells. BACKGROUND

[0002] Surface antigens are involved in a variety of physiological activities of cells, and determine the interaction between cells and the outside world. They are also closely related to diseases, such as tumor-related antigens, which are closely related to the occurrence, malignant transformation, diffusion and metastasis of cancer cells, and are widely used as tumor-related markers. At present, the detection of antigens in clinical practice is mainly focused on traditional methods, such as chemiluminescence, enzyme-linked immunosorbent assay, radioimmunoassay, etc., which belong to the analysis of cell populations. Only the amount can provide a relationship with the disease, and the information obtained is not comprehensive. In addition, it is increasingly clear that the distribution and aggregation of surface antigens on the nanoscale are crucial to enhancing their interactions. Therefore, in order to more comprehensively and truly reveal the relationship between antigens and diseases and understand the role of antigens in complex cellular activities, we need to directly observe the antigens on the cell surface and study the distribution of antigens at the single-cell level.

[0003] Super-resolution imaging is a powerful tool for exploring the microscopic world, and has important significance in the field of life sciences. For the study of antigens on single cells, the current method is mainly focused on single-cell super-resolution imaging. In order to achieve nanoscale single-cell imaging, scientists have creatively developed super-resolution fluorescence microscopes such as stimulated emission depletion microscopy (STED), photoactivated localization microscopy (PALM) and stochastic optical reconstruction microscopy (STORM), which have broken through the diffraction limit of optical imaging and greatly promoted the development of single-cell analysis. However, the high background emission of fluorescence and the characteristics of probe molecules prone to bleaching will affect the signal-to-noise ratio and quantitative ability of the measurement process. Therefore, in order to better evaluate the actual distribution of single-cell antigens, it is necessary to develop a single-cell super-resolution imaging strategy that does not require optical excitation.

[0004] Scanning ion conductance microscopy (SICM) is an emerging electrochemical imaging technique with nanoscale spatial resolution, whose signal comes from the ion current between two electrodes. With the help of piezoelectric ceramic to control the position of nanocapillary electrode, the surface topography information of the sample can be obtained. In addition, since the ion current is related to the ion concentration, and the ion concentration can reflect the state of the diffusion layer on the surface of the sample. Therefore, the fine analysis of the ion current can obtain a lot of information other than topography. According to the electric neutrality theory, on both sides of the electrode, the net charge contributed by ions in the diffusion layer on the solution side should be equal to the charge contributed by electrons on the electrode side, so the description of the diffusion layer can reflect the charge density on the surface of the scanned sample. In summary, SICM can become an effective means for surface charge density characterization, and the charge density of the corresponding site can be obtained while the nanoscale topography of the sample surface is obtained. SICM also has many applications in the field of biology, but this method still has limitations in identifying special molecules on cells. Therefore, imaging the distribution of specific molecules in a single living cell is the goal of SICM.

[0005] Carcinoembryonic antigen (CEA), also known as CEA-related cell adhesion molecule 5 (CEACAM5) or CD66e, is a glycosylphosphatidylinositol-anchored membrane glycoprotein anchored in specific membrane microdomains, which is overexpressed in many human cancers including colon cancer, breast cancer and lung cancer. CEA is not only related to respiratory or digestive tract cancer, but also related to some infectious diseases such as gonorrhea or chronic inflammatory diseases such as interstitial lung disease (ILD). Recent literature reports that CEA may be a potential biomarker for assessing the severity and prognosis of COVID-19. Therefore, in-depth study of it helps researchers further understand the mechanism and development process of disease pathology, and has important significance for the development of new disease diagnosis and treatment schemes. SUMMARY

[0006] To solve the problems existing in the prior art, the present application provides a kind of single living cell surface antigen super-resolution electrochemical imaging device, including nanocapillary electrode, scanning ion conductance microscope (SICM) imaging system, Ag / AgCl electrode, current amplifier, control software, optical microscope and data processing system;Wherein,

[0007] The Ag / AgCl electrode comprises two, as a quasi-reference electrode;The SICM imaging system includes a positioning module, a scanning module and a control module;Wherein, the positioning module and the scanning module are connected together, and the positioning module includes at least one motor for preliminary positioning of the sample;The scanning module includes at least one piezoelectric ceramic;The control module is composed of an acquisition board and a control board, the acquisition board is used to collect current signal, and the control board is used to control the voltage applied to the nanocapillary and the sample, the motor and the movement of the piezoelectric ceramic.

[0008] wherein one of the two Ag / AgCl electrodes is placed in the nanocapillary electrode and the other is placed in a solution pool on the culture dish where the cell sample to be measured is placed.

[0009] In an embodiment, the nanocapillary electrode has a tip opening inner diameter of 30 nm to 180 nm.

[0010] In an embodiment, the data processing system comprises Matlab 2018a and Origin95, and the data is processed by drawing the approximation curve obtained by taking current as the Y axis and probe-sample distance as the X axis.

[0011] The application also provides a detection method of the single living cell surface antigen super-resolution electrochemical imaging device, comprising the following steps:

[0012] Step one, preparation of antigen markers, the fluorescent substance and the antibody corresponding to the target antigen are used to obtain the target antigen markers by using the streptavidin-biotin technology;

[0013] Step two, preparation of cell samples, the cells to be measured are placed in the culture medium on the culture dish and cultured at 37°C, the culture dish is taken out of the incubator and washed, the cells are fixed in the dark to obtain the cell sample before modification, the PBS solution containing the target antigen markers of step one is added to the culture dish, and the cells are incubated for 30 minutes to modify the cells, and the PBS solution is washed for three times to obtain the modified cell sample on the culture dish;

[0014] Step three, SICM characterization, the culture dish obtained in step two is installed on the substrate, the substrate is connected to the piezoelectric ceramic in the horizontal direction, and the nanocapillary is installed on the piezoelectric ceramic in the vertical direction; the nanocapillary is positioned to a specific position on the surface of the sample with the help of an optical microscope; two Ag / AgCl electrodes are used as quasi-reference electrodes, one is placed in the nanocapillary electrode and the other is placed in the solution pool on the culture dish, a bias voltage is applied between the two electrodes to obtain a stable ionic current, the SICM scanning adopts a hopping mode to complete the scanning of the entire set area, and the antigen information on the surface of the cell sample is obtained, wherein the antigen imaging information on the surface of the cell is obtained by a scanning ion conductance microscope (SICM) imaging system, and the content information of the antigen on the surface of the cell is obtained according to a data processing system.

[0015] In an embodiment, the fluorescent substance is bis(2,2'-bipyridine)-4'-methyl-4-carboxyl bipyridine ruthenium-succinimidyl ester-bis(hexafluorophosphate), and the antibody is a CEA antibody.

[0016] In one embodiment, the preparation of the antigen-labeled marker is stirring 1 mg / mL bis(2,2'-bipyridine)-4'-methyl-4-carboxy bipyridine ruthenium-succinimidyl ester-bis(hexafluorophosphate) and 0.1 mg / mL streptavidin at 4 °C for 2 hours; the mixed solution is purified by ultrafiltration membrane; the concentrated streptavidin-modified Ru(bpy)3 2+ The complex is diluted to 20 μg / mL with PBS at pH 7.4 and stored at 4 °C; the streptavidin-modified Ru(bpy)3 2+ The complex is incubated with the biotinylated CEA antibody at 37 °C for 30 min to obtain the antigen-labeled marker Ru(bpy)3 2+ - CEA antibody conjugate.

[0017] In one embodiment, the fixation of the cells in the dark is immersing the culture dish in 4% paraformaldehyde and treating in the dark for 30 min.

[0018] In one embodiment, the SICM scanning is moving the nanocapillary downward after the vertical direction Z-axis piezoelectric ceramic controls the movement of the nanocapillary to a safe height before scanning at each scanning point; the detection limit is set to 1% of the current value, and the downward speed of the probe is 20 μm / s; when the ion current difference between the ion current when the probe is at the safe height and the ion current when the probe is at the sample position reaches the set detection limit, it is considered that the probe reaches the sample position; then, the capillary is lifted to the safe height again, and the horizontal direction XY-axis piezoelectric ceramic controls the sample to reach the next scanning point, and the cycle is repeated until the scanning of the entire set area is completed.

[0019] In one embodiment, in the scanning detection, the curve obtained by taking the current as the Y-axis and the probe-sample distance as the X-axis is the approach curve, and the expression of the approach curve is shown in Formula 1-1:

[0020]

[0021] wherein U is the applied voltage, Λ m is the molar conductivity of the solution, d is the probe diameter, is the potential at a certain distance from the electrode surface in the double layer, c - , c + are the concentrations of negative ions and positive ions at the potential , k is the Boltzmann constant, and T is the thermodynamic temperature; is the potential energy, which represents the work done for moving a charge from infinity to the potential at a certain distance from the electrode surface. According to the difference in charge density on the surface of the cell before and after modification, the number of antibodies modified to the surface of the sample can be calculated, and since the antibody and the antigen are one-to-one, the antigen content on the surface of the cell can be obtained accordingly.

[0022] In an embodiment, the simplified approximation curve expression is seen in Equation 1-2;

[0023]

[0024] Advantages

[0025] Compared with various fluorescence imaging, the electrochemical imaging process relies on the charge on the immune complex, which can provide a stable signal for membrane antigens, making up for the lack of quantitative measurement in fluorescence imaging. In addition, SICM can easily realize three-dimensional imaging of molecules, without the need for any complex three-dimensional optical imaging equipment. Therefore, SICM provides a new tool for analyzing the spatial organization of nanoscale molecules, and is an alternative super-resolution microscopy technology that can be used for single-cell imaging, which helps to promote the development of biology and electrochemistry.

[0026] The work herein introduces SICM into the process of single-cell research, and constructs a brand-new electrochemical analysis system for single-cell surface antigens. The system has high stability and spatial resolution, with a spatial resolution of 30 nm in the XY direction and less than 5 nm in the Z direction. Using this system, the application of SICM in biological imaging is realized, a method for high-spatial-resolution imaging and characterization of single-cell surface antigens based on electrochemical signals is developed, and the foundation for the simultaneous characterization of multiple antigens is laid. The current research results provide detailed spatial information of CEA antigens and related potential organization mechanisms on the cell membrane, which will help better understand the relationship between molecular aggregation and its function, as well as the overall structure of the cell membrane. This is of great significance to single-cell biology.

[0027] In addition, in addition to improving stability and spatial resolution, the present application realizes quantitative detection of cell surface antigens by constructing an approximation curve, which provides important information for subsequent research related to cells, antigens, and tumor-related pathogenesis, and has great significance. BRIEF DESCRIPTION OF DRAWINGS

[0028] Figure 1 Schematic diagram of the principle of SICM for single-cell surface CEA antigen super-resolution characterization. Among them, (A) before modification of Ru(bpy)3 2+ -CEA antibody; (B) after modification of Ru(bpy)3 2+ -CEA antibody.

[0029] Figure 2 Schematic diagram of the SICM system for single-cell surface CEA antigen super-resolution characterization.

[0030] Figure 3 Approximation curve results fitted by Equation 1-1 and Equation 1-2.

[0031] Figure 4 Approximation curve fitting results. Wherein (A) nanocapillary SEM image; (B) Theoretical approximation curves drawn according to the formula at different values; (C) measured and fitted approximation curves before and after modification of the substrate and cells, respectively; (D) measured and fitted approximation curves before and after modification of the substrate and cells, respectively.

[0032] Figure 5 Fluorescence images and SICM characterization results of MCF-7 cells. Wherein (A) bright field image of cells; (B) fluorescence image of cells before modification; (C) fluorescence image of cells after modification; (D) cell morphology; (E) surface potential before modification; (F) surface potential after modification.

[0033] Figure 6 (A) real-time current and probe running track; (B) Figure 5 Statistical analysis results of the surface potential of the substrate along the dashed line in (A); (C) Figure 5 Statistical analysis results of the surface potential of the cells along the green dashed line in (A); (D) morphology distribution along the white dashed line in (A) and potential distribution at the green dashed line in (B) and (C). Figure 5 Statistical analysis results of the surface potential of the cells along the white dashed line in (D) and the green dashed line in (E) and (F). Figure 5 Potential distribution at the green dashed line in (E) and (F).

[0034] Figure 7 Fluorescence images of A549 cells. Wherein (A) bright field image of cells; (B) fluorescence image of cells before modification; (C) fluorescence image of cells after modification.

[0035] Figure 8 SICM characterization results of A549 cells. (A) cell morphology; (B) surface potential before modification; (C) surface potential after modification; (D) superimposed graph of the surface potential of (B) and (C). (E) morphology distribution along the white dashed line in (A) and potential distribution at the green dashed line in (B) and (C); (F) statistical analysis results of the surface potential of the substrate along the green dashed line in (B) and (C).

[0036] Figure 9 Fine characterization results of the surface antigen of the cell membrane by SICM. Wherein (A) cell membrane morphology; (B) surface potential of the cells after modification; (C) superimposed graph of (A) and (B); (D) morphology distribution along the white dashed line in (A) and potential distribution at the green dashed line in (B).

[0037] Figure 10 Specific simulation equations and parameters used in COMSOL 5.4 for simulation in an embodiment of the present application. DETAILED DESCRIPTION

[0038] The application will be further clarified by the following examples which are intended to be purely exemplary of the application. It is understood that these examples in no way limit the scope of the application as defined in the claims. Various modifications of the application in addition to those described herein will become apparent to those skilled in the art from the foregoing description. Such modifications are intended to fall within the scope of the appended claims.

[0039] Reagents and chemicals

[0040] Breast cancer cells (MCF-7) were purchased from the Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences. Human lung cancer cells (A549) were purchased from the China Center for Type Culture Collection. Dulbecco’s Modified Eagle Media (DMEM) / High Glucose medium was purchased from Thermo Fisher Biochemical Products (Beijing) Co., Ltd. F-12K medium was purchased from the China Center for Type Culture Collection. Fetal Bovine Serum (FBS), Antibiotic (penicillin + streptomycin), Trypsin were purchased from Gibco. Paraformaldehyde was purchased from Sigma-Aldrich. Phosphate Buffered Saline (PBS) was purchased from Beijing Solabio Science and Technology Co., Ltd. Gold nanocolloids were purchased from Nanjing Xianfeng Nanometer Material Technology Co., Ltd. Bis(2,2'-bipyridine)-4'-methyl-4-carboxy bipyridine ruthenium-succinimidyl ester-bis(hexafluorophosphate), streptavidin, 1-ethyl-(3-dimethylaminopropyl) carbodiimide (EDC) were purchased from Sigma-Aldrich. Biotinylated CEA antibody was purchased from Beijing Bays Bio-technology Co., Ltd. Glass capillary (BF100-58-10) was provided by Sutter Instrument Co. (Novato, CA). The resistivity of the ultra-pure water used was 18.2 MΩ / cm. All chemicals and reagents were used without further purification.

[0041] Instruments

[0042] The SICM imaging system was built independently and can be basically divided into three modules: a positioning module, a scanning module, and a control module. The positioning module is connected to the scanning module. The positioning module consists of three motors, primarily used for initial sample positioning; the scanning module is constructed from three piezoelectric ceramics. The control module consists of a self-developed acquisition board and a control board. The acquisition board collects current signals, while the control board controls the voltage applied to the nanocapillary and sample, the motors, and the movement of the piezoelectric ceramics. Two Ag / AgCl electrodes are used as quasi-reference electrodes. A Nikon-Ti optical microscope is used to assist in sample positioning. Scanning data is processed using Matlab 2018a and Origin95.

[0043] Nanocapillary electrodes were prepared using a P-2000 needle puller (Sutter Instruments). The inner diameter of the electrode tip opening was approximately 180 nm (parameters: Line 1: Heat 350Fil 3Vel 30Del 220Pul; Line 2: Heat 350Fil 3Vel40Del 180Pul 150) and 30 nm (parameters: Heat 950Fil 3Vel 16Del 125Pul160). An S-4800 field emission scanning microscope (Hitachi, Japan) was used to characterize the nanocapillary electrodes. Bright field and fluorescence microscopy images were obtained using an optical microscope (Nikon-Ti). COMSOL 5.4 was used for simulation, and the physical field used was dilute species transport. For specific simulation equations and parameters, see [ 14 ]. Figure 10 .

[0044] Cell culture

[0045] Breast cancer cells (MCF-7) were cultured in DMEM high-glucose medium containing 10% (v / v) fetal bovine serum and 1% (v / v) double-antibody (penicillin + streptomycin) at 37° C. and a humidified environment of 5% CO 2 .

[0046] Human lung cancer cells (A549) were cultured in F-12K medium containing 10% (v / v) fetal bovine serum and 1% (v / v) double-antibody (penicillin + streptomycin) at 37°C in a humidified atmosphere of 5% CO2. 2+ -CEA antibody synthesis

[0047] 1 mg / mL bis(2,2'-bipyridyl)-4'-methyl-4-carboxylbipyridyl ruthenium-succinimidyl ester-bis(hexafluorophosphate) and 0.1 mg / mL streptavidin were stirred at 4°C for 2 hours. The mixed solution was purified by ultrafiltration using a 10K molecular weight cutoff ultrafiltration membrane (Millipore). The concentrated streptavidin-modified Ru(bpy)32+ The complex was diluted with PBS (pH 7.4) to 20 μg / mL and stored at 4 °C. To obtain Ru(bpy)3 2+ The complex was attached to the antigen on the surface of the cells, and the streptavidin-modified Ru(bpy)3 2+ The complex was incubated with the biotinylated CEA antibody at 37 °C for 30 min to obtain Ru(bpy)3 2+ - CEA antibody conjugate.

[0048] Preparation of cell samples

[0049] MCF-7 cells and A549 cells were incubated at 37 °C on a dish. After 48 h of adhesion, the dish was taken out of the incubator and washed with PBS three times to obtain a sample of living cells before modification. After washing with PBS three times, 5 mM Ru(bpy)3 2+ - CEA antibody conjugate was added to the dish and incubated for 30 min. After washing with a PBS solution three times, a sample of cells after modification was obtained.

[0050] Preparation of gold particle samples

[0051] The surface of the colloidal gold was activated with 1% EDC for 30 min, and then centrifuged to remove the supernatant. After washing with deionized water, the mixture was centrifuged to remove the supernatant. 5 mM Ru(bpy)3 2+ - CEA antibody conjugate was added to the dish and incubated for 30 min. After washing with a PBS solution three times, a sample of cells after modification was obtained.

[0052] SICM characterization

[0053] During the SICM scanning, the culture dish was mounted on a base, which was connected to the XY piezoelectric ceramic (P-621, PI, Germany). The nanocapillary was mounted on the Z piezoelectric ceramic (P-753, PI, Germany). The nanocapillary was positioned to the specific position on the sample surface with the help of the optical microscope. The SICM scanning was performed in the hopping mode, i.e. before the scanning of each point, the Z piezoelectric ceramic controlled the nanocapillary to move to a set safe height, and then moved downward. As the distance between the capillary and the sample decreased, the ion current also gradually decreased. The detection limit was set to 1% of the current value, and the downward speed of the probe was 20 μm / s. When the real-time ion current and the ion current when the probe was at the safe height reached the set detection limit, it was considered that the probe reached the sample position. Then, the capillary was lifted to the safe height again, and the XY piezoelectric ceramic controlled the sample to reach the next scanning point, and so on, until the scanning of the entire set area was completed.

[0054] As shown in Figure 1 , after modification, the charge density of the specific region on the cell surface CEA antigen changed, which would make Ru 3+ adsorb a large number of anions around. The high concentration of anions and the resistance at the capillary hole counteracted each other, resulting in high ion current and thus smaller probe-sample distance. Through the fine analysis of the ion current, the information of the charge density of the sample surface was obtained, and finally the visualization of the single cell surface antigen distribution was realized.

[0055] Characterization of whole cell antigen

[0056] The fluorescence images of MCF-7 cells before and after modification are shown in Figure 5 B and C, it can be seen that the whole cell shows fluorescence after modification, indicating that CEA antigen is distributed everywhere on the cell surface, but only weak fluorescence is emitted in the pseudopod part, indicating that the cell surface antigen distribution is uneven. The morphology imaging results of a single MCF-7 cell by SICM are shown in Figure 5 D, the cell is polygonal, indicating that the cell can grow and spread on the surface of the culture dish. Through fitting, the surface potential of the cell before and after modification was calculated and plotted into an image. Before modification, the surface potential of the cell was uniformly distributed around -20 mV, as shown in Figure 5 E. After modification, Ru(bpy)3 2+ -CEA antibody combined with CEA antigen on the cell surface through immune reaction, the potential of most regions on the cell surface increased significantly to about -3 mV, but the surface potential change before and after modification in the cell pseudopod and the region close to the pseudopod was small, indicating that the CEA antigen in these regions was less, as shown in Figure 5This is the same as the fluorescence imaging result, which verifies the accuracy of this method. Figure 5 The white dotted line and the green dotted line are used for line analysis. Figure 6 A in the middle shows the real-time current and probe running track of this part. Figure 6 Middle B and Figure 6 Figures C and B are the results of statistical analysis of the surface potential of the substrate and cells along the line. It can be seen that the surface potential of the substrate area does not change significantly before and after modification. A single point on the substrate is used to fit the approximate curve, as shown in Figure 4. Figure 4 As shown by the red curves in C and D, the approximation curves before and after modification are almost identical. However, statistical analysis of the surface potential on the cells before and after modification reveals two distinct bands, indicating the presence of the antigen. Both the cell and the substrate are negatively charged, but the cell surface potential is lower. Figure 6 Figure D shows the morphology and surface potential distribution of this part. The cell surface potential is significantly lower than the substrate surface potential, which is consistent with the actual situation. Regarding the difference in potential before and after modification, it can be clearly seen that the surface potential on the substrate is almost the same before and after modification, but on the cells, the potential after modification is significantly higher than before modification, which is consistent with the results of previous theoretical analysis, indicating the distribution of the antigen and verifying the accuracy of this antigen analysis method.

[0057] controlled trials

[0058] A control experiment was conducted on A549 cells. Studies have shown that the concentration of CEA antigen on A549 cells is very low. Fluorescence microscopy results also show that the fluorescence intensity of modified A549 cells is weak, such as Figure 7 As shown, the influence of nonspecific adsorption was excluded. SICM results showed that the surface potential difference before and after modification was very small, suggesting that there was less CEA antigen on the cells, which was consistent with the actual situation and positively verified the accuracy of the method. Figure 8 shown. Figure 8 Figure D in the middle is the superposition of the surface potential before and after modification. The base, white circle, and pseudopods in the figure are areas of relatively high potential. Because they exhibit high potential both before and after modification, they appear yellow. Areas with lower potential both before and after modification appear black. It can be seen that, with the exception of the antigen-distributed region, the potential changes in other areas before and after modification are similar. Figure 8 It can be clearly seen in E that this is Figure 8 The topographic and potential distributions at the dotted line show minimal differences in potential before and after modification, both on cells and substrates, and consistent changes in characteristics. This fully demonstrates the stability and reliability of this antigen analysis method.

[0059] According to the literature, the diameter of CEA antigen is about 14.72 nm, in order to realize the analysis of single antigen, the local cell antigen is characterized by using the probe with the opening diameter of 30 nm Figure 5 The scanning step is 50 nm and the scanning range is 5 μm x 5 μm, and such scanning conditions make there is no repeated scanning area in adjacent scanning points. The scanning results obtained by using such scanning mode are more continuous, and the antigen distribution characterization results of cell surface are also more continuous and stable. As shown in Fig. 2A, Figure 9 As shown in Fig. 2A, in this area, the cell surface fluctuation reaches 700 nm, the cell membrane surface is rough, and is composed of protrusions with great difference in size such as circle, triangle, strip and irregular shape, the protrusions are densely distributed, and the structure is smooth and full, the arrangement is moderate, and each protrusion structure is close to each other to form a gap-like depression. Figure 9 Fig. 2B is a surface potential distribution map after modification, according to the full cell characterization results, the surface potential before modification is distributed near -20 mV, and the surface potential after modification increases to about -3 mV. The surface potential of the blue area in the figure is also about -20 mV, indicating that the surface potential of these areas does not change after modification, and the CEA antigen density is basically 0. The surface potential of the white area in the figure increases to about -15 mV, and the CEA antigen density is low. The surface potential of the red area in the figure is distributed in -6 to -3 mV, and the CEA antigen density is high. The results show that the CEA antigen tends to be distributed and gathered on the surface of MCF-7 cells, and there are high antigen density areas and low antigen density areas on the cell surface.

[0060] In the process of participating in various physiological activities, the biological molecules on the cell membrane often need specific distribution position or aggregation form to realize the interaction with different molecules, so as to better complete its physiological function. Figure 9 As shown in Fig. 2C, the topography results are superimposed with the surface potential results, Figure 9 There are four colors in Fig. 2C, the red color represents the area with high topography and low potential, the green color represents the area with low topography and high potential, the yellow color represents the area with high topography and high potential, and the black color represents the area with low topography and low potential. Along Figure 9 The topography distribution of the white dotted line in Fig. 2A is consistent with Figure 9 The potential distribution of the green dotted line in Fig. 2B is shown in Fig. 2D. Figure 9 As shown in Fig. 2D, the potential of the green dotted line in Fig. 2B is consistent with Figure 9 Fig. 2C and Fig. 2D Figure 9It can be seen from the middle D that the CEA antigen presents a characteristic of clustering accumulation on the cell membrane surface rather than scattered distribution. According to the literature, CEA is one of the members of the immunoglobulin superfamily, which is anchored to the "lipid raft" structure region on the cell membrane surface through glycosylphosphatidylinositol (GPI). After binding with antibodies or physiologically related ligands, the aggregation of CEA molecules causes the repositioning and activation of related signal molecules, thereby leading to changes in the state of cells and tissues, such as differentiation, endocytosis, and anoikis, etc. Therefore, this analysis technique can be used to conduct in-depth research on the molecular mechanisms involved in these biological activities and find new cancer targets.

[0061] Further analysis of the results can find that most of the CEA clusters are distributed at the protrusions of the cell membrane surface, and are mainly distributed at the base and top of these protrusions, of which the distribution range is wider at the base. According to the literature, the membrane protrusions are rich in "lipid rafts", which not only provide an important driving force for the formation of many membrane protein domains, but also help a variety of cellular processes, including endocytosis and cell signaling. Actin is mainly located at the base and top of the membrane protrusions, and the actin cytoskeleton can promote the formation of independent and compact protein domains. In addition, the "lipid rafts" and actin cytoskeleton have a greater stabilizing effect on protein clusters at the base than at the top. Moreover, there are a large number of carbohydrate molecules distributed on the cell surface, which not only maintain the stability of molecules as an important component of glycoproteins, but also participate in the distribution of membrane biomolecules by interacting with endogenous lectins. We speculate that the differential distribution of CEA clusters on the membrane may be the result of the combined effects of "lipid rafts", actin cytoskeleton, and carbohydrate chains on the membrane. In summary, through the developed analysis method, the density and spatial distribution of CEA on the cell membrane surface are accurately characterized, which will have important reference value for exploring new cancer cell targets and designing reasonable targeted nanotherapy.

[0062] Approach curve

[0063] The structure of the SICM system for single cell characterization is as follows Figure 2The two Ag / AgCl electrodes were used as quasi-reference electrodes, one inside the nanocapillary and the other in the bulk solution pool. A stable ionic current can be obtained when a bias is applied between the two electrodes. During the scanning at each point, the nanocapillary gradually approaches the sample from a safe height. When the distance between the capillary and the sample is small enough, the ionic current will decrease due to the steric hindrance. The curve obtained by plotting the current as the Y axis and the probe-sample distance as the X axis is the approach curve. The current here is normalized, which is the ratio of the steady-state current in the bulk solution to the instantaneous current when the threshold is reached in the approach process. When the surface charge density of the sample changes, the concentration of the adsorbed counterions in the double layer will also change, which will lead to changes in the approach curve. Therefore, the charge density distribution on the surface of the sample can be obtained from the approach curve by fitting. The mathematical relationship between the approach curve and the surface charge of the sample is established directly in this application, and the expression of the approach curve is shown in formula 1-1, which lays the foundation for using SICM to characterize the surface charge density of the sample. Due to the complexity of the formula and the feasibility of fitting, the formula 1-1 is normalized, and the final expression of the simplified approach curve is shown in formula 1-2.

[0064]

[0065]

[0066] The specific theoretical derivation process and simplification process of the mathematical expression of the approach curve:

[0067] The potential drop and the non-uniform distribution of positive and negative ion concentrations are important characteristics in the double layer. According to the Boltzmann distribution, when a voltage is applied to the surface of the electrode, the concentration distribution of positive and negative ions in the double layer can be obtained:

[0068]

[0069]

[0070] wherein, is the potential at a certain distance from the surface of the electrode, c - , c + are the negative ion and positive ion concentrations at the potential of , k is the Boltzmann constant, and T is the thermodynamic temperature. is the potential energy, which represents the work done when moving the charge from infinity to the potential of .

[0071] From formula 2-1, it can be found that only the expression of is obtained, and the concentration of positive and negative ions in the double layer can be obtained. The expression can be derived from the Poisson equation in electromagnetism:

[0072]

[0073] in, is the surface potential of the sample, which is also the basis for quantifying the charge density in this work. Negative, CEA-Ru 3+ After modification, In addition, due to Ru 3+ The charge is relatively large, resulting in obvious differences after modification, which ensures the signal-to-noise ratio of the characterization results. x is the distance between a point in the double layer and the electrode surface, and κ is a constant with a unit of m -1 , the physical meaning is the reciprocal of the double layer thickness, which can be obtained from the following relationship:

[0074]

[0075] Among them, N A is Avogadro's constant, and ε is the dielectric constant of the solution. At room temperature, once the solution concentration is determined, the κ value can be determined. According to Pauling's principle of electroneutrality, the charge density on the electrode surface is equal to the net charge density carried by the ions between the double membranes. This can be expressed as:

[0076]

[0077] Where σ is the charge density and n is the valence of the corresponding ion. For SICM, since the probe diameter is small enough, most of the resistance is concentrated at the probe tip. According to Ohm's law:

[0078]

[0079] Where I is the current value recorded by SICM; U is the voltage applied between the intercapillary electrode and the reference electrode. p The resistance inherent in the probe. Under the condition that the probe size is determined, R p is a fixed value. t It is an additional resistance introduced by the reduction of the distance between the probe and the sample. Its value becomes non-negligible when the probe-sample distance is reduced to several hundred nanometers, and it also increases as the probe-sample distance is further reduced. Combining the above derivation process, we can get the expression of the approximation curve:

[0080]

[0081] Among them, Λ m is the molar conductivity of the solution. In formula 2-6, only This is an unknown quantity and is also a parameter that needs to be obtained by fitting the approximated curve with this expression. After obtaining The positive and negative ion concentration distribution at this point can be obtained, and the charge density at this point can be obtained by integrating equation 2-4. The results of electrochemical theory lay the foundation for the characterization of the charge density on the sample surface using SICM.

[0082] Due to the complexity of the formula and the feasibility of fitting, the formula is simplified. First, the formula 2-6 is normalized to obtain:

[0083]

[0084] Where parameter C is used to describe the relative size between R p and R t .

[0085]

[0086] Substituting equation 2-8 into equation 2-7 gives the following relationship:

[0087]

[0088] Substituting equation 2-8 into equation 2-2 gives the following relationship:

[0089]

[0090] Substituting numerical values into equation 2-10 gives:

[0091]

[0092] Substituting equation 2-11 into equation 2-9 gives the final expression of the simplified approximated curve:

[0093]

[0094] Before and after modification, the surface charge density of the cell will change, and according to the amount of change in the charge density, a quantitative relationship between the electric potential and the number of antigens on the cell surface can be established, and the specific process is shown as follows:

[0095]

[0096] After integration, we get:

[0097]

[0098] Where A is related to , and after obtaining , the charge density value can be further obtained.

[0099] First, the fitting of the approximation curve was performed using formula 1-1 and formula 1-2 respectively, and the results are shown in Figure 3 There is no difference between the fitting results before and after simplification, which fully verifies the feasibility and accuracy of the simplified formula. In the formula, the surface potential is the only parameter that needs to be determined, which is determined by the surface conditions of the sample. The surface charge density of the cell will change before and after modification, and a quantitative relationship can be established between the potential and the number of antigens on the cell surface according to the change in charge density. According to the research, the cell is usually negatively charged, and a large number of cations are adsorbed in the surface double layer. After Ru(bpy)3 2+ modification, a large number of anions will be adsorbed in the specific region of the CEA antigen on the cell surface. Therefore, the CEA antigen can be identified by the difference in charge density. According to the formula, the approximation curve of the surface potential from -10mV to 10mV was fitted, and the potential difference between the adjacent two curves was 1mV. The fitting results are shown in Figure 4 It can be seen that at the same tip-sample distance, the current increases with the increase of , which is the basis for antigen recognition in this paper.

[0100] As shown in Figure 4 , the potential before modification is -21.79mV, the charge density is -1.58mC / m 2 , the potential after modification is -3.15mV, the charge density is -0.23mC / m 2 , and the difference in charge density before and after modification is 1.35mC / m 2 According to the difference in charge density on the surface of the cell before and after modification, the number of antibodies modified to the surface of the sample can be obtained, and since the antibody and antigen are one-to-one, the antigen content on the surface of the cell can be obtained. The diameter of CEA antigen is about 14.8nm, and when a probe with a diameter of 200nm is used for scanning, a scanning point can contain a maximum of about 183 CEA antigens. According to the SICM scanning results, each antibody carries eight positive charges, so the actual number of antigens measured is about 33.

[0101] For scanning of smaller areas, as shown in Figure 9 , it can be observed that the potential after modification rises from about -28mV to about -6mV, and the difference in charge density is about 1.73mC / m 2 At this time, the diameter of the probe used is about 30nm, and the number of CEA antigens calculated from the scanning results is about 2.

[0102] The above merely is the preferred embodiment of the present application, it should be pointed out that for the ordinary skilled in the technical field, several improvements and refinements can be made without departing from the principles of the present application, these improvements and refinements should also be considered as the protection scope of the present application.

Claims

1. A method for detecting a single living cell surface antigen by a super-resolution electrochemical imaging device, the device comprising a nanocapillary electrode, a scanning ion conductance microscope (SICM) imaging system, two Ag / AgCl electrodes, a current amplifier, control software, an optical microscope, and a data processing system, wherein the two Ag / AgCl electrodes are used as quasi-reference electrodes; the SICM imaging system comprises a positioning module, a scanning module, and a control module; the positioning module and the scanning module are connected together, the positioning module comprises at least one motor for the preliminary positioning of a cell sample, the scanning module comprises at least one piezoelectric ceramic, and the control module comprises a collection board and a control board, the collection board is used to collect current signals, and the control board is used to control the voltage applied to the nanocapillary electrode and the cell sample, the motor, and the movement of the piezoelectric ceramic. The method comprises the following steps: Step 1: preparing an antigen label, using a fluorescent substance and an antibody corresponding to a target antigen to obtain a target antigen label by means of streptavidin-biotin technology; Step 2: preparing a cell sample, placing a cell to be tested in a culture medium on a culture dish, culturing at 37℃, washing the culture dish after it is taken out of the incubator, fixing the cell in the dark, obtaining a cell sample before modification, adding a PBS solution containing the target antigen label of step 1 to the culture dish, incubating for 30 minutes to modify the cell, and washing the PBS solution three times to obtain a modified cell sample on the culture dish; Step 3: SICM characterization, installing the culture dish obtained in step 2 on a substrate, connecting the substrate to a piezoelectric ceramic in the horizontal direction, installing the nanocapillary electrode on a piezoelectric ceramic in the vertical direction, positioning the nanocapillary electrode to a specific position on the surface of the cell sample with the help of an optical microscope, using the two Ag / AgCl electrodes as quasi-reference electrodes, one of which is placed in the nanocapillary electrode and the other of which is placed in a solution pool on the culture dish, applying a bias voltage between the two electrodes to obtain a stable ion current, using a hopping mode for SICM scanning, completing the scanning of the entire set area, and obtaining antigen information on the surface of the cell sample, wherein the imaging information of the cell surface antigen is obtained by the SICM imaging system, and the content information of the cell surface antigen is obtained by the data processing system. The fluorescent substance is bis(2,2'-bipyridine)-4'-methyl-4-carboxy bipyridine ruthenium-succinimidyl ester-bis(hexafluorophosphate), and the antibody is a CEA antibody. The cell is fixed in the dark by immersing the culture dish in 4% paraformaldehyde and placing it in the dark for 30 minutes.

2. The detection method according to claim 1, characterized in that, ​ 3. The method of claim 1, wherein, The preparation of the antigen marker is as follows: 1 mg / mL bis(2,2'-dipyridyl)-4'-methyl-4-carboxylate pyridine ruthenium-succinimidyl ester-bis(hexafluorophosphate) and 0.1 mg / mL streptavidin are stirred at 4 °C for 2 hours; the mixed solution is purified by ultrafiltration membrane; the concentrated streptavidin-modified Ru(bpy)3 2+ The complex is diluted with PBS at pH 7.4 to 20 μg / mL and stored at 4 °C; the streptavidin-modified Ru(bpy)3 2+ The complex is incubated with the biotinylated CEA antibody at 37 °C for 30 min to obtain the antigen marker Ru(bpy)3 2+ - CEA antibody conjugate.

4. The method of claim 1, wherein, ​ 5. The method of claim 1, wherein, The SICM scanning is moving downward after the nanocapillary electrode is moved to a safe height by the vertical Z-axis piezoelectric ceramic before scanning at each scanning point; the detection limit is set as 1% of the current value, and the descending speed of the nanocapillary electrode is 20 μm / s; when the ion current difference between the ion current and the nanocapillary electrode at the safe height reaches the set detection limit, it is considered that the nanocapillary electrode reaches the cell sample position; then, the nanocapillary electrode is lifted to the safe height again, the horizontal XY-axis piezoelectric ceramic controls the cell sample to reach the next scanning point, and the cycle is repeated until the scanning of the entire set area is completed.

6. The method of claim 1, wherein, The inner diameter of the nanocapillary electrode tip opening is 30 nm to 180 nm.

7. The method of claim 1, wherein, The data processing system comprises Matlab2018a and Origin95, and the data is processed by drawing an approximate curve with the current as the Y-axis and the nanocapillary electrode-cell sample distance as the X-axis.

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