A method for detecting single cell surface antigen and its application

Abstract

The present application provides a detection method and application of single cell surface antigens. By constructing a new ASS‑SECCM system, antigen recognition and detection at the single cell level are successfully achieved. Compared with traditional liquid SECCM, the system constructed in this application has higher stability and spatial resolution, with the spatial resolution in the XY direction reaching 20nm and the spatial resolution in the Z direction less than 5nm. Using this system, the application of SECCM in biological imaging was realized for the first time, and a method for imaging and characterizing single cell surface antigens based on electrochemical signals was proposed. The imaging results show the fine distribution of CEA antigen on the surface of MCF‑7 cells, which plays an important role in understanding the various life activities of cells and the diagnosis and treatment of cancer.

Description

Technical Field

[0001] The present application relates to the field of detection, and specifically to a method and application for detecting single cell surface antigens. Background Art

[0002] Carcinoembryonic antigen (CEA) is a tumor-associated antigen first isolated from colon cancer and embryonic tissue by Gold and Freedman in 1965. It is an acidic glycoprotein with properties of a human embryonic antigen. It is present on the surface of cancer cells differentiated from endoderm cells and is a structural protein of the cell membrane. It is formed in the cytoplasm, secreted through the cell membrane, and then enters the surrounding body fluids. Therefore, it can be detected in a variety of body fluids and excretions, including serum, cerebrospinal fluid, breast milk, gastric juice, pleural effusion, ascites, urine, and feces.

[0003] Previously, CEA was used as a specific marker for the early diagnosis of colon and rectal cancer. However, extensive clinical experience has revealed that CEA is not only upregulated in gastrointestinal malignancies, but also elevated in the serum of breast, lung, and other malignancies. Therefore, carcinoembryonic antigen is a broad-spectrum tumor marker. Although it cannot be used as a specific indicator for diagnosing a specific malignancy, it still has important clinical value in the differential diagnosis, disease monitoring, and therapeutic efficacy evaluation of malignant tumors.

[0004] Studying antigens at the single-cell level is crucial for understanding their roles in complex cellular processes. Currently, fluorescence microscopy is a commonly used tool for visualizing cellular antigens. Antigen recognition is achieved by fluorescently labeling antibodies and then exploiting specific antigen-antibody binding. However, the resolution of conventional optical microscopes is limited by the optical diffraction limit, with the lowest resolution exceeding 200 nm. The size of individual antigens is typically a few to tens of nanometers, far below the diffraction limit. To overcome this problem, researchers have designed a number of super-resolution fluorescence microscopes, such as stimulated emission depletion microscopy (STED), photoactivated localization microscopy (PALM), stochastic optical reconstruction microscopy (STORM), and scanning near-field optical microscopy (SNOM), achieving resolutions down to 10 nm. Although fluorescence imaging has achieved significant success in single-cell antigen analysis, fluorescence-based detection methods are still subject to several challenges, including photobleaching and high background emission, which can compromise measurement accuracy. Therefore, to better evaluate single-cell antigens, it is necessary to develop super-resolution strategies that do not require optical excitation.

[0005] Analysis based on electrochemical signals has become an alternative, but traditional electrochemical techniques lack spatial resolution. To address this limitation, researchers have introduced microelectrodes and combined electrochemical detection methods with nanoscale positioning systems. This has led to the development of scanning electrochemical probe microscopes such as scanning electrochemical microscopy (SECM), scanning ion conductivity microscopy (SICM), and electrochemical atomic force microscopy (EC-AFM). These techniques have demonstrated significant value in electrocatalysis and bioanalysis. However, a major challenge with these techniques is how to correlate the detected signals with the surface characteristics of the scanned sample. In the case of SICM, the signal is based on the ionic current between two electrodes. Although the ionic current is affected by characteristics such as the surface topography and surface charge of the scanned sample, isolating the influence of individual variables from the changes in the ionic current has always been a challenge. Furthermore, even if the influence of individual variables is isolated, the resolution is extremely limited. In contrast, SECM and EC-AFM rely on the diffusion of redox species between two electrodes. While the acquired signal is closely related to the electrochemical characteristics of the sample surface, the diffusion of substances in the solution significantly affects the true spatial resolution and also introduces non-Faradaic processes. These issues limit the application of these techniques for single-cell antigen analysis. Summary of the Invention

[0006] CEA antigen is an important cancer marker. In-depth research on it will help researchers further understand the mechanisms and development of cancer. Due to the limitations of detection limits, resolution, and stability of existing technologies, it is impossible to identify and detect antigens at the single cell level. To address these issues, this application provides a method for detecting single cell surface antigens, wherein the method comprises the following steps:

[0007] Step 1: Prepare solid-state nanocapillaries and construct an all-solid-state scanning electrochemical cell microscope detection system. Use a needle puller to prepare nanocapillaries. The opening diameter of the nanocapillary is about 20nm. Monomers, water, and photoinitiators are injected into the above nanocapillaries for ultraviolet polymerization. Under pressure, solid-state nanocapillaries are obtained. The prepared solid-state nanocapillaries are used as probes and form an all-solid-state scanning electrochemical cell microscope detection system (ASS-SECCM) with a scanning electrochemical cell microscope. The monomers are composed of acrylamide, hydroxyethyl acrylate, and polyethylene glycol diacrylate.

[0008] Step 2: Prepare target antigen markers and add fluorescent substance Ru(bpy)3 2+ The antibody corresponding to the target antigen is labeled with the target antigen using streptavidin-biotin technology;

[0009] Step 3: Prepare cell samples. Place the cells on an ITO electrode in a culture medium and culture at 37°C. Remove the ITO from the culture medium and wash it three times with PBS solution. Fix the cells. Immerse the electrode in a PBS solution containing the target antigen marker for 30 minutes to modify the cells. Wash the electrodes three times with ultrapure water and dry them.

[0010] Step 4: An indium tin oxide (ITO) electrode is used as a working electrode, a copper wire is connected to the working electrode, and a silver wire coated with AgCl is used as a reference electrode. The modified cell sample is scanned using the all-solid-state scanning electrochemical cell microscope detection system using the chronoamperometry method to detect the target antigen on the cell.

[0011] In one embodiment, the parameters of the needle pulling instrument in step 1 are: Line 1: Heat 350 Fil 3 Vel 30 Del 220 Pul\, Line 2: Heat 350 Fil 3 Vel 40 Del 180 Pul 150.

[0012] In one embodiment, the monomers in step 1 are composed of 50 wt% acrylamide, 20 wt% hydroxyethyl acrylate and 30 wt% polyethylene glycol diacrylate, wherein the ratio of water to monomers is 6:4, and the photoinitiator is 2-hydroxy-2-methyl-1-phenylpropanone, with a content of 1 vol%.

[0013] In one embodiment, the light source for photopolymerization in step 1 is 365 nm ultraviolet light with a power of 6 W; before polymerization, the nanocapillaries are pressurized to 0.2 MPa by an air pump and polymerized under ultraviolet light under pressurized conditions; after 5 minutes of ultraviolet illumination, the light source is turned off and the pressure is released.

[0014] In one embodiment, the antibody in step 2 is a CEA carcinoembryonic antigen antibody.

[0015] In one embodiment, the target antigen marker is prepared in step 2 by mixing bis(2,2'-bipyridyl)-4'-methyl-4-carboxylbipyridyl-ruthenium N-succinimidyl ester-bis(hexafluorophosphate) (Ru(bpy)3 2+ ) and 0.1 mg / mL streptavidin were reacted at 4°C for 2 hours under stirring to obtain concentrated streptavidin-modified Ru(bpy)3 2+ complex;

[0016] The concentrated streptavidin-modified Ru(bpy)3 2+ The complex was diluted to 20 μg / mL and stored at 4°C;

[0017] Streptavidin-modified Ru(bpy)3 2+The complex was incubated with biotinylated CEA antibody at 37°C for 30 minutes to obtain the target antigen marker CEA antigen marker.

[0018] In one embodiment, the step 3 of fixing the cells comprises immersing the cells in 4% paraformaldehyde for 30 minutes in the dark to fix the cells.

[0019] In one embodiment, in step three, drying the cells comprises washing the modified cells with ultrapure water three times and then drying the electrodes at 37°C.

[0020] In one embodiment, the scanning parameters of the all-solid-state scanning electrochemical cell microscope detection system are: a bias voltage of -2 V applied to the ITO electrode, a step size of 500 nm and 10 nm, and a controllable pause of 30 milliseconds when the nanocapillary contacts the sample.

[0021] The present application also provides an application of the single cell surface antigen detection method in the detection of cancer-specific markers, wherein the cancers include: colorectal cancer, pancreatic cancer, gastric cancer, breast cancer, medullary thyroid cancer, liver cancer, lung cancer and ovarian cancer.

[0022] Beneficial effects

[0023] Unlike existing liquid SECCM, the all-solid-state scanning electrochemical cell microscope detection system (ASS-SECCM) constructed in this application can freeze-dry the all-solid-state nanocapillaries, and the morphology of the all-solid-state beads at the needle tip can be roughly maintained. Therefore, SEM can be used for characterization to more intuitively determine its lateral spatial resolution.

[0024] The ASS-SECCM system constructed in this application can distinguish morphological height differences of about 1 nm, has extremely high Z-axis resolution, can achieve a low detection limit, and realize antigen recognition and detection at the single-cell level.

[0025] In this application, solid electrolytes were introduced into SECCM to construct a new ASS-SECCM system. Compared with traditional liquid SECCM, this system has higher stability and spatial resolution, with a spatial resolution of 20nm in the XY direction and less than 5nm in the Z direction. Using this system, the application of SECCM in biological imaging was realized for the first time, and a method for imaging and characterizing single cell surface antigens based on electrochemical signals was proposed. The imaging results show the fine distribution of CEA antigens on the surface of MCF-7 cells, which plays an important role in understanding the various life activities of cells and the diagnosis and treatment of cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 Schematic diagram of detecting single cell surface antigens provided in this application

[0027] Figure 2 (A) SEM image of nanocapillary; (B) SEM image of nanocapillary tip; (C) SEM image of solid-state nanocapillary; (D) SEM image of nanosphere at the solid-state nanocapillary tip.

[0028] Figure 3 (A) SEM image of ASS-SECCM footprint array on Au / ITO surface; (B) statistical analysis of footprint diameter.

[0029] Figure 4 .Mass spectrometry analysis of HMPP solution.

[0030] Figure 5 (A) Under illumination, the solution containing AM and HMPP in the presence or absence of Ru(bpy)3 3+ Comparison of states under different conditions; (B) Before illumination, HMPP / Ru(bpy)3 3+ / Ru(bpy)3 2+ XPS test results of the mixed solution; (C) After 5 minutes of illumination, HMPP / Ru(bpy)3 3+ / Ru(bpy)3 2+ XPS test results of mixed solutions; (D) HMPP / H2SO4 / Ru(bpy)3 before and after illumination 2+ CV results of the solution on an ITO electrode.

[0031] Figure 6 .Ru(bpy)3 on ITO 2+ XRD test results of crystals.

[0032] Figure 7 (A) ASS-SECCM on ITO and Ru(bpy)3 2+ CA results on the surface; (B) Ru(bpy)3 on ITO surface 2+ ASS-SECCM morphology of the crystal layer; (C) Ru(bpy)3 on the ITO surface 2+ ASS-SECCM Faraday charge diagram of the crystal layer; (D) Statistical diagram of the Faraday charge distribution in the boxed area in Figures B and C.

[0033] Figure 8 (A) SEM image of MCF-7 cell sample; (B) fluorescence image of MCF-7 cell sample.

[0034] Figure 9(A) ASS-SECCM morphology of MCF-7 cell sample; (B) ASS-SECCM morphology of J-774 cell sample; (C) ASS-SECCM Faraday charge diagram of MCF-7 cell sample; (D) ASS-SECCM Faraday charge diagram of J-774 cell sample.

[0035] Figure 10 (A) is Figure 9 ASS-SECCM three-dimensional morphology of the cell area marked by the white arrow in (A); (B) Figure 9 ASS-SECCM three-dimensional morphology of the cell area marked by the gray arrow in (A); (C) Figure 9 CEA antigen distribution in the cell area marked by white arrows in (A); (D) Figure 9 CEA antigen distribution in the cell area marked by the grey arrow in middle (A). DETAILED DESCRIPTION

[0036] The present invention will be further illustrated below with reference to specific examples. It should be understood that these examples are only used to illustrate the present invention and are not used to limit the scope of the present invention. After reading the present invention, modifications of various equivalent forms of the present invention made by those skilled in the art all fall within the scope defined by the claims attached to this application.

[0037] Example 1 Preparation and characterization of all-solid-state nanocapillaries and resolution test of the ASS-SECCM system.

[0038] Nanocapillaries were prepared using a P-2000 micropipette puller (Sutter Instrument) to create openings with a diameter of approximately 20 nm. The parameters were: Line 1: Heat 350 Fil 3 Vel 30 Del 220 Pul\, Line 2: Heat 350 Fil 3 Vel 40 Del 180 Pul 150. Solid electrolyte nanocapillaries were prepared by photoinitiated free radical polymerization. Monomers were injected into the capillaries prior to polymerization. The monomers consisted of 50 wt% AM (acrylamide), 20 wt% HEA (hydroxyethyl acrylate), and 30 wt% PEGDA (polyethylene glycol diacrylate). The water-to-monomer ratio was 6:4, and the photoinitiator was 2-hydroxy-2-methyl-1-phenylpropanone at a content of 1 vol%. The light source was 365 nm UV light at a power of 6 W. Before polymerization, the nanocapillaries were pressurized to 0.2 MPa using an air pump. Polymerization was performed under UV light under sufficient pressure. After 5 minutes of illumination, the light source was turned off and the pressure was released. The morphology of the nanocapillaries was characterized by scanning electron microscopy (SEM, Hitachi, Japan, S-4800). The solid electrolyte nanocapillaries were freeze-dried before SEM characterization.

[0039] The schematic diagram of the prepared all-solid-state nanocapillary used to construct an all-solid-state scanning electrochemical cell microscope detection system (ASS-SECCM) is shown in the figure. Figure 1 As shown. The interior of the nanocapillary is a solid electrolyte obtained by photopolymerization. In order to verify the stability and spatial resolution of this system, the surface imprints of the all-solid-state nanocapillary and the scanned sample were analyzed. Figure 2 As shown in Figures A and B, the diameter of the nanocapillary tip used in this embodiment is about 20 nm. After polymerization under light irradiation, a nanosphere protrudes from the tip of the nanoprobe, which acts as an electrochemical cell in the ASS-SECCM ( Figure 2 (C) Unlike existing liquid SECCM, the ASS-SECCM system constructed in this application can freeze-dry the all-solid nanocapillary tube, and the morphology of the all-solid sphere at the needle tip can be roughly maintained. Therefore, SEM characterization can be used to more intuitively determine its lateral spatial resolution.

[0040] like Figure 2 As shown in D, the size of the nanospheres is about 20nm. Before polymerization, the solid electrolyte precursor in the nanocapillary is in liquid state. After being processed by the present application, it becomes solid and basically leaves no trace on the sample surface during the scanning process. In order to measure the actual contact area between the all-solid-state nanocapillary and the sample, the embodiment of the present application used the electrochemical reduction process of Au / ITO for investigation. According to the scheme we developed previously, the nanospheres are in contact with the Au-coated ITO electrode, and a voltage of 1.4V is applied to the Au / ITO electrode for corrosion. When a positive potential is applied, Au undergoes the following reaction:

[0041] Au-3e - +4Cl - →[AuCl4] -

[0042] A 6×6 regular array was obtained on the ITO surface using the ASS-SECCM system, such as Figure 3 As shown in (A), the spacing between adjacent points in this array is 50 nm. The average diameter of the corrosion holes on the electrode surface, representing the contact area, is 13.6 nm. This demonstrates the high spatial resolution of the ASS-SECCM system established in this application.

[0043] Example 2: Verification of the photoreduction-assisted redox cycle mechanism and CA function test of the ASS-SECCM system

[0044] Applying a positive potential on ITO, Ru(bpy)3 on the electrode surface 2+ Electrochemically oxidized to Ru(bpy)33+ Under continuous ultraviolet irradiation, the remaining 2-hydroxy-2-methyl-1-phenylpropanone (HMPP) generates isopropyl alcohol radical and benzaldehyde radical, among which the isopropyl alcohol radical loses proton to generate acetone. It is reported that benzaldehyde radical has strong reducing ability and can convert Ru(bpy)3 3+ Reduction to Ru(bpy)3 2+ Benzaldehyde radical itself is oxidized to benzoic acid. The generated Ru(bpy)3 2+ Participate in the subsequent electrochemical oxidation process, complete the redox cycle, and ultimately achieve the amplification of the electrochemical current.

[0045] To support the proposed photoreduction-assisted redox cycling mechanism, a preliminary verification was first performed using a solution containing HMPP. Under UV illumination, mass spectrometry analysis showed the consumption of HMPP and the generation of benzoic acid in the solution ( Figure 4 ), which confirmed that the decomposition of HMPP follows the path of isopropyl alcohol and benzaldehyde. 3+ Mix and expose to light. 3+ The solution is different, adding Ru(bpy)3 3+ The solution did not polymerize ( Figure 5 A). This indicates that Ru(bpy)3 3+ The presence of can scavenge free radicals, thus inhibiting the photopolymerization process. X-ray photoelectron spectroscopy (XPS) analysis shows that the HMPP, Ru (bpy) 3 3+ (77%) and Ru(bpy)3 2+ After 5 minutes of light irradiation of the mixed solution of Ru(bpy)3 3+ and Ru(bpy)3 2+ The contents of Figure 5 B and C), which is Ru(bpy)3 3+ The free radicals generated by HMPP decomposition are reduced to Ru(bpy)3 2+ Sufficient evidence was provided.

[0046] In the presence of Ru(bpy)3 2+ The Ru(bpy)3 2+ / 3+ Redox cycle current on ITO electrode. Before illumination, cyclic voltammetry was used to record the Ru(bpy)3 2+ and Ru(bpy)3 3+ Reversible redox peaks related to electrochemical redox ( Figure 5(D, black curve). The peak current of the oxidation process is about 4 μA. After the solution is illuminated, the oxidation current increases to 12 μA and approaches a steady state, while the reduction current is almost invisible ( Figure 5 (D, red curve). This increase in oxidation current confirms that Ru(bpy)3 2+ Oxidized to Ru(bpy)3 3+ From the above results, we can see that Ru(bpy)3 2+ / 3+ The redox cycle should proceed according to the photoreduction-assisted mechanism proposed above.

[0047] In order to verify the CA function of the constructed ASS-SECCM system, a thin layer of Ru(bpy)3 was deposited on ITO. 2+ Crystal. XRD data showed that the minimum peak position of the crystal was 7.68°( Figure 6 ). According to the formula:

[0048] 2Dsinθ=nλ

[0049] Where D is the interlayer distance, θ is the diffraction angle, and λ is the wavelength n is the reflection energy level number (n=1). Get Ru(bpy)3 2+ The interlayer spacing is 1.12nm, which is close to the results in the literature. 2+ The area of ​​the molecule was calculated to be 1.25 nm 2 Since the contact area of ​​the nanospheres at the electrode is determined to be 13.6 nm, there are 180 Ru(bpy)3 in each area. 2+ molecular.

[0050] Polyacrylamide (PAM) with HMPP and H2SO4 as solid electrolyte was filled into the nanocapillary and nanospheres were prepared at the tip of the nanocapillary. 2+ When the crystal layer is in contact, a voltage of 1.5 V is applied to the ITO surface and the probe is kept hovering on the sample surface for 30 ms. During this time, the Ru(bpy)3 2+ / 3+ Redox cycle current. Compared with the single-point measurement of cyclic voltammetry (300ms to complete a scan from 0 to 1.5V, scan rate 10V / s), CA measurement single-point contact takes 30ms, which can improve the imaging speed. Therefore, for a 64×64 imaging array, the imaging time can be shortened from 20 minutes (CV) to 2.1 minutes (CA). At a voltage of 1.5V, when the nanospheres are in contact with Ru(bpy)3 2+ When the Ag / AgCl in the nanoprobe contacts the ITO surface, a circuit is formed between the Ag / AgCl in the nanoprobe and the ITO. The initial pulse current is mainly a non-Faraday current ( Figure 7 A, blue curve). After 20ms, it is observed that the non-Faradaic current does not decay completely and no Faradaic current is collected. This is because no Ru(bpy)3 2+ / 3+ Redox cycle. When exposed to light, the current increases significantly ( Figure 7 (A, red curve). Under the assistance of photoreduction, Ru(bpy)3 appears during 20-30ms. 2+ / 3+ The redox cycle current. This result shows that the ASS-SECCM system can distinguish between faradaic and non-faradaic processes through CA function. After removing the initial non-faradaic charge within 20ms, the remaining charge collected is 240fC, corresponding to 1.5×10 6 There are 180 Ru(bpy)3 in each region. 2+ molecules, so one molecule undergoes about 8300 cycles in 10ms, and one cycle time is 1.2μs. Considering the Ru(bpy)3 2+ to Ru(bpy)3 3+ The electron transfer time is 0.64 μs, and 1.2 μs to complete a cycle is reasonable.

[0051] Based on this, Ru(bpy)3 2+ The morphology and electrochemical characteristics of the crystal layer were imaged by ASS-SECCM. The average height of the crystal layer is 25 nm, with fluctuations within 10 nm ( Figure 7 (B) Under illumination, the Faradaic charge of all single points is recorded to obtain Ru(bpy)3 2+ Electrochemical image of the layer ( Figure 7 C). The Faraday charge is independent of the thickness of a single point ( Figure 7 D), which shows that only the single layer of Ru(bpy)3 on the top of the film 2+ Contact with nanospheres for electrochemical oxidation. In summary, the detection limit of the established photoreduction-assisted ASS-SECCM can reach the single-molecule level.

[0052] Example 3 Detection of CEA antigen on MCF-7 cells

[0053] Ru(bpy)3 2+ -CEA antibody synthesis

[0054] Bis(2,2'-bipyridyl)-4'-methyl-4-carboxybipyridyl-ruthenium N-succinimidyl ester-bis(hexafluorophosphate) and streptavidin were purchased from Sigma-Aldrich. Biotinylated CEA antibody (mouse anti-CEA(B5) / biotin) was purchased from Bioss Biotechnology Co., Ltd. (Beijing, China). 1 mg / mL bis(2,2'-bipyridyl)-4'-methyl-4-carboxybipyridyl-ruthenium N-succinimidyl ester-bis(hexafluorophosphate) (Ru(bpy)3 2+ ) 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 cut-off membrane (Millipore). The concentrated streptavidin-modified Ru(bpy)3 2+ The complex was diluted to 20 μg / mL and stored at 4°C. Streptavidin-linked Ru(bpy)3 2+ The complex was incubated with biotinylated CEA antibody at 37°C for 30 minutes.

[0055] Preparation of cell samples

[0056] MCF-7 cells and J-774 cells were cultured on ITO electrodes at 37°C. The ITO electrodes were removed from the culture medium and washed three times with 10X PBS. They were then immersed in 4% paraformaldehyde for 30 minutes in the dark to fix the cells. After washing three times with 10X PBS, the electrodes were immersed in a solution containing 5mM Ru(bpy)3 2+ The cells were modified by incubating the electrodes in 10X PBS containing CEA antibody for 30 minutes. After washing three times with ultrapure water, the electrodes were dried at 37°C. Finally, copper wires were connected to the electrodes. The samples were characterized by optical microscopy (Nikon, Ti).

[0057] In this example, Ru(bpy)3 2+ The electrochemical probe is used to modify the CEA antibody. The CEA antibody is then bound to the CEA antigen through an immune reaction to achieve electrochemical probe labeling of the CEA antibody. This method is also effective for the analysis of other antigens.

[0058] In this example, the cells were fixed and air-dried. Using this method, the cell morphology and surface antigen location can be well preserved. The most important thing is that the cells now become an electron conductor. MCF-7 cells were cultured on the surface of the ITO electrode and fixed and dried to obtain a sample that meets the requirements. The cells were characterized using a scanning electron microscope and a fluorescence microscope. SEM image ( Figure 8 Middle A) shows that the fixed cells have intact morphology and pseudopod features at the edge. 2+With fluorescent properties, fluorescence imaging shows obvious fluorescence in the whole cell ( Figure 8 B), confirmed that Ru(bpy)3 2+ - Binding of CEA antibodies on the plasma membrane.

[0059] The cell morphology was imaged using the established ASS-SECCM ( Figure 9 In Figure A), the imaging results clearly show the cell surface features, including the high area of ​​the cell nucleus (~800nm), the low cell area around the cell nucleus (less than 400nm), and pseudopodia. This image is very similar to the SEM observation results, confirming the accuracy of ASS-SECCM imaging. Following the same protocol as above, Ru(bpy)3 was collected under illumination. 2+ -The Faradaic charge on the surface of MCF-7 cells modified with CEA antibody ranges from 4 to 12 fC ( Figure 9 (C) Based on the principle, scanning points with Faradaic charge indicate the distribution of CEA antigen. Therefore, the resulting cell surface Faradaic charge map provides a qualitative picture of the CEA antigen distribution on the cell surface. The image shows that CEA antigen is unevenly distributed across the cell membrane. The low concentration of antigen in the cell nucleus, likely due to the higher electrical resistance in this region, does not reflect the true distribution of CEA and is therefore excluded from the analysis. CEA antigen is more abundant in the perinuclear cell region, particularly on either side of the pseudopodia. This result is consistent with previous reports that CEA is enriched in areas of cell migration.

[0060] According to Ru(bpy)3 2+ The charge collected by the film (1.33fC / molecule), the Ru(bpy)3 contained in a single contact point on the cell membrane 2+ -The number of Ab-Ag complexes is about 0 to 9. Considering that each CEA antibody consists of 4 Ru(bpy)3 2+ Molecular labeling, the number of CEA antigens on a single contact point is approximately 0 to 2, reaching the single-molecule level. The radius of the CEA antigen is known to be approximately 7.9 nm, which matches the radius of the solid electrolyte probe (7.5 nm). Therefore, a contact point contains a maximum of 1 CEA antigen, supporting the use of the established ASS-SECCM method for single-molecule imaging. The total charge of the entire cell surface is 370 pC, corresponding to 23 fg of CEA antigen per cell. This value is consistent with the literature results (18-27 fg) measured using other methods, verifying the accuracy of the ASS-AECCM antigen analysis method.

[0061] Control test J774 cells

[0062] As a control, this example also performed ASS-SECCM scanning on J774 cells. J774 cells are macrophages that do not contain CEA antigen on their surface. Figure 9 As shown in D, almost no Faradaic charge was collected on the ASS-SECCM image, indicating that there was no Ru(bpy)3 on the surface of J774 after modification. 2+ probe, eliminating the influence of nonspecific adsorption.

[0063] Example 4: Fine Scanning of CEA Antigen on MCF-7 Cells

[0064] From the above results, it can be seen that CEA antigen tends to aggregate and distribute on the surface of MCF-7 cells, and there are high antigen density areas and low antigen density areas on the cell surface. Figure 9 In Figure A, the area with sparse cell surface antigen distribution (marked by white arrows) and the area with dense cell surface antigen distribution (marked by gray arrows) were scanned in detail. Since the all-solid-state nanoprobe does not leave traces on the sample surface when scanning, the scans between two adjacent points will not interfere with each other. Based on this, this embodiment makes the scanning step smaller than the actual resolution to obtain a more detailed scanning result. Figure 10 As shown in the figure, the spatial resolution is 20nm, the step is 10nm, and the scanning range is 640nm×640nm, which means that there are repeated scanning areas in several adjacent scanning points. The scanning results obtained by this scanning method are more continuous, and the results of the antigen distribution characterization on the cell surface can also be more stable. The fine scanning results show the fine structure of the cell membrane, such as protrusions and depressions, with a height difference of about 13nm ( Figure 10 A and B), while the charge image shows the location of individual CEA antigens ( Figure 10 (C and D) The average antigen density in the white arrow region is ∼89 / μm 2 The average antigen density in the gray arrow area is ~752 / μm 2 Although the density of CEA antigen varies from region to region, the image clearly shows that there is more antigen in the convex areas than in the concave areas. Although the reason for this uneven distribution is still unclear, the manifestation of this uneven distribution can provide more information about the role of the antigen and help understand these roles. In summary, due to the signal amplification effect of photoreduction-assisted redox cycling, the single-cell electrochemical detection capability is improved, which is conducive to providing more accurate information on protein distribution.

[0065] The above are only preferred embodiments of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as protection of the present invention.

Claims

1. A method for detecting a single cell surface antigen, wherein: The method comprises the following steps: Step 1, prepare solid nanocapillaries, construct a full solid-state scanning electrochemical cell microscope detection system, use a needle puller to prepare nanocapillaries, the opening diameter of the nanocapillaries is 20nm, inject monomers, water and photoinitiators into the nanocapillaries for ultraviolet polymerization, and obtain solid nanocapillaries under pressure. The prepared solid nanocapillaries are used as probes to form a full solid-state scanning electrochemical cell microscope detection system with a scanning electrochemical cell microscope, and the monomers are composed of acrylamide, hydroxyethyl acrylate and polyethylene glycol diacrylate; Step 2: Prepare the target antigen marker and add the fluorescent substance Ru(bpy)3 2+ The antibody corresponding to the target antigen is labeled with the target antigen using streptavidin-biotin technology; Step 3: Prepare cell samples, place cells on ITO electrodes in culture medium at 37°C, remove ITO from culture medium and wash three times with PBS solution, fix cells, immerse electrodes in PBS solution containing target antigen marker for 30 minutes to modify cells, wash three times with ultrapure water and dry cells; Step 4: Using an indium tin oxide electrode as a working electrode, connecting a copper wire to the working electrode, and using a silver wire coated with AgCl as a reference electrode, the modified cell sample is scanned using the all-solid-state scanning electrochemical cell microscope detection system using the chronoamperometry method to detect the target antigen on the cell.

2. The detection method according to claim 1, characterized in that The parameters of the needle pulling instrument in step 1 are: Line 1: Heat 350Fil 3Vel 30Del 220Pul\, Line 2: Heat 350Fil 3Vel 40Del 180Pul150.

3. The detection method according to claim 1, characterized in that The monomers in step 1 are composed of 50 wt% acrylamide, 20 wt% hydroxyethyl acrylate and 30 wt% polyethylene glycol diacrylate, wherein the ratio of water to monomer is 6:4, and the photoinitiator is 2-hydroxy-2-methyl-1-phenylpropanone, with a content of 1 vol%.

4. The detection method according to claim 1, characterized in that In the step 1, the light source for photopolymerization is 365nm ultraviolet light with a power of 6W; before polymerization, the nanocapillary is pressurized to 0.2MPa by an air pump and polymerized under ultraviolet light; after 5 minutes of ultraviolet light illumination, the light source is turned off and the pressure is released.

5. The detection method according to claim 1, characterized in that In the step 2, the antibody is CEA carcinoembryonic antigen antibody.

6. The detection method according to claim 5, characterized in that In the step 2, the target antigen marker is prepared by mixing bis(2,2'-bipyridine)-4'-methyl-4-carboxylbipyridine-ruthenium N-succinimide ester-bis(hexafluorophosphate) (Ru(bpy)3 2+ ) and 0.1 mg / mL streptavidin were reacted at 4°C for 2 hours under stirring to obtain concentrated streptavidin-modified Ru(bpy)3 2+ Complex; The concentrated streptavidin-modified Ru(bpy)3 2+ The complex was diluted to 20 μg / mL and stored at 4°C; Streptavidin-modified Ru(bpy)3 2+ The complex was incubated with biotinylated CEA antibody at 37°C for 30 minutes to obtain the target antigen marker CEA antigen marker.

7. The detection method according to claim 1, characterized in that In the step 3, the cells are fixed by immersing the cells in 4% paraformaldehyde for 30 minutes in the dark.

8. The detection method according to claim 1, characterized in that In the step 3, the cells are dried by washing the modified cells with ultrapure water for 3 times and then drying the electrodes at 37°C.

9. The detection method according to claim 1, characterized in that The scanning parameters of the all-solid-state scanning electrochemical cell microscope detection system are as follows: the bias voltage applied to the ITO electrode is -2V, the step length is set to 500nm and 10nm, and the nanocapillary is controlled to pause for 30 milliseconds when contacting the sample.

10. Use of the method for detecting single cell surface antigens according to claim 1 in detecting cancer-specific markers, wherein the cancer includes: Colorectal cancer, pancreatic cancer, stomach cancer, breast cancer, medullary thyroid cancer, liver cancer, lung cancer and ovarian cancer.

Images