Circulating tumor cell immunofluorescence detection method based on thermosensitive adhesive and application of circulating tumor cell immunofluorescence detection method

Abstract

The invention discloses a circulating tumor cell immunofluorescence detection method based on thermosensitive glue and application thereof, the thermosensitive glue used in the method is denatured at a specific temperature, the interface adhesive force is greatly reduced, an antibody-fluorescent compound can be stripped, but cell retention can still be maintained under the electrostatic interaction of a cell glass slide, and the cell retention rate is greater than 90%. After stripping, residual fluorophores are oxidized with low-concentration H2O2, and after double bonds are broken, signals are reduced to the background. The whole process is free of strong acid, strong alkali and high salt, damage to cells and protein is small, and operation is easy. According to the method, the circulating tumor cell detection material is repeatedly utilized, and multiple rounds of detection are realized, so that more detection information is obtained; the method has no limitation on the species of the antibody, improves the experiment flexibility and reduces the cost.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biomedical detection, and relates to a detection method for circulating tumor cells in peripheral blood, in particular to a circulating tumor cell immunofluorescence detection method based on heat-sensitive glue and application thereof. BACKGROUND

[0002] Circulating tumor cells (CTCs) are the vanguard signal of tumor metastasis, which are derived from the spread of primary or metastatic tumors, and CTCs are direct evidence of tumor occurrence and development. At present, the detection of CTCs in the clinic has the following difficulties and defects: 1. Difficulty in detecting CTCs: the content of CTCs in blood is very low, and the shape and nature of CTCs are similar to those of normal blood cells. How to efficiently identify CTCs in the enrichment and separation process and ensure accuracy is a great challenge. 2. Interference of false negative and false positive in CTC detection: in the process of recognizing CTCs by using antibody-dependent methods such as surface markers EpCAM, CTCs expressing low or no markers may be missed, resulting in false negative results; in the process of separating CTCs by using physical properties (such as density gradient, microfluidic method), false positive results may be caused due to the similarity of CTCs to other cells in blood.

[0003] In addition, due to the extremely low content of CTCs in blood and the similar shape to normal cells, multiple protein targets need to be detected by immunofluorescence for further determination. However, the elution process of antibody complexes easily damages cells, affects cell activity and subsequent detection, and limits the reuse of valuable CTC samples. In addition, traditional immunofluorescence detection needs to use different species of antibodies or one-antibody coupled with fluorescence, which increases the complexity and cost of experiments, reduces the flexibility and repeatability of experiments. These shortcomings restrict the wide application and in-depth research of CTC detection technology.

[0004] The main problems to be solved in the current multi-round immunofluorescence detection are: 1. The use of hot elution or strong acid and alkali solution elution easily leads to cell or protein loss in multi-round staining; 2. The use of high-salt or formamide solution elution easily leads to high residual fluorescence signal; 3. Cell shape distortion and other problems. SUMMARY

[0005] The technical problem to be solved is to overcome the deficiencies of the prior art, improve the flexibility of the experiment, and realize the reuse of circulating tumor cell samples to obtain more detection information. In view of this, the application provides a circulating tumor cell immunofluorescence detection method based on heat-sensitive glue and application thereof.

[0006] Technical scheme: The circulating tumor cell immunofluorescence detection method based on heat-sensitive glue comprises the following steps: S1, based on the difference in glucose metabolism between circulating tumor cells and normal cells, a charge-modified nanomagnetic bead is designed to have selective affinity for circulating tumor cells, and then the specific binding of the nanomagnetic bead and the surface of the circulating tumor cells is used to achieve the capture and enrichment of the circulating tumor cells; wherein the charge-modified nanomagnetic bead is the material disclosed in Chinese Patent ZL202210784391.5 A polymer-modified magnetic nanomaterial, a preparation method thereof and applications.

[0007] S2, drop the cell suspension obtained in S1 to form a wet cell sheet, and let it stand at room temperature to dry, so that the cell layer is firmly attached to the adhesive glass slide; add fixing solution to the glass slide to fix the cell morphology; S3, the glass slide obtained in S2 is washed with PBS buffer, then soaked in sodium citrate antigen repair solution, and the antigen epitopes are exposed in a boiling water bath. S4, after cooling to room temperature, wash with PBS buffer, and block with blocking solution for 30-60 minutes; wherein the blocking solution is 5% BSA by mass fraction.

[0008] S5, add the primary antibody solution, incubate overnight at 4°C or incubate at 37°C for 2 hours; wherein the primary antibody solution is a dilution of anti-human pan-CK rabbit monoclonal antibody or a dilution of CK7 mouse antibody, and the dilution ratio is 1 / 500-1 / 1000. S6, wash the glass slide treated in S5 with PBST buffer, add the fluorescent secondary antibody solution, and incubate at room temperature for 1 hour in the dark; wherein the secondary antibody solution is goat anti-mouse IgG conjugated with Alexa Fluor® 488 fluorescent dye, and the dilution ratio of the 488 anti-mouse secondary antibody is 1 / 500-1 / 1000.

[0009] S7, wash the glass slide treated in S6 with PBST buffer, add the anti-fluorescence quenching mounting medium containing DAPI, and observe and take pictures under a fluorescence microscope; S8, wash the glass slide treated in S7 with PBST buffer, dry it, cover the cell layer surface with heat-sensitive glue, press it gently to make it fit tightly, and maintain it at room temperature for >10 minutes. S9, immerse the glass slide treated in S8 in 90°C sodium citrate buffer for 2 minutes, then remove the heat-sensitive glue after it loses its stickiness, and wash it with PBS for 3 times. S10, repeat steps S4-S7, and use the remaining types of primary antibodies for the next round of fluorescent staining according to actual needs.

[0010] Preferably, in S3, the antigen epitopes masked by the fixing solution are recovered by high-pressure pot treatment for 2 minutes or atmospheric boiling water bath treatment for 10 minutes, to provide binding sites for antibodies and improve the signal-to-noise ratio by 3-5 times.

[0011] Preferably, in S9, after the thermal adhesive loses its adhesiveness and is peeled off, the glass slide is treated with 0.5% H2O2 at room temperature for 10 minutes to oxidize residual fluorescent groups, improve the removal rate of residual fluorescence, and reduce background fluorescence noise.

[0012] Preferably, in S1, a microfluidic chip method is used to replace the magnetic bead method for enriching circulating tumor cells, in order to accommodate large-sized circulating tumor cells.

[0013] Preferably, in S1, density gradient centrifugation is used instead of magnetic beads to enrich circulating tumor cells, thereby reducing the enrichment cost of circulating tumor cells.

[0014] Preferably, in S9, an enzymatic cleavage-oxidation combination method is used to replace the thermosensitive gel removal method to remove the antibody complex. Specifically, the enzymatic cleavage-oxidation combination method involves cleaving the antibody with pepsin, neutralizing it, and then bleaching the residual fluorescence with H2O2.

[0015] Preferably, in S9, a reversible binding method of the primary antibody is used to replace the heat-sensitive gel removal method to remove the antibody complex. Specifically, the primary antibody is pre-coupled with desulfurized biotin, and after staining, it is competitively eluted with free biotin. This does not damage the antibody structure, and the cells and proteins do not lose their peeling force, resulting in a higher retention rate.

[0016] The above-described thermosensitive gel-based immunofluorescence detection method for circulating tumor cells is applied to the reuse of circulating tumor cell samples.

[0017] The application of any of the above-described thermosensitive gel-based immunofluorescence detection methods for circulating tumor cells in multi-round immunofluorescence detection of circulating tumor cells.

[0018] The principle of the method described in this invention lies in the fact that thermal adhesive, as a special medium, has temperature-dependent viscosity properties. At lower temperatures, the thermal adhesive exhibits high viscosity, which can effectively remove antibody complexes without damaging CTC cells fixed on the slide; as the temperature increases, the viscosity of the thermal adhesive decreases significantly, thereby allowing for easy removal of excess thermal adhesive without affecting the cell state; thus enabling the sample to be reused and obtaining more protein detection information.

[0019] In traditional immunofluorescence assays, the elution of antibody complexes often damages cells, affecting the accuracy of the results and the reusability of the cells. The temperature-responsive properties of thermosensitive gels allow for antibody complex elution under gentle conditions, avoiding cell damage caused by chemical reagents or harsh physical manipulations in traditional methods. Traditional immunofluorescence assays typically require the use of antibodies from different species, which increases experimental complexity and cost, and limits flexibility and reproducibility. This invention, through a multi-round immunofluorescence assay using a thermosensitive gel, allows the use of antibodies from the same species. This innovation leverages the temperature-responsive properties of thermosensitive gels to remove antibody complexes at different temperatures, thus avoiding cross-reactions between antibodies from different species. By using antibodies from the same species, this invention simplifies the experimental procedure, reduces costs, and improves flexibility and reproducibility, making the experiment more convenient and efficient.

[0020] Beneficial effects: (1) The thermosensitive adhesive used in this invention denatures at a specific temperature, significantly reducing the interfacial adhesion and allowing the antibody-fluorescent complex to be peeled off. However, the electrostatic effect of the cell slide can still maintain cell retention, with a cell retention rate of >90%. After peeling, the residual fluorophores are oxidized with a low concentration of H2O2, and the signal drops to the background after the double bonds break. The entire process is free of strong acids and bases and high salts, causing less damage to cells and proteins, and is simple to operate. (2) The method enables the reuse of circulating tumor cell samples, allowing for multiple rounds of detection to obtain more detection information. (3) The method does not restrict the species of antibodies, improving experimental flexibility and reducing costs. Attached Figure Description

[0021] Figure 1 This is a graph showing the results of the first round of immunofluorescence detection in Example 1; Figure 2 This is a diagram showing the results of Example 1 after the removal of the heat-sensitive adhesive and oxidation with hydrogen peroxide. Figure 3 This is a graph showing the results of the second round of immunofluorescence detection in the example. Detailed Implementation

[0023] Example 1: Using HT29 cells and HeLa cells as examples An immunofluorescence detection method for circulating tumor cells based on thermosensitive gel, the method comprising the following steps: S1. Based on the difference in glucose metabolism between circulating tumor cells and normal cells, charge-modified magnetic nanobeads are designed to give them selective affinity for circulating tumor cells. Then, the specific binding of the magnetic nanobeads to the surface of circulating tumor cells is used to capture and enrich them. Among them, the charge-modified magnetic nanobeads are the materials disclosed in Chinese Patent ZL202210784391.5, "A Polymer-Modified Magnetic Nanomaterial, Its Preparation Method and Application".

[0024] S2. Take the cell suspension obtained in S1 and drop it onto a wet cell sheet. Let it air dry at room temperature to allow the cell layer to adhere firmly to the adhesive slide. Add fixative to the slide to fix the cell morphology. S3. Wash the glass slide obtained in S2 with PBS buffer, then soak it in sodium citrate antigen retrieval solution and expose the antigen epitopes in a boiling water bath. S4. After cooling to room temperature, wash with PBS buffer and block with blocking solution for 30-60 minutes; the blocking solution is 5% BSA by mass.

[0025] S5. Add the primary antibody solution and incubate overnight at 4°C or for 2 hours at 37°C. The primary antibody solution is a dilution of the anti-human pan-CK rabbit monoclonal antibody, with a dilution ratio of 1 / 500-1 / 1000. S6. Wash the slides treated in S5 with PBST buffer, add fluorescent secondary antibody solution, and incubate at room temperature in the dark for 1 hour. The secondary antibody solution is goat anti-mouse IgG conjugated with Alexa Fluor® 488 fluorescent dye, and the dilution ratio of 488 anti-mouse secondary antibody is 1 / 500-1 / 1000.

[0026] S7. Wash the glass slides treated in S6 with PBST buffer, add anti-fluorescence quenching mounting medium containing DAPI to mount the slides, and observe and photograph them under a fluorescence microscope. S8. Wash the glass slides treated in S7 with PBST buffer, air dry, cover the cell layer surface with thermal adhesive, press gently to make it adhere tightly, and maintain at room temperature for >10 min. S9. Place the glass slide treated in S8 into a sodium citrate buffer solution at 90°C for 2 minutes. After the heat-sensitive adhesive loses its stickiness, peel it off and wash it 3 times with PBS. S10. Repeat steps S4-S7. If necessary, use the primary antibody diluted with CK7 mouse antibody for the second round of fluorescent staining. The secondary antibody is the same as in the first round.

[0027] In addition, in S3, a normal pressure boiling water bath was used for 10 minutes to restore the antigen epitopes that were blocked by the fixative cross-linking, providing binding sites for the antibody and improving the signal-to-noise ratio by 3-5 times. In S9, after the thermosensitive adhesive lost its stickiness and was peeled off, the slide was treated with 0.5% H2O2 at room temperature for 10 minutes to oxidize residual fluorescent groups, improve the clearance rate of residual fluorescence, and reduce background fluorescence noise.

[0028] This embodiment uses a slide prepared from a mixture of HT29 cells and HeLa cells. HT29 cells (relatively small) are panCK-positive and CK7-negative; HeLa cells (relatively large) are panCK-negative and CK7-positive. During the first round of immunofluorescence detection of panCK, HT29 cells emitted green fluorescence (…). Figure 1 After treatment with heat-sensitive adhesive and hydrogen peroxide, most of the green fluorescence disappeared. Figure 2During the second round of immunofluorescence detection of CK7, HeLa cells emitted green fluorescence ( Figure 3 There was almost no interference between the two rounds of immunofluorescence assays.

Claims

1. A heat-sensitive glue-based immunofluorescence detection method for circulating tumor cells, characterized in that, The method comprises the following steps: S1, based on the difference in glucose metabolism between circulating tumor cells and normal cells, design charge-modified nanomagnetic beads, so that they have selective affinity for circulating tumor cells, then use the specific binding of nanomagnetic beads and the surface of circulating tumor cells to achieve their capture and enrichment; S2, the cell suspension obtained in S1 is spun to form a wet cell sheet, and the cell layer is firmly attached to the adhesive glass slide after air drying at room temperature; add fixing solution to the glass slide to fix the cell morphology; S3, the glass slide obtained in S2 is washed with PBS buffer, then soaked in sodium citrate antigen repair solution, and the antigen epitopes are exposed in a boiling water bath; S4, after cooling to room temperature, wash with PBS buffer, and block with blocking solution for 30-60 min; S5, add the first antibody solution, incubate overnight at 4°C or incubate at 37°C for 2 hours; S6, wash the glass slide treated in S5 with PBST buffer, add the fluorescent second antibody solution, and incubate at room temperature in the dark for 1 hour; S7, wash the glass slide treated in S6 with PBST buffer, add the anti-fluorescence quenching mounting agent containing DAPI, and mount under a fluorescence microscope for observation and photography; S8, wash the glass slide treated in S7 with PBST buffer, air dry, cover the cell layer surface with heat-sensitive glue, and press gently to make it adhere tightly, and maintain at room temperature for >10 min; S9, immerse the glass slide treated in S7 in 90°C sodium citrate buffer for 2 min, then remove the heat-sensitive glue after it loses adhesion, and wash with PBS for 3 times; S10, repeat steps S4-S7, and use the remaining types of first antibodies for the next round of fluorescent staining according to actual needs.

2. The heat-sensitive glue-based immunofluorescence detection method for circulating tumor cells according to claim 1, wherein, In S3, use a pressure cooker for 2 min or a normal pressure boiling water bath for 10 min to restore the antigen epitopes masked by the fixing solution, provide binding sites for antibodies, and improve the signal-to-noise ratio by 3-5 times.

3. The heat-sensitive glue-based immunofluorescence detection method for circulating tumor cells according to claim 1, wherein, In S9, after the heat-sensitive glue loses adhesion and is removed, treat the glass slide with 0.5% H2O2 at room temperature for 10 min to oxidize the residual fluorescent groups, improve the removal rate of residual fluorescence, and reduce background fluorescence noise.

4. The heat-sensitive glue-based immunofluorescence detection method for circulating tumor cells according to claim 1, wherein, In S1, replace the magnetic bead method with a microfluidic chip method to enrich circulating tumor cells, which is suitable for large-sized circulating tumor cells.

5. The heat-sensitive glue-based immunofluorescence detection method for circulating tumor cells according to claim 1, wherein, In S1, replace the magnetic bead method with a density gradient centrifugation method to reduce the enrichment cost of circulating tumor cells.

6. The heat-sensitive glue-based immunofluorescence detection method for circulating tumor cells according to claim 1, wherein, In S9, replace the heat-sensitive glue removal method with an enzyme cutting and oxidation combination method to remove the antibody complex, wherein the enzyme cutting and oxidation combination method specifically comprises: cutting the antibody with pepsin, neutralizing, and then bleaching the residual fluorescence with H2O2.

7. The heat-sensitive glue-based immunofluorescence detection method for circulating tumor cells according to claim 1, wherein, In S9, replace the heat-sensitive glue removal method with a first antibody reversible binding method to remove the antibody complex, specifically: pre-couple desulfurized biotin to the first antibody, and then use free biotin to compete and elute after staining, without damaging the antibody structure, and the cells and proteins are not peeled off, which has a higher retention rate.

8. Application of the circulating tumor cell immunofluorescence detection method based on heat-sensitive glue according to any one of claims 1-7 in the repeated use of circulating tumor cell samples.

9. Use of the thermal glue based immunofluorescence assay for circulating tumor cells according to any one of claims 1 to 7 for multiple rounds of immunofluorescence assay for circulating tumor cells.

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