Method for detecting positive circulating tumor cells of intestinal cancer HER2

Abstract

The invention provides a method for detecting intestinal cancer HER2 positive circulating tumor cells, and relates to the technical field of medical detection, the sensitivity of the method to intestinal cancer patient detection reaches 100%, which means that the circulating tumor cells in the intestinal cancer patient can be extremely accurately detected, a reliable basis is provided for early diagnosis and timely treatment, and the method is suitable for clinical application. According to the method, the survival rate and life quality of patients can be improved, the polyp sample detection specificity is as high as 100%, intestinal cancer patients and polyp control samples can be accurately distinguished, misdiagnosis and misjudgment are effectively avoided, a highly accurate result is provided for clinical diagnosis, the method is highly consistent with a clinical diagnosis result, and the method is suitable for clinical diagnosis. And the reliability and the practicability are further verified. The method has a good effect in the aspect of auxiliary diagnosis of intestinal cancer patients, important diagnosis information can be provided for medical workers, formulation of treatment schemes can be guided, the detection method is clear and definite in operation steps and easy to implement and popularize, and repeatability and stability of the method are ensured.

Description

Technical Field

[0001] The present invention belongs to the field of medical detection technology, and more specifically, particularly relates to a method for detecting HER2-positive circulating tumor cells in colorectal cancer. Background Art

[0002] Intestinal cancer is a malignant tumor originating from the epithelial tissue of the large intestine, including colon cancer and rectal cancer, also known as colorectal cancer, which is one of the most common malignant tumors. Colorectal cancer (CRC) is a common cancer that occurs in the gastrointestinal tract, and the causes of the disease are mostly related to bad living habits and aging. Certain familial hereditary diseases such as colorectal polyposis syndrome and hereditary non-polyposis colorectal cancer may cause colorectal cancer, but these hereditary diseases account for less than 5% of all colorectal cancer cases. Studies have found that with the development of intestinal cancer, symptoms such as blood in the stool, changes in bowel habits, abdominal masses, persistent pain in the pelvis or lower abdomen, and weight loss will appear. According to statistics, the incidence of colorectal cancer ranks third among malignant tumors in the world, and the mortality rate ranks second in developed countries and fifth in my country. With the changes in people's lifestyles and dietary structures, its incidence and mortality rates have a tendency to increase year by year. It usually takes about 10 years for colorectal adenomas to become cancerous, so patients also have enough time to block their cancerous process at an early stage through screening and subsequent adenoma removal surgery. In addition to surgery, chemotherapy is also required for the treatment of advanced colorectal cancer. Commonly used chemotherapy drugs include oxaliplatin and fluorouracil, and the cost of treatment is high. Therefore, early screening and diagnosis of colorectal cancer and detection of cancer can greatly reduce the cost of colorectal cancer treatment and reduce the burden on patients' families.

[0003] Circulating tumor cells (CTC) are tumor cells that are released from solid tumor lesions and enter the peripheral blood circulation. They are the key factors leading to tumor metastasis. Most CTCs undergo apoptosis or are phagocytosed after entering the peripheral blood, and a few can escape and anchor to develop into metastatic lesions, increasing the risk of death in patients with malignant tumors. CTC detection monitors the trend of its type and quantity changes through capture detection, so as to monitor tumor dynamics in real time, evaluate the treatment effect, and achieve real-time individual treatment. At present, the detection of CTC mainly relies on the expression of various markers on the surface of tumor cells (such as epithelial cell marker EpCAM), and the corresponding antibodies or peptides coupled to solid phase carriers (such as chips, magnetic beads, etc.) are used to directly capture circulating tumor cells, and specific target proteins are used to identify CTCs. Human epidermal growth factor receptor 2 (HER2, also known as ERBB2) is a member of the receptor tyrosine kinase EGFR family, and its carcinogenic potential has been confirmed in a variety of human malignancies, such as breast cancer, intestinal cancer, gastric cancer, urothelial carcinoma, etc. The main mechanism of HER2 oncogenic activation is that HER2 gene amplification leads to overexpression of HER2 protein on the cell membrane. This abnormal expression leads to cascade activation of downstream signaling pathways, thereby promoting uncontrolled tumor cell proliferation. For this reason, targeted drugs targeting HER2, such as Trastuzumab, have achieved great results in clinical cancer treatment.

[0004] At present, the most widely used methods for colorectal cancer detection in clinical practice are fecal occult blood test and colonoscopy. Among them, although the fecal occult blood test is fast, simple, painless and non-invasive, its sensitivity and specificity are relatively low, with a sensitivity of less than 30%, which cannot meet the early diagnosis of CRC. Colonoscopy is the gold standard for diagnosing CRC, but because its operation is damaging, patients need to endure pain, and it is easy to cause intestinal bleeding, perforation and infection. These factors limit its application to a certain extent. Therefore, there is an urgent need for a detection method with high sensitivity and specificity and low damage to perform early diagnosis on colorectal cancer patients in clinical practice. To this end, the present invention combines the characteristics of colorectal cancer epithelial tumors to develop a method for detecting circulating tumor cells that are HER2-positive for colorectal cancer, which can not only perform early screening and auxiliary diagnosis of colorectal cancer, but also provide medication guidance to patients and provide treatment plans. Summary of the invention

[0005] In order to solve the above technical problems, the present invention provides a method for detecting HER2-positive circulating tumor cells in colorectal cancer to solve the above problems.

[0006] A method for detecting HER2-positive circulating tumor cells in colorectal cancer, comprising the following steps:

[0007] S1: capture chip preparation;

[0008] S2: Clinical sample processing;

[0009] S3: Reagent preparation;

[0010] S4: sample testing and interpretation;

[0011] S5: Statistics of sample test results.

[0012] Preferably, the preparation of the S1 capture chip comprises the following steps:

[0013] S11: Select a stainless steel chip with a pore size of 18×18 μm and a diameter of 8.8 μm;

[0014] S12: Gold plating of stainless steel chips;

[0015] S13: After the gold plating is completed, the chip is rinsed with ethanol for 2 minutes, and then transferred to a clean glass beaker containing a mixed solution of water, 25% ammonia water, and 30% hydrogen peroxide (the cleaning solution is prepared and used immediately) in a volume ratio of 5:1:1. After the beaker is sealed, it is placed in an oil bath pot and heated at 75°C for 5 minutes. After the cleaning is completed, the chip is rinsed with deionized water, ethanol, and ethanol in turn, and then the chip is taken out and blown dry with high-purity nitrogen;

[0016] S14: Take 3 mL of self-synthesized thiolated sodium hyaluronate and add it to a 5 mL EP tube. Then add 15-18 washed gold-plated chips to the EP tube and incubate on an oscillator at 200 rpm / min for 2 hours;

[0017] S15: After the incubation, the thiolated sodium hyaluronate was aspirated, and then 3 mL of PBS solution was added and mixed, and the chips were placed on an oscillator at 200 rpm / min for 3 minutes, and the washing was repeated twice. After the washing was completed, the chips were taken out one by one using tweezers and placed on the surface of lens cleaning paper to dry naturally, and then the dried chips were placed in a 24-well plate for use;

[0018] S16: Weigh 0.609 mg of EDC (need to be taken out from -20°C and equilibrated to room temperature 30 min in advance before use) and dissolve it in 35 μL of MES buffer (0.05 M, pH=6). Weigh 0.348 mg of Sulfo-NHS and dissolve it in 20 μL MES solution. Dissolve them separately and then mix them to obtain 55 μL of solution. Use a pipette to transfer 55 μL of activator to the chip surface in the well plate and incubate at room temperature in the dark for 30 min.

[0019] S17: After activation, take 1 mL of PBS solution in the well plate to wash the activated chip, shake the well plate back and forth 3-4 times, discard the PBS, and repeat the washing twice. After washing, use tweezers to take out the chips one by one and place them on the surface of lens cleaning paper to dry naturally. Then, place the dried chips in a new 24-well plate for use;

[0020] S18: Add 53.5 μL of antibody incubation solution (a total of 1.75 μg EpCAM antibody) to each well plate where the chip is located, incubate at 37°C for 2 h, and after the incubation is completed, take 1 mL of PBS solution to the well plate to wash the chip, shake the well plate back and forth 3-4 times, and discard the PBS;

[0021] S19: Add 500 μL of 1% BSA to the well plate containing the chip and block at room temperature for 1 h. After blocking, take 1 mL of PBS solution to the well plate to wash the chip, shake the well plate back and forth 3-4 times, and discard the PBS;

[0022] S110: Assemble the chip and add 300 μL of freeze-drying protective agent to the sample wells and pre-cool at -80°C for 1-2 hours. Then, place the assembled chip in a freeze dryer for freeze drying. After freeze drying, store the chip at 4°C for future use.

[0023] Preferably, S2 clinical sample processing comprises the following steps:

[0024] S21: Select colorectal cancer patient / polyp control samples, take 4 mL of PBS in a 15 mL centrifuge tube, add 4 mL of whole blood to dilute the blood 1:1, and mix after dilution;

[0025] S22: Take 4 mL of sample density separation solution and place it in a 15 mL centrifuge tube. Slowly add 8 mL of diluted blood to the centrifuge tube, and the total volume after addition is 12 mL.

[0026] S23: centrifuging the centrifuge tube in step 2) for 20 min at a centrifugal speed of 2500 rpm / min;

[0027] S24: Take out the centrifuge tube, use a pipette to discard the plasma layer (the top layer), draw the PBMC layer (the buffy coat layer) into a 15 mL centrifuge tube (about 2 mL), and add 10 mL PBS to wash the PBMC thoroughly;

[0028] S25: After washing, centrifuge at 1500 rpm / min for 5 min;

[0029] S26: Take out the centrifuge tube and discard the supernatant;

[0030] S27: Add 5 mL of PBS to wash PBMCs for the second time;

[0031] S28: After washing, centrifuge at 1500 rpm / min for 5 min;

[0032] S29: Take out the centrifuge tube, discard the supernatant, add 300 μL PBS to resuspend the precipitate, mix thoroughly and set aside.

[0033] Preferably, in S3, the prepared reagents include a fixative, a membrane permeabilizing agent, a blocking solution, a staining solution, and a cleaning solution. The fixative is 250 mL of 4% paraformaldehyde, and the membrane permeabilizing agent is 250 mL of 0.1% Triton. TM X-100, blocking solution is 250mL (1.5mg / mL glycine + 50mg / mL BSA + 3% goat serum + 2% Tween20 dissolved in PBS), staining solution is 250mL (1μg Alexa Fluor 647 fluorescent Anti-ErbB2 / HER2 antibody + 1μg Alexa 488 fluorescent Anti-panCytokeratin antibody + 5μg PE anti-human CD45 antibody dissolved in PBS), and the washing solution was PBS buffer.

[0034] Preferably, S4 comprises the following steps:

[0035] S41: Take out the staining reagent and the prepared chip;

[0036] S42: Use a pipette to place all 300 μL of sample into the chip loading wells;

[0037] S43: Sample cell capture and staining using an automated sampler;

[0038] S44: After the instrument runs, the reagent strip is taken out for film reading, that is, the green light channel (AF488) is first used to determine the green bright spot, and then the green light channel (AF488), blue light channel (DAPI), orange light channel (PE), and red light channel (AF647) are used to take pictures in sequence;

[0039] S45: After the interpretation is completed, data statistics are performed, among which DAPI+, PanCK+, CD45-, and HER2+ are interpreted as HER2-positive circulating tumor cells.

[0040] Compared with the prior art, the present invention has the following beneficial effects:

[0041] 1. The sensitivity of this method in detecting colorectal cancer patients reaches 100%, which means that it can detect circulating tumor cells in colorectal cancer patients extremely accurately, providing a reliable basis for early diagnosis and timely treatment, and helping to improve patients' survival rate and quality of life.

[0042] 2. The specificity of polyp sample detection is as high as 100%, which can accurately distinguish colorectal cancer patients and polyp control samples, effectively avoid misdiagnosis and misjudgment, and provide highly accurate results for clinical diagnosis.

[0043] 3. This method is highly consistent with clinical diagnosis results, which further verifies its reliability and practicality. It has a good effect in auxiliary diagnosis of colorectal cancer patients, can provide important diagnostic information for medical workers, and guide the formulation of treatment plans.

[0044] 4. The detection method has clear operation steps and is easy to implement and promote. The repeatability and stability of the method are ensured through detailed capture chip preparation, sample processing, reagent preparation, and detection interpretation process. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 It is a result schematic diagram of sample detection result statistics in the present invention;

[0046] Figure 2 These are some representative pictures of positive and negative in the present invention. DETAILED DESCRIPTION

[0047] The following embodiments of the present invention are described in further detail in conjunction with the accompanying drawings and examples. The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention.

[0048] See also Figure 1-Figure 2 The present invention provides a method for detecting HER2-positive circulating tumor cells in colorectal cancer, comprising the following steps:

[0049] S1: Capture chip preparation: A chip specifically designed for capturing circulating tumor cells was constructed, and its specific structure and material selection facilitated efficient binding and capture of target cells;

[0050] S2: Clinical sample processing: By diluting and centrifuging clinical samples, the samples are initially separated and purified, so that circulating tumor cells can be better detected in the subsequent tests.

[0051] S3: Reagent preparation: ensure that appropriate fixative, membrane permeabilizing agent, blocking solution, staining solution, cleaning solution and other reagents are available in the subsequent detection process, providing a material basis for the processing and labeling of sample cells;

[0052] S4: Sample detection and interpretation: Use specific instruments and methods to capture and stain sample cells, and then use multi-channel photography and interpretation to accurately identify and characterize HER2-positive circulating tumor cells, thereby achieving detection and property determination of target cells;

[0053] S5: Statistics of sample test results; organize and compile statistics on the test data to facilitate analysis and evaluation of the test results and provide data support for subsequent clinical diagnosis and research.

[0054] S1 capture chip preparation includes the following steps:

[0055] S11: Selecting a stainless steel chip with a pore size of 18×18 μm and a diameter of 8.8 μm can effectively screen out target cells while reducing the capture of other non-target cells or impurities, improving the specificity and accuracy of capture, and can better adapt to the size and morphological characteristics of circulating tumor cells, thereby improving capture efficiency;

[0056] S12: Gold plating is performed on stainless steel chips. Gold plating can improve the conductivity of the chip, which is very important for some detection methods based on electrical principles or when used in conjunction with other electronic devices. It helps to transmit signals and improve the accuracy of detection. Gold has good biocompatibility and can reduce adverse effects on biological samples such as cells and reduce interference such as nonspecific adsorption, which is more conducive to the capture and detection of circulating tumor cells. Gold is relatively stable and not easily oxidized or corroded, which can keep the chip stable in performance during long-term use and extend its service life. It can optimize the physical and chemical properties of the chip surface, making it more suitable for interaction with subsequent reagents or cells, thereby improving capture efficiency and detection effect.

[0057] S13: After the gold plating is completed, the chip is rinsed with ethanol for 2 minutes, and then transferred to a clean glass beaker containing a mixed solution of water, 25% ammonia water, and 30% hydrogen peroxide (the cleaning solution is prepared and used immediately) in a volume ratio of 5:1:1. After the beaker is sealed, it is placed in an oil bath and heated at 75°C for 5 minutes. After the cleaning is completed, the chip is rinsed with deionized water, ethanol, and ethanol in turn. The chip is then taken out and blown dry with high-purity nitrogen. The use of ethanol for soaking can preliminarily remove impurities and contaminants that may remain during the gold plating process. The mixed solution of water, ammonia water and hydrogen peroxide can have a strong cleaning effect and remove the surface of the chip. The possible stubborn attachments or contaminants can further improve the cleanliness of the chip. The freshly prepared cleaning solution can ensure the effectiveness and activity of the cleaning solution. The timed heating at 75°C can enhance the cleaning effect and promote the reaction. The subsequent soaking and drying with high-purity nitrogen can further remove the possible residual cleaning solution to ensure the cleanliness of the chip. The high-purity nitrogen drying can avoid the introduction of new impurities and quickly dry the chip so that it can be put into subsequent use as soon as possible. The purity, surface quality and performance of the capture chip can be guaranteed to the greatest extent, thus laying a good foundation for the subsequent accurate and reliable detection of circulating tumor cells.

[0058] S14: Take 3mL of self-synthesized sodium thiolated hyaluronate and add it to a 5mL EP tube. Then add 15-18 cleaned gold-plated chips to the EP tube and incubate on an oscillator at 200rpm / min for 2h. By incubating sodium thiolated hyaluronate with the cleaned gold-plated chips, sodium thiolated hyaluronate can interact specifically with the chip surface, which helps to improve the binding affinity of the chip to certain components, thereby better capturing target cells. This incubation process may allow the chip surface to acquire specific chemical properties or functional groups, which helps to achieve more specific detection of circulating tumor cells. Oscillating on the oscillator at a certain speed can promote the uniform distribution of sodium thiolated hyaluronate on the chip surface, ensuring the performance consistency of the entire chip surface. The 2-hour incubation time allows the reaction to proceed fully to achieve the ideal modification effect;

[0059] S15: After the incubation is completed, the thiolated sodium hyaluronate is aspirated, and then 3 mL of PBS solution is added to mix, and the chip is placed on an oscillator at 200 rpm / min for 3 minutes, and the washing is repeated twice; after the washing is completed, the chips are taken out one by one with tweezers and placed on the surface of lens paper to dry naturally, and then the dried chips are placed in a 24-well plate for use. The thiolated sodium hyaluronate can remove unbound or excess substances and reduce interference. Adding PBS solution to wash and repeating the operation can further remove possible residual impurities and unreacted substances to ensure the cleanliness of the chip surface. Oscillating and washing at a certain speed can improve the cleaning efficiency and effect. Taking out the chip and drying it naturally can avoid the adverse effects of residual moisture on subsequent use, and also avoid damage that may be caused by other improper drying methods. The dried chip is placed in a 24-well plate for use, which is convenient for accurate and orderly use in subsequent operations, and is also conducive to maintaining the stability and operability of the chip, and preparing for subsequent sample testing and other steps;

[0060] S16: Weigh 0.609 mg of EDC (need to be taken out from -20°C and equilibrated to room temperature 30 min in advance before use) and dissolve it in 35 μL of MES buffer (0.05 M, pH=6). Weigh 0.348 mg of Sulfo-NHS and dissolve it in 20 μL MES solution. Dissolve them separately and then mix them to obtain 55 μL of solution. Use a pipette to transfer 55 μL of activator to the chip surface in the well plate and incubate at room temperature in the dark for 30 min.

[0061] S17: After activation, take 1 mL of PBS solution in the well plate to wash the activated chip, shake the well plate back and forth 3-4 times, discard the PBS, and repeat the washing twice. After washing, use tweezers to take out the chips one by one and place them on the surface of lens cleaning paper to dry naturally. Then, place the dried chips in a new 24-well plate for use;

[0062] S18: Add 53.5 μL of antibody incubation solution (a total of 1.75 μg EpCAM antibody) to each well plate where the chip is located, incubate at 37°C for 2 h, and after the incubation is completed, take 1 mL of PBS solution to the well plate to wash the chip, shake the well plate back and forth 3-4 times, and discard the PBS;

[0063] S19: Add 500 μL of 1% BSA to the well plate containing the chip and block at room temperature for 1 h. After blocking, take 1 mL of PBS solution to the well plate to wash the chip, shake the well plate back and forth 3-4 times, and discard the PBS;

[0064] S110: Assemble the chip and add 300 μL of freeze-drying protective agent to the sample wells and pre-cool at -80°C for 1-2 hours. Then, place the assembled chip in a freeze dryer for freeze drying. After freeze drying, store the chip at 4°C for future use.

[0065] S2 clinical sample processing includes the following steps:

[0066] S21: Select colorectal cancer patient / polyp control samples, take 4 mL of PBS in a 15 mL centrifuge tube, add 4 mL of whole blood to dilute the blood 1:1, and mix after dilution;

[0067] S22: Take 4 mL of sample density separation solution and place it in a 15 mL centrifuge tube. Slowly add 8 mL of diluted blood to the centrifuge tube, and the total volume after addition is 12 mL.

[0068] S23: centrifuging the centrifuge tube in step 2) for 20 min at a centrifugal speed of 2500 rpm / min;

[0069] S24: Take out the centrifuge tube, use a pipette to discard the plasma layer (the top layer), draw the PBMC layer (the buffy coat layer) into a 15 mL centrifuge tube (about 2 mL), and add 10 mL PBS to wash the PBMC thoroughly;

[0070] S25: After washing, centrifuge at 1500 rpm / min for 5 min;

[0071] S26: Take out the centrifuge tube and discard the supernatant;

[0072] S27: Add 5 mL of PBS to wash PBMCs for the second time;

[0073] S28: After washing, centrifuge at 1500 rpm / min for 5 min;

[0074] S29: Take out the centrifuge tube, discard the supernatant, add 300 μL PBS to resuspend the precipitate, mix thoroughly and set aside.

[0075] S4 includes the following steps:

[0076] S41: Take out the staining reagent and the prepared chip;

[0077] S42: Use a pipette to place all 300 μL of sample into the chip loading wells;

[0078] S43: Sample cell capture and staining using an automated sampler;

[0079] S44: After the instrument runs, the reagent strip is taken out for film reading, that is, the green light channel (AF488) is first used to determine the green bright spot, and then the green light channel (AF488), blue light channel (DAPI), orange light channel (PE), and red light channel (AF647) are used to take pictures in sequence;

[0080] S45: After the interpretation is completed, data statistics are performed, among which DAPI+, PanCK+, CD45-, and HER2+ are interpreted as HER2-positive circulating tumor cells.

[0081] In S3, the prepared reagents include fixative, membrane permeabilization agent, blocking solution, staining solution, and cleaning solution. The fixative is 250mL 4% paraformaldehyde, and the membrane permeabilization agent is 250mL 0.1% Triton TM X-100, blocking solution is 250mL (1.5mg / mL glycine + 50mg / mL BSA + 3% goat serum + 2% Tween20 dissolved in PBS), staining solution is 250mL (1μg Alexa Fluor 647 fluorescent Anti-ErbB2 / HER2 antibody + 1μg Alexa 488 fluorescent Anti-panCytokeratin antibody + 5μg PE anti-human CD45 antibody dissolved in PBS), the washing solution was PBS buffer;

[0082] Fixative (4% paraformaldehyde) is used to fix cells to maintain their original morphology and structure and prevent them from deforming or disintegrating during subsequent treatment. TM X-100) helps to destroy the integrity of the cell membrane, allowing antibodies and other reagents to enter the cell for staining and other operations. The blocking solution (1.5 mg / mL glycine + 50 mg / mL BSA + 3% goat serum + 2% Tween20 dissolved in PBS) is mainly used to block nonspecific binding sites, reduce background staining, and improve the specificity of detection. In the staining solution, Alexa 647 fluorescent Anti-ErbB2 / HER2 antibody specifically recognizes and binds to HER2 protein and is used to detect HER2 positive cells. 488 fluorescent Anti-panCytokeratin antibody labels cytokeratin and can be used to identify cells of epithelial origin and assist in determining circulating tumor cells. PE anti-human CD45 antibody binds to the leukocyte surface marker CD45 and is used to distinguish circulating tumor cells from leukocytes. The cleaning solution (PBS buffer) is used to wash cells between each step and remove unbound reagents, etc., to keep the detection environment clean and reduce interference.

[0083] See the statistical graph of sample test results in S5. Figure 1 , some representative pictures of positive and negative are shown in Figure 2 .

[0084] HRE2-positive circulating tumor cells were detected in 5 colorectal cancer patients and 5 polyp controls. According to the test results, the kit has a sensitivity of 100% for colorectal cancer patients and a specificity of 100% for polyp samples, which are consistent with the clinical diagnosis results and have a good effect in auxiliary diagnosis of colorectal cancer patients.

[0085] The embodiments of the present invention are given for the purpose of illustration and description, and are not intended to be exhaustive or to limit the invention to the disclosed forms. Many modifications and variations will be apparent to those of ordinary skill in the art. The embodiments are selected and described in order to better illustrate the principles and practical applications of the present invention and to enable those of ordinary skill in the art to understand the present invention and thereby design various embodiments with various modifications suitable for specific uses.

Claims

1. A method for detecting HER2-positive circulating tumor cells in colorectal cancer, characterized in that: The following steps are involved: S1: capture chip preparation; S2: Clinical sample processing; S3: Reagent preparation; S4: sample testing and interpretation; S5: Statistics of sample test results.

2. A method for detecting HER2-positive circulating tumor cells in colorectal cancer as claimed in claim 1, characterized in that: S1 capture chip preparation includes the following steps: S11: Select a stainless steel chip with a pore size of 18×18 μm and a diameter of 8.8 μm; S12: Gold plating of stainless steel chips; S13: After the gold plating is completed, the chip is rinsed with ethanol for 2 minutes, and then transferred to a clean glass beaker containing a mixed solution of water, 25% ammonia water, and 30% hydrogen peroxide (the cleaning solution is prepared and used immediately) in a volume ratio of 5:1:

1. After the beaker is sealed, it is placed in an oil bath pot and heated at 75°C for 5 minutes. After the cleaning is completed, the chip is rinsed with deionized water, ethanol, and ethanol in turn, and then the chip is taken out and blown dry with high-purity nitrogen; S14: Take 3 mL of self-synthesized thiolated sodium hyaluronate and add it to a 5 mL EP tube. Then add 15-18 washed gold-plated chips to the EP tube and incubate on an oscillator at 200 rpm / min for 2 hours; S15: After the incubation, the thiolated sodium hyaluronate was aspirated, and then 3 mL of PBS solution was added and mixed, and the chips were placed on an oscillator at 200 rpm / min for 3 minutes, and the washing was repeated twice. After the washing was completed, the chips were taken out one by one using tweezers and placed on the surface of lens cleaning paper to dry naturally, and then the dried chips were placed in a 24-well plate for use; S16: Weigh 0.609 mg of EDC (need to be taken out from -20°C and equilibrated to room temperature 30 min in advance before use) and dissolve it in 35 μL of MES buffer (0.05 M, pH=6). Weigh 0.348 mg of Sulfo-NHS and dissolve it in 20 μL MES solution. Dissolve them separately and then mix them to obtain 55 μL of solution. Use a pipette to transfer 55 μL of activator to the chip surface in the well plate and incubate at room temperature in the dark for 30 min. S17: After activation, take 1 mL of PBS solution in the well plate to wash the activated chip, shake the well plate back and forth 3-4 times, discard the PBS, and repeat the washing twice. After washing, use tweezers to take out the chips one by one and place them on the surface of lens cleaning paper to dry naturally. Then, place the dried chips in a new 24-well plate for use; S18: Add 53.5 μL of antibody incubation solution (a total of 1.75 μg EpCAM antibody) to each well plate where the chip is located, incubate at 37°C for 2 h, and after the incubation is completed, take 1 mL of PBS solution to the well plate to wash the chip, shake the well plate back and forth 3-4 times, and discard the PBS; S19: Add 500 μL of 1% BSA to the well plate containing the chip and block at room temperature for 1 h. After blocking, take 1 mL of PBS solution to the well plate to wash the chip, shake the well plate back and forth 3-4 times, and discard the PBS; S110: Assemble the chip and add 300 μL of freeze-drying protective agent to the sample wells and pre-cool at -80°C for 1-2 hours. Then, place the assembled chip in a freeze dryer for freeze drying. After freeze drying, store the chip at 4°C for future use.

3. A method for detecting HER2-positive circulating tumor cells in colorectal cancer as claimed in claim 1, characterized in that: S2 clinical sample processing includes the following steps: S21: Select colorectal cancer patient / polyp control samples, take 4 mL of PBS in a 15 mL centrifuge tube, add 4 mL of whole blood to dilute the blood 1:1, and mix after dilution; S22: Take 4 mL of sample density separation solution and place it in a 15 mL centrifuge tube. Slowly add 8 mL of diluted blood to the centrifuge tube, and the total volume after addition is 12 mL. S23: centrifuging the centrifuge tube in step 2) for 20 min at a centrifugal speed of 2500 rpm / min; S24: Take out the centrifuge tube, use a pipette to discard the plasma layer (the top layer), draw the PBMC layer (the buffy coat layer) into a 15 mL centrifuge tube (about 2 mL), and add 10 mL PBS to wash the PBMC thoroughly; S25: After washing, centrifuge at 1500 rpm / min for 5 min; S26: Take out the centrifuge tube and discard the supernatant; S27: Add 5 mL of PBS to wash PBMCs for the second time; S28: After washing, centrifuge at 1500 rpm / min for 5 min; S29: Take out the centrifuge tube, discard the supernatant, add 300 μL PBS to resuspend the precipitate, mix thoroughly and set aside.

4. A method for detecting HER2-positive circulating tumor cells in colorectal cancer as claimed in claim 1, characterized in that: In S3, the prepared reagents include fixative, membrane permeabilization agent, blocking solution, staining solution, and cleaning solution.

5. A method for detecting HER2-positive circulating tumor cells in colorectal cancer as claimed in claim 1, characterized in that: S4 includes the following steps: S41: Take out the staining reagent and the prepared chip; S42: Use a pipette to place all 300 μL of sample into the chip loading wells; S43: Sample cell capture and staining using an automated sampler; S44: After the instrument runs, the reagent strip is taken out for film reading, that is, the green light channel (AF488) is first used to determine the green bright spot, and then the green light channel (AF488), blue light channel (DAPI), orange light channel (PE), and red light channel (AF647) are used to take pictures in sequence; S45: After the interpretation is completed, data statistics are performed, among which DAPI+, PanCK+, CD45-, and HER2+ are interpreted as HER2-positive circulating tumor cells.

6. A method for detecting HER2-positive circulating tumor cells in colorectal cancer as claimed in claim 4, characterized in that: The fixative solution is 250 mL 4% paraformaldehyde.

7. A method for detecting HER2-positive circulating tumor cells in colorectal cancer as claimed in claim 4, characterized in that: The permeabilization agent is 250 mL 0.1% Triton TM X-100.

8. A method for detecting HER2-positive circulating tumor cells in colorectal cancer as claimed in claim 4, characterized in that: The blocking solution is 250 mL (1.5 mg / mL glycine + 50 mg / mL BSA + 3% goat serum + 2% Tween 20 dissolved in PBS).

9. A method for detecting HER2-positive circulating tumor cells in colorectal cancer as claimed in claim 4, characterized in that: The staining solution is 250mL (1μg Alexa 647 fluorescent Anti-ErbB2 / HER2 antibody + 1μg Alexa 488 fluorescent anti-panCytokeratin antibody + 5μg PE anti-human CD45 antibody dissolved in PBS).

10. A method for detecting HER2-positive circulating tumor cells in colorectal cancer as claimed in claim 4, characterized in that: The washing solution was PBS buffer.

Images