CTC cell detection method based on micro-fluidic chip

Abstract

The invention discloses a CTC cell detection method based on a micro-fluidic chip, and belongs to the technical field of cell treatment. In order to solve the problems of high false positive and false negative rates, time consumption and low efficiency caused by difficulty in accurately distinguishing CTC from other cell types, the invention can efficiently and specifically capture CTC cells, reduce the possibility of false positive and false negative and improve the detection accuracy by constructing a specific capture chamber and filling specific antibodies and immunomagnetic beads. The multi-channel design allows a plurality of samples to be processed at the same time, the detection efficiency is improved, the micro-fluidic chip technology integrates a plurality of steps on one platform, automation and standardization of operation are achieved, personal errors are reduced, the stability and repeatability of detection are improved, the uniformity and stability of the detection process are ensured, and the detection efficiency is improved. The fluorescence imaging technology clearly displays CTC and obtains key parameters such as quantity, form and fluorescence intensity of CTC, thereby providing powerful support for cancer diagnosis and research.

Description

Technical Field

[0001] The present invention relates to the technical field of cell processing, and in particular to a CTC cell detection method based on a microfluidic chip. Background Art

[0002] CTC refers to circulating tumor cells, which is a general term for various types of tumor cells present in the peripheral blood. They fall off from solid tumor lesions (primary lesions, metastatic lesions) spontaneously or during diagnostic and treatment operations. Most CTCs undergo apoptosis or are phagocytosed after entering the peripheral blood, while a few can escape and anchor to develop into metastatic lesions, increasing the risk of death in patients with malignant tumors.

[0003] CTC detection has a wide range of clinical applications, such as predicting the risk of cancer metastasis, liquid biopsy, predicting patients' response to treatment and prognosis, etc. By monitoring the trend of changes in the type and quantity of CTCs, the dynamics of the tumor can be reflected in real time and the treatment effect can be evaluated to achieve individual treatment.

[0004] However, there are still the following problems in actual operation:

[0005] Traditional methods often have difficulty accurately distinguishing CTCs from other cell types, resulting in high false positive and false negative rates. They are also often time-consuming and inefficient, and cannot meet the needs of rapid, high-throughput testing. For samples with a small number of CTCs, such as those from early-stage cancer patients, traditional methods often have difficulty effectively detecting them. Summary of the invention

[0006] The object of the present invention is to provide a CTC cell detection method based on a microfluidic chip to solve the problems raised in the above background technology.

[0007] To achieve the above object, the present invention provides the following technical solution: a CTC cell detection method based on a microfluidic chip, comprising the following steps:

[0008] Chip design and production, construction of a multi-layer microfluidic chip, construction of micrometer-scale channels for blood inflow and CTC cell capture on the microfluidic chip, construction of capture chambers on the microfluidic chip, each capture chamber is filled with specific antibodies and immunomagnetic beads for binding to and capturing CTC cells, and construction of inlets and outlets for blood inflow and outflow on the microfluidic chip;

[0009] CTC capture and enrichment: collect blood samples from patients, pre-treat the blood samples, remove red blood cell and white blood cell impurities from the blood samples, load the blood samples into the microfluidic chip, control the flow rate and pressure to make the blood flow in the chip, and the blood sample to be tested enters the chip through the injection port. The pre-treated blood sample enters the capture area and binds to the specific antibodies and ligands in the capture area to specifically capture CTCs. CTCs in multiple samples are captured simultaneously through multiple channels, and the captured CTCs enter the washing area;

[0010] CTC washing detection: washing solution is introduced into the microfluidic chip. The washing solution is introduced into the washing area to take away non-specifically bound cells. The washed CTC enters the detection area, and the CTC is specifically marked by combining immunofluorescence labeling, and observed and analyzed using a microscope and fluorescence imaging;

[0011] Data analysis output, import CTC images obtained by microscopy and fluorescence imaging, and after image processing and analysis, obtain the characteristic information of CTC, including quantity, morphology, and expression, and output the analysis results in the form of data and images.

[0012] Furthermore, the microfluidic chip is a multi-layer structure, and the microfluidic chip includes an injection port, a capture zone, a washing zone and a detection zone, wherein the injection port is used to introduce a blood sample to be tested, the capture zone is used to specifically capture CTCs using specific antibodies and ligands, the washing zone is used to remove non-specifically bound cells, and the detection zone is used to observe and analyze captured CTCs.

[0013] Furthermore, the injection port is provided with an interface compatible with an external device, and the external device includes a syringe pump and a peristaltic pump. The capture area is provided with a capture chamber, each capture chamber is filled with specific antibodies and immunomagnetic beads, the washing area is provided with a flow washing channel and a fixed washing chamber, and the detection area is provided with an observation window and a marking area for marking the captured CTC cells.

[0014] Furthermore, the CTC capture and enrichment further includes the following steps:

[0015] Collect blood samples from patients. After collection, ensure that the blood collection tube is well sealed and send the samples for testing. Centrifuge the blood samples to separate plasma, white blood cells and red blood cells.

[0016] Transfer the plasma and white blood cells to a new centrifuge tube and remove the red blood cells;

[0017] Add phosphate buffer to the centrifuge tube, mix well with the white blood cells and centrifuge. Repeat 2-3 times to remove plasma and impurities.

[0018] Furthermore, the CTC capture and enrichment further includes the following steps:

[0019] Check that the chip is connected correctly and leak-free, load the pretreated blood sample into the injection port of the microfluidic chip, and control the flow rate and pressure to ensure that the blood flows stably in the chip;

[0020] When the blood sample flows through the capture zone, the CTCs therein will bind to specific antibodies and ligands in the capture zone for specific capture, wherein epithelial cell adhesion molecules and CD45 are selected as antibodies, and peptides and aptamers are selected as ligands.

[0021] Furthermore, the washing solution in the CTC washing detection is selected from phosphate buffer.

[0022] Furthermore, the CTC washing detection specifically includes the following steps:

[0023] The washing liquid is introduced into the washing liquid inlet of the microfluidic chip through the automatic fluid control system, and the flow rate and pressure of the washing liquid in the chip are adjusted through the pressure regulator so that the washing liquid flows evenly through the washing area;

[0024] The washing process was monitored by a visualization device to ensure that non-specifically bound cells were carried away by the wash solution while CTCs remained in the capture zone.

[0025] Furthermore, the CTC washing detection specifically includes the following steps:

[0026] Select fluorescent secondary antibody fluorescent dye as the labeling reagent, introduce the labeling reagent into the labeling reagent inlet of the microfluidic chip, so that the reagent can flow evenly through the capture area and bind to the specific marker on the CTC;

[0027] Incubate at 24-30°C for 45-60 minutes to allow the antibody to fully bind to the specific marker on the CTC. After binding, wash the chip again with washing solution to remove unbound antibodies and fluorescent dyes.

[0028] Furthermore, the CTC washing detection specifically includes the following steps:

[0029] Place the microfluidic chip on the stage of a microscope, adjust the focus and light intensity of the microscope to make the CTC clearly visible, use a fluorescent imaging system to image the CTC, and record the image data;

[0030] The acquired image data was identified and analyzed to count the number, morphology, and fluorescence intensity parameters of CTCs.

[0031] Furthermore, the data analysis output specifically includes the following steps:

[0032] Import CTC images acquired through microscopy and fluorescence imaging, and preprocess the CTC images;

[0033] Define the CTC area in the image as ROI, distinguish CTC from other cells by shape, size, and fluorescence intensity characteristics, and extract quantity, morphology, and expression feature information;

[0034] Performing statistical analysis on the extracted feature information, the statistical analysis includes calculating the mean, standard deviation, and median, and presenting the analysis results in the form of charts, tables, and histograms;

[0035] Output the analysis results in the form of data, and output the original image, ROI definition, and feature extraction results in the form of images;

[0036] Generate detailed analysis reports including images, charts, tables and text descriptions.

[0037] Compared with the prior art, the present invention has the following beneficial effects:

[0038] 1. The present invention constructs a specific capture chamber on a microfluidic chip and fills it with specific antibodies and immunomagnetic beads. The method can efficiently capture CTC cells and has a high specificity in identifying CTC cells. This high specificity reduces the possibility of false positives and false negatives and improves the accuracy of detection. The multi-channel design allows the simultaneous capture and detection of CTCs in multiple samples, thereby improving the efficiency of detection and saving time and cost. The microfluidic chip technology integrates multiple steps such as sample processing, cell capture, washing and detection on a single platform, realizing automation and standardization of operations, which not only simplifies the operating process and reduces human errors, but also improves the stability and repeatability of detection. Combined with microscopy and fluorescence imaging technology, the captured CTC cells can be intuitively observed, and the characteristic information of the cells can be obtained through image processing and analysis.

[0039] 2. The present invention removes red blood cells by centrifugation, and uses phosphate buffer to wash and repeatedly centrifuge white blood cells, which can effectively reduce impurities and nonspecific components in the blood, thereby reducing nonspecific capture during the capture process and improving the capture purity of CTCs. The flow rate and pressure of blood samples in the microfluidic chip are precisely controlled to ensure that the blood flows stably in the chip, thereby increasing the contact opportunity and binding time between CTCs and the capture agent and improving the capture efficiency. Due to the high-throughput characteristics of the microfluidic chip and the efficient capture capability of CTCs, this technical solution can achieve efficient enrichment and detection of CTCs in a smaller sample volume, which is particularly suitable for situations where the number of CTCs is small, such as blood sample detection in early cancer patients.

[0040] 3. The present invention can accurately control the flow rate and pressure of the washing solution and labeling reagent in the microfluidic chip through an automatic fluid control system and a pressure regulator, thereby ensuring that the washing and labeling processes are uniform and stable, and improving the accuracy and reliability of detection. The use of phosphate buffer as a washing solution, combined with an automatic fluid control system, can effectively flush and remove non-specifically bound cells, retain specifically captured CTCs, and improve the specificity of detection. The use of fluorescent secondary antibody fluorescent dyes as labeling reagents can specifically bind to markers on CTCs. CTCs can be clearly observed through a fluorescent imaging system, and parameters such as their quantity, morphology, and fluorescence intensity can be obtained, providing important information for the diagnosis and research of cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 Schematic diagram of the process of CTC cell detection method of the present invention. DETAILED DESCRIPTION

[0042] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0043] In order to solve the technical problems that traditional methods often have difficulty in accurately distinguishing CTCs from other cell types, resulting in high false positive and false negative rates, and are often time-consuming and inefficient, and cannot meet the needs of rapid and high-throughput detection, for samples with a small number of CTCs such as early-stage cancer patients, traditional methods are often difficult to effectively detect, please refer to Figure 1 , the present invention provides the following technical solutions:

[0044] A CTC cell detection method based on a microfluidic chip comprises the following steps:

[0045] Chip design and production, construction of a multi-layer microfluidic chip, construction of micrometer-scale channels for blood inflow and CTC cell capture on the microfluidic chip, construction of capture chambers on the microfluidic chip, each capture chamber is filled with specific antibodies and immunomagnetic beads for binding to and capturing CTC cells, and construction of inlets and outlets for blood inflow and outflow on the microfluidic chip;

[0046] CTC capture and enrichment: collect blood samples from patients, pre-treat the blood samples, remove red blood cell and white blood cell impurities from the blood samples, load the blood samples into the microfluidic chip, control the flow rate and pressure to make the blood flow in the chip, and the blood sample to be tested enters the chip through the injection port. The pre-treated blood sample enters the capture area and binds to the specific antibodies and ligands in the capture area to specifically capture CTCs. CTCs in multiple samples are captured simultaneously through multiple channels, and the captured CTCs enter the washing area;

[0047] CTC washing detection: washing solution is introduced into the microfluidic chip. The washing solution is introduced into the washing area to take away non-specifically bound cells. The washed CTC enters the detection area, and the CTC is specifically marked by combining immunofluorescence labeling, and observed and analyzed using a microscope and fluorescence imaging;

[0048] Data analysis output, import CTC images obtained by microscopy and fluorescence imaging, and after image processing and analysis, obtain the characteristic information of CTC, including quantity, morphology, and expression, and output the analysis results in the form of data and images.

[0049] In the above embodiment, by constructing a specific capture chamber on the microfluidic chip and filling it with specific antibodies and immunomagnetic beads, the method can efficiently capture CTC cells and has a high specificity in the identification of CTC cells. This high specificity reduces the possibility of false positives and false negatives and improves the accuracy of detection. The multi-channel design allows the simultaneous capture and detection of CTCs in multiple samples, thereby improving the efficiency of detection and saving time and cost.

[0050] In the above embodiments, the microfluidic chip technology integrates multiple steps such as sample processing, cell capture, washing and detection on a single platform, thereby realizing automation and standardization of operations. This not only simplifies the operation process and reduces human errors, but also improves the stability and repeatability of detection. Combined with microscopy and fluorescence imaging technology, the captured CTC cells can be visually observed, and the characteristic information of the cells can be obtained through image processing and analysis.

[0051] In the above embodiment, since this method has the advantages of high sensitivity, specificity, high throughput and integration, it has broad clinical application potential in early diagnosis of cancer, efficacy evaluation, recurrence monitoring, etc. By pre-treating the blood sample, impurities such as red blood cells and white blood cells are effectively removed, reducing sample waste and improving sample utilization.

[0052] The microfluidic chip is a multi-layer structure, which includes an injection port, a capture area, a washing area and a detection area, wherein the injection port is used to introduce a blood sample to be tested, the capture area is used to specifically capture CTCs using specific antibodies and ligands, the washing area is used to remove non-specifically bound cells, and the detection area is used to observe and analyze captured CTCs.

[0053] The injection port is provided with an interface compatible with an external device, and the external device includes a syringe pump and a peristaltic pump. The capture area is provided with a capture chamber, each capture chamber is filled with specific antibodies and immunomagnetic beads, the washing area is provided with a flow washing channel and a fixed washing chamber, and the detection area is provided with an observation window and a marking area for marking the captured CTC cells.

[0054] In the above embodiments, the multi-layer microfluidic chip integrates multiple steps such as injection, capture, washing and detection. External devices such as syringe pumps or peristaltic pumps can realize automatic fluid control, which greatly improves the convenience and accuracy of operation.

[0055] In the above embodiment, by setting a capture chamber filled with specific antibodies and immunomagnetic beads in the capture area, CTC cells can be efficiently and specifically captured, the interference of non-specific cells can be reduced, and the accuracy of detection can be improved. The design of the multi-layer structure makes the size of the internal channels and capture chambers of the microfluidic chip reach the micron level, thereby improving the capture efficiency of CTC cells, which is particularly suitable for situations where the number of CTC cells is small. Through the multi-channel design, multiple samples can be processed simultaneously, greatly improving the detection efficiency, and suitable for large-scale screening and clinical testing.

[0056] In the above embodiments, the observation window and marking area set in the detection area make the captured CTC cells easy to observe and mark. Combined with microscopy and fluorescence imaging technology, the morphology, quantity and expression of CTC cells can be intuitively obtained, providing strong support for the diagnosis and research of cancer. The design of the microfluidic chip can be customized and optimized according to specific needs to adapt to different sample types and detection requirements. At the same time, this technology can also be combined with other biotechnologies to achieve more complex biological analysis and detection. Through the fine design of the microfluidic chip, efficient cell capture and detection can be achieved in a very small sample volume, thereby saving sample resources.

[0057] CTC capture and enrichment also includes the following steps:

[0058] Collect blood samples from patients. After collection, ensure that the blood collection tube is well sealed and send the samples for testing. Centrifuge the blood samples to separate plasma, white blood cells and red blood cells.

[0059] Transfer the plasma and white blood cells to a new centrifuge tube and remove the red blood cells;

[0060] Add phosphate buffer to the centrifuge tube, mix well with the white blood cells and centrifuge, repeat 2-3 times to remove plasma and impurities.

[0061] Check that the chip is connected correctly and leak-free, load the pretreated blood sample into the injection port of the microfluidic chip, and control the flow rate and pressure to ensure that the blood flows stably in the chip;

[0062] When the blood sample flows through the capture zone, the CTCs therein will bind to specific antibodies and ligands in the capture zone for specific capture, wherein epithelial cell adhesion molecules and CD45 are selected as antibodies, and peptides and aptamers are selected as ligands.

[0063] In the above embodiments, epithelial cell adhesion molecules and peptides, aptamers are used as capture agents to efficiently and specifically capture CTCs from the blood. In the pretreatment stage, red blood cells are removed by centrifugation, and white blood cells are washed and repeatedly centrifuged using phosphate buffer, which can effectively reduce impurities and nonspecific components in the blood, thereby reducing nonspecific capture during the capture process and improving the capture purity of CTCs.

[0064] In the above embodiment, the flow rate and pressure of the blood sample in the microfluidic chip are precisely controlled to ensure that the blood flows stably in the chip, thereby increasing the contact opportunities and binding time between CTC and the capture agent and improving the capture efficiency. Due to the high throughput characteristics of the microfluidic chip and the efficient capture capability of CTC, this technical solution can achieve efficient enrichment and detection of CTC in a smaller sample volume, which is particularly suitable for situations where the number of CTCs is small, such as blood sample detection in early cancer patients. The entire CTC capture and enrichment process is implemented through a microfluidic chip, which is easy to operate and highly automated, reducing errors and interference in manual operations and improving the stability and reliability of detection.

[0065] CTC washing detection specifically includes the following steps:

[0066] A phosphate buffer solution is selected as the washing solution, and the washing solution is introduced into the washing solution inlet of the microfluidic chip through an automatic fluid control system, and the flow rate and pressure of the washing solution in the chip are adjusted through a pressure regulator so that the washing solution flows evenly through the washing area;

[0067] The washing process was monitored by a visualization device to ensure that non-specifically bound cells were carried away by the wash solution while CTCs remained in the capture zone.

[0068] Select fluorescent secondary antibody fluorescent dye as the labeling reagent, introduce the labeling reagent into the labeling reagent inlet of the microfluidic chip, so that the reagent can flow evenly through the capture area and bind to the specific marker on the CTC;

[0069] Incubate at 24-30°C for 45-60 minutes to allow the antibody to fully bind to the specific marker on the CTC. After binding, wash the chip again with washing solution to remove unbound antibodies and fluorescent dyes.

[0070] Place the microfluidic chip on the stage of a microscope, adjust the focus and light intensity of the microscope to make the CTC clearly visible, use a fluorescent imaging system to image the CTC, and record the image data;

[0071] The acquired image data was identified and analyzed to count the number, morphology, and fluorescence intensity parameters of CTCs.

[0072] In the above embodiments, the flow rate and pressure of the washing solution and labeling reagent in the microfluidic chip can be accurately controlled by the automatic fluid control system and the pressure regulator, ensuring that the washing and labeling processes are uniform and stable, thereby improving the accuracy and reliability of the detection.

[0073] In the above embodiment, phosphate buffer is used as a washing liquid, and combined with an automatic fluid control system, non-specifically bound cells can be effectively washed and removed, and specifically captured CTCs can be retained, thereby improving the specificity of detection. Fluorescent secondary antibody fluorescent dyes are used as labeling reagents, which can specifically bind to markers on CTCs. CTCs can be clearly observed through a fluorescent imaging system, and parameters such as their number, morphology, and fluorescence intensity can be obtained, providing important information for the diagnosis and research of cancer. The high-throughput characteristics of the microfluidic chip and its efficient capture capability of CTCs, combined with fluorescent labeling and imaging technology, make this technical solution have high sensitivity and low detection limit, and can detect CTCs in a small amount of samples.

[0074] Data analysis output also includes the following steps:

[0075] Import CTC images acquired through microscopy and fluorescence imaging, and pre-process the CTC images;

[0076] Define the CTC area in the image as ROI, distinguish CTC from other cells by shape, size, and fluorescence intensity characteristics, and extract quantity, morphology, and expression feature information;

[0077] Performing statistical analysis on the extracted feature information, the statistical analysis includes calculating the mean, standard deviation, and median, and presenting the analysis results in the form of charts, tables, and histograms;

[0078] Output the analysis results in the form of data, and output the original image, ROI definition, and feature extraction results in the form of images;

[0079] Generate detailed analysis reports including images, charts, tables and text descriptions.

[0080] In the above embodiment, by importing and preprocessing CTC images, ROI can be accurately defined to distinguish CTC from other cells, further extract quantity, morphology and expression feature information, and ensure the detail and accuracy of data analysis. Statistical analysis of the extracted feature information, such as calculating the mean, standard deviation and median, can fully reflect the characteristics of CTC, which not only helps to understand the overall distribution of CTC, but also reveals potential biological information.

[0081] In the above embodiments, the analysis results are displayed in the form of charts, tables, histograms, etc., making the data more intuitive and easy to understand. The analysis results are output in the form of data, and the original image, ROI definition and feature extraction results are output in the form of images, which helps to preserve the integrity and traceability of the original data, and generate a detailed analysis report including images, charts, tables and text descriptions, which can comprehensively display the results and process of CTC detection, which not only helps researchers understand the characteristics of CTCs, but also provides important diagnosis and treatment basis for clinicians.

[0082] In the above embodiments, the tediousness and errors of manual operations can be greatly reduced through automated and standardized data analysis processes, thereby improving research efficiency. At the same time, standardized data analysis methods also help to ensure the accuracy and reliability of the results.

[0083] The above description is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes according to the technical scheme and inventive concept of the present invention within the technical scope disclosed by the present invention, which should be covered by the protection scope of the present invention.

Claims

1. A CTC cell detection method based on a microfluidic chip, characterized in that: The following steps are involved: Chip design and production, construction of a multi-layer microfluidic chip, construction of micrometer-scale channels for blood inflow and CTC cell capture on the microfluidic chip, construction of capture chambers on the microfluidic chip, each capture chamber is filled with specific antibodies and immunomagnetic beads for binding to and capturing CTC cells, and construction of inlets and outlets for blood inflow and outflow on the microfluidic chip; CTC capture and enrichment: collect blood samples from patients, pre-treat the blood samples, remove red blood cell and white blood cell impurities from the blood samples, load the blood samples into the microfluidic chip, control the flow rate and pressure to make the blood flow in the chip, and the blood sample to be tested enters the chip through the injection port. The pre-treated blood sample enters the capture area and binds to the specific antibodies and ligands in the capture area to specifically capture CTCs. CTCs in multiple samples are captured simultaneously through multiple channels, and the captured CTCs enter the washing area; CTC washing detection: washing solution is introduced into the microfluidic chip. The washing solution is introduced into the washing area to take away non-specifically bound cells. The washed CTC enters the detection area, and the CTC is specifically marked by combining immunofluorescence labeling, and observed and analyzed using a microscope and fluorescence imaging; Data analysis output, import CTC images obtained by microscopy and fluorescence imaging, and after image processing and analysis, obtain the characteristic information of CTC, including quantity, morphology, and expression, and output the analysis results in the form of data and images.

2. A CTC cell detection method based on a microfluidic chip as claimed in claim 1, characterized in that: The microfluidic chip is a multi-layer structure, comprising an injection port, a capture zone, a washing zone and a detection zone, wherein the injection port is used to introduce a blood sample to be tested, the capture zone is used to specifically capture CTCs using specific antibodies and ligands, the washing zone is used to remove non-specifically bound cells, and the detection zone is used to observe and analyze captured CTCs.

3. A CTC cell detection method based on a microfluidic chip as claimed in claim 2, characterized in that: The injection port is provided with an interface compatible with an external device, and the external device includes a syringe pump and a peristaltic pump. The capture area is provided with a capture chamber, each capture chamber is filled with specific antibodies and immunomagnetic beads, the washing area is provided with a flow washing channel and a fixed washing chamber, and the detection area is provided with an observation window and a marking area for marking the captured CTC cells.

4. A CTC cell detection method based on a microfluidic chip as claimed in claim 1, characterized in that: The CTC capture and enrichment further comprises the following steps: Collect blood samples from patients. After collection, ensure that the blood collection tube is well sealed and send the samples for testing. Centrifuge the blood samples to separate plasma, white blood cells and red blood cells. Transfer the plasma and white blood cells to a new centrifuge tube and remove the red blood cells; Add phosphate buffer to the centrifuge tube, mix well with the white blood cells and centrifuge, repeat 2-3 times to remove plasma and impurities.

5. A CTC cell detection method based on a microfluidic chip as claimed in claim 4, characterized in that: The CTC capture and enrichment further comprises the following steps: Check that the chip is connected correctly and leak-free, load the pretreated blood sample into the injection port of the microfluidic chip, and control the flow rate and pressure to ensure that the blood flows stably in the chip; When the blood sample flows through the capture zone, the CTCs therein will bind to specific antibodies and ligands in the capture zone for specific capture, wherein epithelial cell adhesion molecules and CD45 are selected as antibodies, and peptides and aptamers are selected as ligands.

6. A CTC cell detection method based on a microfluidic chip as claimed in claim 1, characterized in that: The washing solution in the CTC washing detection is selected as phosphate buffer.

7. A CTC cell detection method based on a microfluidic chip as claimed in claim 1, characterized in that: The CTC washing detection specifically further comprises the following steps: The washing liquid is introduced into the washing liquid inlet of the microfluidic chip through the automatic fluid control system, and the flow rate and pressure of the washing liquid in the chip are adjusted through the pressure regulator so that the washing liquid flows evenly through the washing area; The washing process was monitored by a visualization device to ensure that non-specifically bound cells were carried away by the wash solution while CTCs remained in the capture zone.

8. A CTC cell detection method based on a microfluidic chip as claimed in claim 7, characterized in that: The CTC washing detection specifically further comprises the following steps: Select fluorescent secondary antibody fluorescent dye as the labeling reagent, introduce the labeling reagent into the labeling reagent inlet of the microfluidic chip, so that the reagent can flow evenly through the capture area and bind to the specific marker on the CTC; Incubate at 24-30°C for 45-60 minutes to allow the antibody to fully bind to the specific marker on the CTC. After binding, wash the chip again with washing solution to remove unbound antibodies and fluorescent dyes.

9. A CTC cell detection method based on a microfluidic chip as claimed in claim 8, characterized in that: The CTC washing detection specifically further comprises the following steps: Place the microfluidic chip on the stage of a microscope, adjust the focus and light intensity of the microscope to make the CTC clearly visible, use a fluorescent imaging system to image the CTC, and record the image data; The acquired image data was identified and analyzed to count the number, morphology, and fluorescence intensity parameters of CTCs.

10. A CTC cell detection method based on a microfluidic chip as claimed in claim 1, characterized in that: The data analysis output specifically includes the following steps: Import CTC images acquired through microscopy and fluorescence imaging, and preprocess the CTC images; Define the CTC area in the image as ROI, distinguish CTC from other cells by shape, size, and fluorescence intensity characteristics, and extract quantity, morphology, and expression feature information; Performing statistical analysis on the extracted feature information, the statistical analysis includes calculating the mean, standard deviation, and median, and presenting the analysis results in the form of charts, tables, and histograms; Output the analysis results in the form of data, and output the original image, ROI definition, and feature extraction results in the form of images; Generate detailed analysis reports including images, charts, tables and text descriptions.

Images