CTC integrated analysis system applying multi-target immunocapture and fluorescence scanning identification and application

Abstract

The invention relates to the technical field of cell fluorescence detection, in particular to a CTC integrated analysis system applying multi-target immunocapture and fluorescence scanning identification and application, and the system comprises a capture module, an analysis module, an image processing module and a signal output module. The efficient capture of EGFR mutant CTC and Kras mutant CTC can be realized by arranging the capture antibody layers of different specific antibodies in the target fitting channel of the micro-channel structure. The analysis module combines a fluorescence labeling probe with target cells and generates fluorescence signals after being excited by an excitation light source, and images are collected by a scanning imaging head. The image processing module realizes CTC classification identification and quantitative analysis through gray level extraction, wavelength identification and position contour matching, and finally the signal output module outputs a detection result. The system is modularized in structure and automatic in analysis process, and has the advantages of high throughput, high sensitivity and adaptability to multi-target detection.

Description

Technical Field

[0001] The present invention relates to the technical field of cell fluorescence detection, and particularly relates to a CTC integrated analysis system applying multi-target immune capture and fluorescence scanning identification and its application. Background Art

[0002] Circulating Tumor Cells (CTCs) are tumor cells derived from primary tumors or metastases and shed into the peripheral blood circulation system. Due to their close relationship with tumor occurrence, development, metastasis, and prognosis, CTCs are considered biomarkers with great clinical application prospects in "liquid biopsy" and are of great significance in early cancer screening, efficacy evaluation, drug resistance monitoring, and individualized treatment decision-making.

[0003] Traditional CTC detection methods mainly include enrichment methods based on physical properties (such as cell size, density) and immune capture methods based on biomarkers (such as EpCAM). Although the immune capture method has high specificity, due to the strong phenotypic heterogeneity of tumor cells, some CTCs may lose the expression of surface markers during the epithelial-mesenchymal transition (EMT) process, resulting in the risk of off-target and missed detection in single-target capture. The currently common CTC detection process usually requires multiple independent devices to separately complete steps such as blood sample pretreatment, cell enrichment, fluorescence staining, image acquisition, and analysis. It is not only complex and time-consuming to operate, but also has large sample loss and poor data consistency, making it difficult to achieve routine clinical application.

[0004] With the development of multi-target labeling, multi-channel fluorescence imaging, and image intelligent recognition technologies, researchers have increasingly emphasized the high-sensitivity, high-specificity, high-throughput detection, and quantitative analysis of CTCs.

[0005] Therefore, there is an urgent need to construct a CTC integrated analysis system applying multi-target immune capture and fluorescence scanning identification, which highly integrates functions such as sample processing, cell enrichment, fluorescence labeling, image acquisition, and automatic recognition and analysis. On the basis of ensuring detection sensitivity and accuracy, it improves the operation simplicity, detection efficiency, and clinical application repeatability. Summary of the Invention

[0006] Aiming at the above problems, the object of the present invention is to propose a CTC integrated analysis system applying multi-target immune capture and fluorescence scanning identification, including: a capture module 1, an analysis module 2, an image processing module 3, and a signal output module 4; The capture module 1 includes: a sample inlet end 11, a shunt channel 12, a target fitting channel 13, a circulation channel 14, a waste liquid end 15, a power pump 16, and a selection valve 17; The sample inlet end 11, the target fitting channel 13, the waste liquid end 15, and the circulation channel 14 are connected to each other through the shunt channel, forming a flow channel system with internal liquid communication; The capture module 1 further includes a capture antibody layer, the capture antibody layer is disposed on the inner sidewall of the target fitting channel 13, and the capture antibody layer contains specific antibodies for capturing cancer cells; The analysis module 2 includes: an identification sequence layer, an excitation light source, a scanning imaging head, and a control circuit; The identification sequence layer is disposed on the inner sidewall of the target fitting channel 13 and is located behind the capture antibody layer. The identification sequence layer contains specific antibodies or fluorescent probes with fluorescent labels; The excitation light source and the scanning imaging head are disposed on both sides of the target fitting channel and reciprocally scan with respect to the target; The control circuit is used to control the excitation light source to emit excitation light, so that the specific antibodies or fluorescent probes with fluorescent labels specifically bind to the target and emit fluorescence, and the fluorescence is collected by the scanning imaging head and forms an image signal; The image processing module 3 is used to process the image signal to obtain data signals for qualitative and quantitative analysis of the target; The signal output module is used to output the data signals.

[0007] In a preferred technical solution, the target fitting channel 13 includes at least one first segment 131 and one second segment 132, and the capture antibody layers of the first segment 131 and the second segment 132 adopt different specific antibodies.

[0008] In a preferred technical solution, the first segment 131 and the second segment 132 that adopt different specific antibodies form a parallel branch.

[0009] In a preferred technical solution, the first segment 131 and the second segment 132 that adopt different specific antibodies form a series branch.

[0010] In a preferred solution, the first segment 131 and the second segment 132 that adopt different specific antibodies form a series-parallel branch.

[0011] In a preferred technical solution, multiple segments that adopt different specific antibodies form one of a series branch, a parallel branch, and a series-parallel branch In a preferred technical solution, the power pump 16 is disposed on the target fitting channel 13; a selection valve is provided at the junction of the target fitting channel 13, the circulation channel 14, and the waste liquid end 15. The two valve positions of the selection valve respectively correspond to: T1: a closed-loop path of the shunt channel 12, the target fitting channel 13, and the circulation channel 14; T2: an open-loop path of the shunt channel 12, the target fitting channel 13, and the waste liquid end 15.

[0012] In a preferred technical solution, the flow channel system is a microchannel structure, and its cross-sectional area ranges from 0.01 to 1 mm 2 , and the length ranges from 10 to 500 mm.

[0013] In a preferred technical solution, the target fitting channel 13 is a detachable structure.

[0014] In a preferred technical solution, the image processing module 3 is used to process the image signal to obtain data signals for qualitative and quantitative analysis of the target object, specifically including: The image processing module 3 extracts the gray value distribution corresponding to the fluorescence intensity in the image signal; According to the known fluorescence wavelengths, the fluorescence signals with different band ranges, emission intensities, and spatial distribution characteristics are compared and analyzed; By matching the central wavelength, light intensity change curve, and its position profile in the target fitting channel 3 of the fluorescence signal, the corresponding fluorescence label types are distinguished; Based on the fluorescence label types, different types of CTCs and their concentrations contained in the sample to be tested are determined.

[0015] The present invention also provides an application of the CTC integrated analysis system according to the above application of multi-target immunocapture and fluorescence scanning identification, and the application includes the following steps: S1. Sample injection: Inject the liquid sample to be tested through the sample inlet end and introduce it into the shunt channel; S2. Target capture: Under the push of the power pump, the sample liquid flows into the target fitting channel provided with the capture antibody layer through the shunt channel, so that the target cells in the liquid react with the target-specific antibody and are captured on the inner side wall of the channel; S3. Fluorescence labeling: Inject the specific antibody or fluorescence probe with a fluorescence label in the identification sequence layer into the target fitting channel to bind it to the captured target cells, forming a complex with a fluorescence signal; S4. Excitation and imaging: Control the excitation light source to emit excitation light with a specific wavelength to irradiate the target fitting channel, and collect the fluorescence response signal through the scanning imaging head to obtain the fluorescence image signal; S5. Image Processing and Recognition: Perform gray-scale analysis and spatial distribution analysis on the collected image signals. According to the known fluorescence wavelengths, compare and analyze the fluorescence signals with different band ranges, luminous intensities, and spatial distribution characteristics to achieve the classification recognition and quantitative analysis of different types of CTCs. S6. Result Output: Output the classified and quantitative information of the targets obtained from the analysis as data signals, and generate the final detection results by the signal output module.

[0016] In a preferred technical solution, the different types of CTCs are EGFR mutant CTCs and Kras mutant CTCs.

[0017] Beneficial Effects: The integrated analysis system for CTCs with multi-target immune capture and fluorescence scanning identification provided by the present invention realizes the continuous enrichment, specific labeling, image acquisition, and quantitative analysis of circulating tumor cells by integrating a capture module, an analysis module, an image processing module, and a signal output module within the same platform, and constructs a complete processing link from sample injection to result output.

[0018] The specially designed target-fitting channels of the present application are respectively provided with a capture antibody layer and a fluorescence labeling sequence layer on the inner sidewalls, which can sequentially complete the selective binding of target cells and multi-channel fluorescence labeling in different segments; with the excitation light sources and scanning imaging heads arranged on both sides of the channels cooperating with the control circuit for reciprocating illumination and imaging, the stability and accuracy of fluorescence acquisition are improved.

[0019] In addition, the flow channel system adopts a microfluidic structure, combined with a precision power pump and a selection valve, which can be flexibly switched between closed-loop and open-loop fluid control modes, significantly enhancing the sample utilization rate and operation stability. The overall device supports the detachable replacement of the target-fitting channels, making it have good maintenance convenience and adaptability.

[0020] In summary, the present invention has the significant advantages of a compact structure, diverse recognition types, high imaging accuracy, and high degree of automation in the processing flow, and is suitable for the efficient screening and fine typing analysis of circulating tumor cells. Brief Description of the Drawings

[0021] Figure 1 It is a schematic diagram of the system structure relationship of the present invention; Figure 2 It is a schematic diagram of the microchannel structure of Embodiment 1 of the present invention; Figure 3 It is a schematic diagram of the microchannel structure of Embodiment 2 of the present invention; Figure 4 It is a schematic diagram of the microchannel structure of Embodiment 3 of the present invention; Figure 5Schematic diagram of the method flow of the present invention. Detailed implementation mode

[0023] To deepen the understanding of the present invention, the present invention will be further described in detail below in conjunction with embodiments. These embodiments are only used to explain the present invention and do not constitute a limitation on the protection scope of the present invention.

[0024] Embodiment 1 According to Figure 1 As shown, this embodiment provides an integrated CTC analysis system applying multi-target immune capture and fluorescence scanning identification, which is used to realize the enrichment, labeling, identification and quantitative analysis of circulating tumor cells (CTC) in peripheral blood.

[0025] The system includes a capture module 1, an analysis module 2, an image processing module 3 and a signal output module 4.

[0026] Among them, the capture module 1 includes a sample inlet end 11, a shunt channel 12, a target fitting channel 13, a circulation channel 14, a waste liquid end 15, a power pump 16 and a selection valve 17. The sample inlet end 11, the target fitting channel 13, and the waste liquid end 15 are connected to the circulation channel 14 through the shunt channel 12 to form an internal flow channel system with liquid circulation. This flow channel system adopts a microfluidic structure, with the cross-sectional area controlled within 0.01 - 1 mm 2 , and the length controlled between 10 - 500 mm, which is beneficial to realizing the fine control of trace liquids.

[0027] The target fitting channel 13 is set as a detachable structure for easy maintenance and replacement. The inner side wall of the target fitting channel 13 is sequentially provided with a capture antibody layer and an identification sequence layer. The capture antibody layer is used to specifically bind to the CTC cell surface antigen, and the identification sequence layer contains a specific antibody or a fluorescent probe with a fluorescent label, which is used to further label the captured cells.

[0028] As Figure 2 shown, the target fitting channel 13 is further divided into at least two structural segments, namely the first segment 131 and the second segment 132, and different types of specific antibodies are set for each of them. Among them, the first segment 131 is used to capture EGFR mutant CTC, and the second segment 132 is used to capture Kras mutant CTC. According to experimental requirements, the segments can be set as a parallel structure. The parallel structure can synchronously realize the shunt identification of multiple CTC subtypes, is suitable for high-throughput screening, can improve the detection efficiency, and is more suitable for the detection of high-concentration samples.

[0029] The power pump 16 is arranged upstream of the target fitting channel 13. Combined with the selection valve 17 arranged at the intersection of the target fitting channel 13, the circulation channel 14 and the waste liquid end 15, the system is supported to flexibly switch between the closed-loop mode T1 and the open-loop mode T2, so as to realize the cyclic enrichment or drainage operation of target cells.

[0030] The analysis module 2 includes an excitation light source and a scanning imaging head arranged on both sides of the channel, as well as a supporting control circuit. The excitation light source emits excitation light with a specific wavelength, which is used to excite the fluorescent label after binding to the target cells. The scanning imaging head collects the fluorescence response signal to form an image signal. The excitation light and the imaging head reciprocate on both sides of the channel to cover the entire field of view and improve the imaging uniformity.

[0031] The image processing module 3 is used to analyze and process the collected image signal. It first extracts the gray value distribution curve corresponding to the fluorescence intensity in the image signal, and according to the known fluorescence wavelength range, compares and analyzes the luminous intensity, spatial distribution and central wavelength of each region in the image, so as to distinguish different types of fluorescent label signals, and based on the light intensity change curve and its position profile in the target fitting channel 13, complete the identification and classification of different CTC subtypes and the concentration estimation.

[0032] The signal output module 4 formats and outputs the above image analysis results, and can realize the output of the identified CTC types and the corresponding quantity results to the backend platform or display device.

[0033] In summary, through microfluidic precise delivery, targeted segmented capture, multi-target fluorescence labeling, spatial resolution imaging and multi-dimensional comparison and analysis of images, the system realizes the simultaneous identification and quantification of multiple CTC types, improves the analysis efficiency and detection accuracy, and is applicable to various clinical application scenarios such as early tumor screening and curative effect monitoring.

[0034] Embodiment 2 As Figure 3 shown, this embodiment is further improved on the basis of Embodiment 1: The target fitting channel 13 is further divided into at least two structural segments, namely the first segment 131 and the second segment 132, and different types of specific antibodies are respectively arranged on the two.

[0035] Among them, the first segment 131 is used to capture EGFR mutant CTCs, and the second segment 132 is used to capture Kras mutant CTCs. According to experimental requirements, the segments can be set in a series structure. Using the series structure can improve the labeling accuracy while ensuring the throughput, realize the full combination of the sample to be tested with different segments, and is more suitable for the detection of low-concentration samples.

[0036] Embodiment 3 As Figure 4As shown in the figure, this embodiment is a further improvement based on Embodiment 1: The target fitting channel 13 is further divided into at least two structural segments. According to experimental requirements, multiple segments can be set in a series-parallel structure. The series-parallel structure means that the shunt channel 12 branches out multiple target fitting channels 13, and each branch of the target fitting channel 13 has multiple segments. A capture antibody layer for detecting different types of CTCs is provided on each segment. When the test sample flows through each branch of the target fitting channel 13, it will contact different specific antibodies. The series-parallel structure can improve the labeling accuracy while ensuring the throughput, and improve the detection efficiency.

[0037] Embodiment 4 As Figure 5 shown, this embodiment provides a specific application method of the CTC integrated analysis system based on the multi-target immunocapture and fluorescence scanning identification. This method is applicable to the efficient capture, targeted recognition and quantitative detection of circulating tumor cells (CTCs) in peripheral blood or other biological samples, and includes the following steps: S1. Sample injection: Inject the test liquid sample (such as collected whole blood or pretreated plasma) through the sample inlet end of the system. The sample first enters the shunt channel and is uniformly diverted to the inlet area of the target fitting channel under the action of the flow channel micro-control system, ensuring the flow velocity stability and distribution uniformity of the subsequent capture process.

[0038] S2. Target capture: Under the continuous drive of the power pump, the test sample enters the target fitting channel along the shunt channel. A capture antibody layer is pre-fixed on the inner side wall of the channel, and the antibody layer contains a variety of specific antibodies with high affinity for tumor cell surface antigens. CTCs in the sample liquid bind to the corresponding antibodies when passing through this area and are stably captured on the channel wall surface. This step can enter the closed-loop mode by switching the selection valve to extend the reaction time and improve the capture efficiency.

[0039] S3. Fluorescent labeling: After the target cells are captured, inject a dye solution containing a fluorescently labeled specific antibody or fluorescent probe into the target fitting channel. The labeling solution further specifically recognizes and binds to the captured cells in the channel, thereby forming a stable fluorescent complex on the surface of the target cells. To enhance the multi-target recognition ability, the wavelengths of the used fluorescent labeling probes can cover multiple recognition channels, and each type of probe labels different CTC subtypes.

[0040] S4. Excitation and imaging: The excitation light source is regulated by a control circuit to emit excitation light of a set wavelength, which irradiates the fluorescent complex in the target fitting channel. After the excitation light irradiation generates a fluorescence response signal, it is collected with high resolution by a scanning imaging head arranged on the opposite side of the channel. Excitation and imaging can cover the entire detection area through a reciprocating scanning method to ensure the integrity of the image and accurate acquisition of spatial information.

[0041] S5. Image processing and recognition: The image processing module receives and processes the image signals collected by the scanning imaging head. First, it extracts the gray value distribution information corresponding to the fluorescence signals in the image, and then analyzes it in combination with the fluorescence intensity, emission center wavelength, and its spatial distribution characteristics in the channel. The system determines the presence of different CTCs in the sample by comparing the intensity integral values, spatial profiles, and fluorescence types of different fluorescence signals, realizing the classification recognition and quantitative analysis of different CTC subtypes.

[0042] S6. Result output: After the system identifies and completes the classification and quantitative analysis, it outputs result data including the type identifiers of various CTC subtypes and their concentration values in the sample, and generates a standardized detection report or transmits it to an external information processing platform through the signal output module.

[0043] The system supports the simultaneous identification of at least two different types of CTCs, including but not limited to: EGFR mutant type, Kras mutant type, HER-2 positive type, CDH-1 positive type, CDKN2A positive type, and CD63 positive type. By configuring identification sequences with different fluorescence emission wavelengths and matching probes, multi-channel synchronous detection is achieved, and the specificity and sensitivity of identification are improved.

[0044] The method described in this embodiment combines targeted immunocapture and multi-band fluorescence imaging analysis technology. Through the precise control of the microfluidic system, while improving the detection efficiency and accuracy, it has good operation flexibility and system scalability, and is applicable to the precise identification and dynamic monitoring of CTCs in a variety of clinical or scientific research scenarios.

[0045] The above shows and describes the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited by the above embodiments. The above embodiments and the descriptions in the specification only illustrate the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention will have various changes and improvements, and these changes and improvements all fall within the scope of the present invention claimed. The scope of protection claimed by the present invention is defined by the appended claims and their equivalents.

Claims

1. An integrated CTC analysis system identified by multi-target immunocapture and fluorescence scanning, characterized in that, Including: a capture module (1), an analysis module (2), an image processing module (3), and a signal output module (4); The capture module (1) includes: a sample inlet end (11), a shunt channel (12), a target attachment channel (13), a circulation channel (14), a waste liquid end (15), a power pump (16), and a selection valve (17); The analysis module (2) includes: an identification sequence layer, an excitation light source, a scanning imaging head, and a control circuit; The identification sequence layer is disposed on the inner sidewall of the target attachment channel (13) and is located behind the capture antibody layer, and the identification sequence layer contains a specific antibody or a fluorescent probe with a fluorescent label; The excitation light source and the scanning imaging head are disposed on both sides of the target attachment channel and reciprocally scan with respect to the target; The control circuit is configured to control the excitation light source to emit excitation light, so that the specific antibody or the fluorescent probe with the fluorescent label specifically binds to the target and emits fluorescence, and the fluorescence is collected by the scanning imaging head and forms an image signal; The image processing module (3) is configured to process the image signal to obtain a data signal for qualitative and quantitative analysis of the target; The signal output module is configured to output the data signal.

2. The CTC integrated analysis system applying multi-target immune capture and fluorescence scanning identification according to claim 1, wherein: The sample inlet end (11), the target attachment channel (13), the waste liquid end (15), and the circulation channel (14) are communicated with each other through the shunt channel to form a flow channel system with internal liquid communication; the capture module (1) further includes a capture antibody layer, and the capture antibody layer is disposed on the inner sidewall of the target attachment channel (13), and the capture antibody layer contains a specific antibody for capturing cancer cells.

3. The CTC integrated analysis system using multi-target immune capture and fluorescence scanning identification according to claim 2, wherein: The target attachment channel (13) includes at least one first segment (131) and one second segment (132), and the capture antibody layers of the first segment (131) and the second segment (132) adopt different specific antibodies.

4. The CTC integrated analysis system applying multi-target immune capture and fluorescence scanning identification according to claim 3, characterized in that: The first segment (131) and the second segment (132) that adopt different specific antibodies form a parallel branch, a series branch, or a series-parallel branch.

5. The CTC integrated analysis system applying multi-target immune capture and fluorescence scanning identification according to claim 4, wherein: The power pump (16) is disposed on the target attachment channel (13); a selection valve is disposed at the junction of the target attachment channel (13), the circulation channel (14), and the waste liquid end (15).

6. The CTC integrated analysis system using multi-target immune capture and fluorescence scanning identification according to any one of claims 5, characterized in that: The two valve positions of the selection valve respectively correspond to: T1: a closed-loop path of the shunt channel (12), the target attachment channel (13), and the circulation channel (14); T2: an open-loop path of the shunt channel (12), the target attachment channel (13), and the waste liquid end (15).

7. The CTC integrated analysis system using multi-target immunocapture and fluorescence scanning identification according to any one of claims 1 to 5, characterized in that: The flow channel system is a microchannel structure with a cross-sectional area ranging from 0.01 to 1 mm 2 , and a length ranging from 10 to 500 mm.

8. The CTC integrated analysis system applying multi-target immunocapture and fluorescence scanning identification according to any one of claims 1 to 5, characterized in that: The target attachment channel (13) is a detachable structure.

9. The CTC integrated analysis system applying multi-target immune capture and fluorescence scanning identification according to claim 1, characterized in that, The image processing module (3) is configured to process the image signal to obtain a data signal for qualitative and quantitative analysis of the target, specifically including: The image processing module (3) extracts the gray value distribution corresponding to the fluorescence intensity in the image signal; According to the known fluorescence wavelength, the fluorescence signals with different band ranges, emission intensities, and spatial distribution characteristics are compared and analyzed; Distinguish the corresponding fluorescence labeling types by matching the central wavelength, the light intensity change curve of the fluorescence signal, and its position profile in the target binding channel (3). Determine the different types of CTCs and their concentrations contained in the sample to be tested based on the fluorescence labeling types.

10. Use of the CTC integrated analysis system applying multi-target immunocapture and fluorescence scanning identification according to any one of claims 1-9, characterized in that The application includes the following steps: S1. Sample injection: Inject the liquid sample to be tested through the sample inlet end and introduce it into the shunt channel. S2. Target capture: Under the push of the power pump, make the sample liquid flow into the target binding channel provided with the capture antibody layer through the shunt channel, so that the target cells in the liquid react with the target-specific antibody and are captured on the inner side wall of the channel. S3. Fluorescence labeling: Inject the specific antibody or fluorescence probe with fluorescence labeling in the identification sequence layer into the target binding channel, and make it bind to the captured target cells to form a complex with fluorescence signal. S4. Excitation and imaging: Control the excitation light source to emit excitation light with a specific wavelength to irradiate the target binding channel, and collect the fluorescence response signal through the scanning imaging head to obtain the fluorescence image signal. S5. Image processing and recognition: Perform gray-scale analysis and spatial distribution analysis on the collected image signal, and compare and analyze the fluorescence signals with different band ranges, light emission intensities, and spatial distribution characteristics according to the known fluorescence wavelengths to achieve the classification recognition and quantitative analysis of different types of CTCs; the different types of CTCs are EGFR mutant CTCs and Kras mutant CTCs. S6. Result output: Output the analyzed target classification and quantity information as a data signal, and generate the final detection result by the signal output module.

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