Multi-channel circulating epithelial cell separation and enrichment device based on microfluidic immune capture and application

Abstract

The application belongs to the technical field of biological detection, and particularly relates to a multi-channel circulating epithelial cell separation and enrichment device based on micro-fluidic immune capture and application. The device comprises a fluid injection unit, a switching valve, a three-layer structure chip body, a reagent bearing module, a barcode scanning module, a temperature control module and an exhaust air cooling system, wherein the chip body is internally integrated with a multi-channel micro-fluidic structure, an interface micro-column regulation area and a three-dimensional structure enrichment cavity. The switching valve is automatically switched by a dynamic scoring algorithm to control sample injection channels, and the target cells are precisely captured in the multi-channel enrichment cavity by combining microstructure regulation and temperature difference induction mechanism. The device has the advantages of high capture efficiency, low cell damage, large throughput and high intelligence, and is suitable for enrichment detection of rare cells such as circulating tumor cells.

Description

Technical Field

[0001] This invention belongs to the technical field of biological detection, specifically relating to a multi-channel circulating epithelial cell separation and enrichment device based on microfluidic immune capture and its application. Background Technology

[0002] Against the backdrop of rapid development in tumor diagnosis and treatment and liquid biopsy technology, circulating tumor cells (CTCs) have attracted much attention due to their important role in early diagnosis, treatment monitoring, and prognostic assessment. CTCs are extremely rare and highly heterogeneous, requiring efficient and stable separation and enrichment techniques for extraction.

[0003] Traditional cell-mediated cell (CTC) separation techniques mainly include density gradient centrifugation, immunomagnetic beading, and membrane filtration. However, these methods generally suffer from drawbacks such as complex operation, high non-specific capture rate, and impaired cell viability, making it difficult to meet the comprehensive clinical requirements for sensitivity, selectivity, and throughput. Microfluidic technology, due to its microscale manipulation capabilities and multifunctional integration characteristics, has been applied to the field of CTC separation. It can utilize CTC surface-specific markers (such as EpCAM and CK) and functionalized interfaces to achieve highly specific cell recognition and fixation while maintaining cell integrity and viability.

[0004] However, most current mainstream microfluidic CTC enrichment devices have a single-channel structure, resulting in low throughput and susceptibility to problems such as clogging and unstable efficiency. This makes them unsuitable for diverse sample processing needs and limits their adoption in high-throughput clinical applications. Therefore, it is necessary to consider introducing multi-channel designs into the chip structure to achieve highly selective enrichment and separation of circulating epithelial cells, providing a more reliable and efficient sample source for downstream molecular analysis and precision diagnosis. This type of technology is becoming an important direction for the development of liquid biopsy platforms. Summary of the Invention

[0005] To address the above problems, the present invention aims to provide a multi-channel circulating epithelial cell separation and enrichment device based on microfluidic immune capture, comprising: a fluid injection unit, a switching valve, a chip body, a reagent carrier module, a barcode scanning module, a coupler temperature control module, a temperature digital display module, and an exhaust air cooling module.

[0006] The chip body has a three-layer composite structure, consisting of a transparent cover layer, a middle microfluidic structure layer, and a bottom support substrate layer.

[0007] The intermediate microfluidic structure layer includes a microfluidic multichannel structure, an interface microstructure control region, and an enrichment cavity;

[0008] The microfluidic multichannel structure has multiple branch channels, and the switching valve has multiple outlets, which are respectively connected to the inlets of the corresponding branch channels, and the outlets of the branch channels are connected to the enrichment chamber.

[0009] The interface microstructure control area is located at the junction of the branch channel outlet and the enrichment cavity inlet. The cross-section has multiple rows of micropillar arrays arranged in a hexagonal pattern. The column diameter is 10-20 μm, the column spacing is no more than 15 μm, and the height is 30-60 μm. It is used to guide cells to slowly enter the enrichment cavity.

[0010] The enrichment cavity is located at the end of each branch channel and consists of a closed cavity. A three-dimensional support skeleton is provided on the inner surface of the cavity. The three-dimensional support skeleton is formed by template-confined silane polymerization reaction and the surface is modified with amino block polymer to introduce immune antibody anchors.

[0011] The surface capture functional layer is located on the surface of the three-dimensional support framework and includes coupled anti-EpCAM antibody and anti-CK antibody. The antibodies are fixed in the form of covalent bonds, and the coupling density is controlled at 20–60 μg / cm².

[0012] As a preferred technical solution, the three-dimensional support framework is formed by confined polymerization of isopropyltriethoxysilane in a polystyrene microsphere template, and a porous interconnected structure is formed after the template is removed by pyrolysis; the pore size distribution of the framework is concentrated in the range of 50 to 150 nm, and the overall framework thickness is 1 to 3 μm, providing stable anchor points and enhancing the capture efficiency in the liquid flow path; the block polymer uses amino end groups as linking groups and introduces carboxyl or epoxy end groups to enhance the multi-point stability of antibody binding.

[0013] As a preferred technical solution, the interface microstructure control zone is provided with a micropillar array to form a transition buffer section. The micropillar structure is integrally formed by micromolding process, and the material used is a polyurethane elastomer compatible with PDMS. The micropillar array is provided with a group of variable diameter columns with gradually increasing diameter in the flow direction to form a deceleration gradient along the flow direction. By intercepting the target cells, it decelerates them and prolongs their residence time in the enrichment cavity.

[0014] As a preferred technical solution, the switching valve uses a weighted scoring algorithm to select the injection branch channel, and the weighted scoring algorithm includes the following steps:

[0015] S101. Before each injection, obtain the last injection time, valve back pressure and cumulative injection volume of all branch channels;

[0016] S102. Calculate the priority score for each channel based on the time interval, valve end resistance, and time interval since the last injection. ,satisfy:

[0017]

[0018] in, For branch channel number, For the injection sequence number; For the serial number The time interval between the current time and the last time fluid was injected into the branch channel. and These represent the shortest and longest time intervals recorded for all branch channels, respectively. For the serial number The valve back pressure of a branch channel is the outlet pressure of the valve channel corresponding to that branch channel. and These are the minimum and maximum valve back pressures recorded for all branch channels, respectively. For the serial number The branch channel in the Sample volume injected in the second injection For the serial number The volume of the enrichment cavity corresponding to the branch channel, This represents the total number of injections into this branch channel; α, β, and γ are adjustable weighting coefficients.

[0019] S103. Inject the samples sequentially into the high-priority branch channels, and recalculate the scores after each round of injection to achieve dynamic channel switching based on load balancing and fluid resistance coordinated control.

[0020] As a preferred technical solution, the width of the branch channel is 80-100 μm, the volume of the enrichment cavity is 50-150 nL, the cavity inlet is designed as a 20-30° tapered flared transition structure, and the inner wall roughness is controlled to Ra≤0.2 μm.

[0021] As a preferred technical solution, the switching valve is an eight-channel rotor valve, which controls the rotation angle through a stepper motor. Different angles connect to branch channels, and the valve body is made of polytetrafluoroethylene material.

[0022] As a preferred technical solution, the fluid injection unit consists of a CNC injection pump, a flexible pipe, and a buffer connector. The maximum flow rate of the injection pump is no more than 5 μL / min, and the accuracy is controlled within the range of ±0.1 μL. A pressure buffer chamber is provided before the flexible pipe is connected to the chip injection port, and the interior of the buffer chamber is a spiral compression tube structure.

[0023] As a preferred technical solution, the coupler temperature control module uses a graphene heating film distributed at the bottom of the chip, and thermistors are arranged according to the location of the enrichment cavity. The thermistors adjust the power supply through the temperature controller to achieve independent temperature zone adjustment for each enrichment cavity. The enrichment cavity is set in a constant temperature zone of 36-38℃, and the branch channels are controlled between 26-28℃. The temperature difference forms a stable heat flow field, which helps cells to move towards the enrichment cavity.

[0024] The present invention also provides an application of the aforementioned device for the separation of circulating epithelial cells, comprising the following steps:

[0025] S1. Sample pretreatment: The collected peripheral blood samples are lysed with red blood cells and resuspended in buffer solution;

[0026] S2. Barcode Recognition: Use the barcode module to recognize the sample number and bind it to the system;

[0027] S3, Fluid loading: Injected into the chip at a rate of 0.2–1.0 μL / min through the injection unit;

[0028] S4. Enrichment and Capture: Cell flow and enrichment are performed under the temperature difference set by the temperature control module, and the target cells bind to the capture antibody in the enrichment chamber.

[0029] S5. Rinse: Inject PBS buffer into the rinse channel to remove unbound cells and impurities;

[0030] S6. Fixation and staining: The captured cells were fixed with formaldehyde and stained with fluorescence.

[0031] S7. Fluorescence Image Acquisition: Image acquisition and data analysis are performed on the fluorescence imaging platform.

[0032] As a preferred technical solution, the enrichment duration is 30-50 minutes, the temperature difference is controlled within 10°C between the branch channel and the enrichment chamber, the length of the microcolumn control zone is not less than 800 μm, the total resistance gradient is controlled within 10-50 Pa, and the image acquisition uses three-channel fluorescence excitation wavelengths of 405 nm, 488 nm and 594 nm to label the nuclear dye, EpCAM and CK signals respectively.

[0033] Beneficial effects:

[0034] This invention introduces a multi-level synergistic "interface microstructure control region" and a "gradually narrowing horn-shaped transition structure" into the chip, which enables orderly deceleration and spatial positioning of cells between the end of the branch channel and the entrance of the enrichment cavity. The micropillar array provides uniform turbulence and slow-release flow rate, so that cells no longer concentrate on impacting the front wall of the cavity, thereby effectively reducing the probability of cell degeneration caused by non-specific binding and shear stress.

[0035] Furthermore, the enrichment cavity is equipped with a three-dimensional support framework structure, and through confined template polymerization and multi-point modification of block polymers, a stable, dense and spatially controllable arrangement of antibody anchor points is achieved. This structure significantly increases the number of cell binding sites per unit volume and forms a fluid-permeable three-dimensional network, which helps cells to be uniformly captured at different depths in the cavity and avoids the enrichment saturation failure problem caused by the "congestion effect" of the surface layer.

[0036] In terms of fluid control strategy, this invention employs a switching valve module control method based on a multi-parameter weighted scoring algorithm. It dynamically prioritizes channels by combining channel injection history, real-time back pressure, and volumetric load status, and implements high-precision liquid injection and channel switching based on this prioritization. This mechanism not only overcomes the channel bias problem caused by traditional sequential switching but also achieves a dynamic optimal balance between enrichment efficiency and uniformity by introducing feedback closed-loop logic.

[0037] Furthermore, this invention proposes a temperature control design that creates a local 10°C temperature difference between the enrichment cavity and the branch channels. By using local graphene heating at the bottom of the chip and feedback regulation from the thermistors on the channel sidewalls, the "temperature-induced migration" behavior of target cells under thermal field induction is controlled, effectively increasing the proportion of target cells shifting towards the enrichment cavity. Combined with the coordinated regulation of spatial flow rate and three-dimensional anchor points, a multi-dimensional cell capture platform is constructed, providing core support for high-sensitivity circulating epithelial cell detection. Attached Figure Description

[0038] Figure 1 This is a schematic diagram of the system structure of the present invention;

[0039] Figure 2 This is a schematic diagram of the method flow of the present invention. Detailed Implementation

[0040] To enhance understanding of the present invention, the present invention will be further described in detail below with reference to embodiments. These embodiments are only used to explain the present invention and do not constitute a limitation on the scope of protection of the present invention.

[0041] Example 1

[0042] like Figure 1 As shown, this embodiment provides a multi-channel circulating epithelial cell separation and enrichment device based on microfluidic immune capture, which is suitable for highly specific separation and enrichment capture of extremely low abundance circulating epithelial cells (CTCs) in peripheral blood samples, facilitating subsequent immunolabeling, morphological analysis and molecular diagnosis.

[0043] The device of this invention integrates a fluid injection unit, a programmable switching valve, a chip body, a reagent carrier module, a barcode recognition module, a coupler temperature control module, an exhaust air cooling module, and a software control platform; it has a compact structure and high functional integration, making it suitable for high-throughput and high-stability sample processing needs.

[0044] The main body of the device is an integrated desktop structure. The outer shell of the device is made of corrosion-resistant ABS material. The overall internal structure is divided into five functional areas: fluid injection unit area, chip loading area, temperature control module area, switching valve control area, and host computer main control board module area.

[0045] The chip loading area is located at the front of the device and features a transparent observation window, allowing operators to easily monitor the fluid flow and enrichment process. The fluid injection unit and switching valve structure are mounted on the left side of the device, connected by a rigid bracket to ensure a stable injection path. The main control unit is integrated at the rear of the device, coordinating all execution modules via a data bus.

[0046] The chip body has a three-layer composite structure, consisting of a transparent cover layer, a middle microfluidic structure layer, and a bottom support substrate layer from top to bottom.

[0047] The transparent cover is made of 0.8 mm thick optical-grade PMMA (polymethyl methacrylate) plate, with an inlet, an outlet, and a waste liquid outlet. It is equipped with a universal plug-in slot to support quick connection with external pipelines.

[0048] The intermediate microfluidic structure layer uses PDMS (polydimethylsiloxane) as the main material and is formed by soft printing using SU-8 photolithography mold. The channel structure and the array microcavity are integrally solidified and permanently bonded to the upper and lower layers through oxygen plasma treatment.

[0049] The support substrate is made of a high-strength glass substrate with a thickness of 1.1 mm. After high-temperature drying, the chip is packaged to ensure the optical transparency and pressure resistance of the channel.

[0050] The chip has an overall size of approximately 60 mm × 30 mm and contains 8 parallel branch channels (80–100 μm wide and 60 μm deep). The main channel runs from left to right, and the branch channels branch off from the main channel like leaf veins and connect to their respective independent enrichment cavities.

[0051] An interface microstructure control zone, approximately 800 μm in length, is set between the end of each branch channel and the inlet of the enrichment cavity. This zone contains an array of hexagonal micropillars arranged along the flow direction. The micropillar diameter gradually transitions from 10 μm at the inlet to 20 μm in the cavity direction, with a spacing not exceeding 15 μm and a height ranging from 30 to 60 μm. The micropillar material is a PDMS-compatible polyurethane elastomer, integrally molded via micromolding to form a multi-level shear control and cell deceleration zone, preventing high-speed cell impact on the cavity surface and increasing the probability of cell adhesion.

[0052] The enrichment chamber employs a closed microcavity design with a volume of 50–150 nL. The internal space is approximately 1.2 mm long, 0.4 mm wide, and 100 μm high. The chamber inlet features a tapered flared opening with an inlet angle of 20–30°, ensuring gradual diffusion of fluid rather than rapid impact. The inner wall of the chamber has a Ra surface roughness of less than 0.2 μm and undergoes plasma treatment to improve biocompatibility.

[0053] Each enrichment chamber contains a three-dimensional support framework for spatial positioning and stable cell capture. This framework structure is polymerized from isopropyltriethoxysilane monomer within a polystyrene microsphere template, and after pyrolysis to remove the template, a porous, interconnected network is formed. The pore size is primarily distributed between 50 and 150 nm, and the framework thickness is controlled between 1 and 3 μm.

[0054] To enhance cell capture capability, the surface of the backbone is modified with amino-terminal block polymers (such as PEG-PAA) to introduce multifunctional sites. Then, anti-EpCAM antibody and anti-CK antibody are covalently fixed to the backbone surface through EDC / NHS coupling reaction, with the coupling density controlled at 20–60 μg / cm².

[0055] This functional layer has a three-dimensional spatial anchor point structure, which increases the bonding capacity per unit volume and avoids the saturation limitations caused by planar bonding.

[0056] The switching valve module employs an eight-channel rotary rotor valve, driven by a stepper motor. The valve body is made of polytetrafluoroethylene (PTFE), and the valve core features a circumferential groove and an angular positioning ring, achieving a channel rotation resolution of 45°. After injection into the main channel, the valve core rotates to connect to the target branch channels, with the connection sequence determined by a dynamic priority scoring algorithm. A priority score is calculated for each channel. ,satisfy:

[0057]

[0058] in, For branch channel number, For the injection sequence number; For the serial number The time interval between the current time and the last time fluid was injected into the branch channel. and These represent the shortest and longest time intervals recorded for all branch channels, respectively. For the serial number The valve back pressure of a branch channel is the outlet pressure of the valve channel corresponding to that branch channel. and These are the minimum and maximum valve back pressures recorded for all branch channels, respectively. For the serial number The branch channel in the Sample volume injected in the second injection For the serial number The volume of the enrichment cavity corresponding to the branch channel, α represents the total number of injections into this branch channel; α, β, and γ are adjustable weighting coefficients.

[0059] The system will The channels are sorted from highest to lowest score, and channels with higher scores are connected first for injection. After injection, the status of each channel is updated and re-scored. This mechanism maintains uniform injection while avoiding resistance overload and flow deviation between channels, thus improving channel utilization efficiency and cell enrichment uniformity.

[0060] A flexible graphene heating film with a thickness of 15 μm is adhered to the bottom of the device chip, and thermistors are distributed according to the location of the enrichment cavity. Each resistor is connected to an independent PID control loop, allowing the enrichment cavity region and the branch channel region to be set with different temperatures: 36–38°C for the enrichment cavity and 26–28°C for the branch channel, forming a thermal gradient with a temperature difference of about 10°C.

[0061] This thermal field-induced mechanism utilizes the cell thermal migration effect to promote the directional migration of target cells to warmer regions, while the fluid disturbance field enhances cell aggregation and enrichment efficiency. The temperature control system error is controlled within ±0.3℃, meeting the requirements for precise capture.

[0062] The injection module employs a high-precision CNC injection pump with a stainless steel piston coated with PTFE. The maximum injection speed is 5 μL / min, and the minimum controllable flow rate is 0.05 μL / min. The tubing is composed of PTFE microtubes, and the interfaces utilize a Luer-lock thread structure to prevent slippage.

[0063] A flexible buffer channel is provided between the chip inlet and the pump outlet. Inside the channel is a spiral compression zone to absorb the initial shock flow and hysteresis backflow generated during the initial injection, thereby achieving continuous, stable, and pulsation-free sample loading.

[0064] The device is equipped with a brushless silent fan at the rear, with an air volume of 2.5 m³ / min. The air duct design adopts a side-extraction channel to guide hot air from the bottom chip loading area and the side circuit area to the rear. With the help of metal heat sinks, the temperature rise of the core parts is effectively controlled, preventing risks such as chip warping and electrical control failure caused by excessive temperature.

[0065] The device is equipped with dedicated control software that runs on the Windows platform, and the interface is divided into five control modules:

[0066] Sample identification and barcode binding;

[0067] Injection pump speed control and injection volume setting;

[0068] Switching valve scoring algorithm scheduling configuration;

[0069] Temperature control parameter setting and operation curve recording;

[0070] System logs, data export, and cloud upload interfaces.

[0071] The software supports remote control, abnormal alarms, process playback and result report generation, and can be connected to external microscopic imaging systems via USB or LAN to realize an integrated cell enrichment and image analysis process.

[0072] In summary, the multi-channel circulating epithelial cell separation and enrichment device based on microfluidic immune capture provided in this embodiment has high structural integration, high operational stability and intelligent sample injection control capabilities. It is particularly suitable for selective enrichment and subsequent analysis of low-abundance target cells, and is a highly practical sample processing platform in the fields of liquid biopsy, tumor screening and personalized medicine.

[0073] Example 2

[0074] like Figure 2 As shown, this embodiment provides an application method for a multi-channel circulating epithelial cell separation and enrichment device based on microfluidic immune capture, which aims to achieve high-throughput, high-selectivity, and high-stability physical separation and immune recognition capture of circulating epithelial cells (CTCs) in peripheral blood samples.

[0075] This method is based on an intelligent enrichment platform that integrates a fluid injection module, a programmable switching valve control system, a three-layer chip body, a fine-grained microstructure control interface, an independent temperature control unit, and a fully automatic injection scheduling algorithm. Specifically, it includes the following steps:

[0076] S1. Sample pretreatment:

[0077] After collecting 2–5 mL of peripheral blood samples from the subject using standard EDTA anticoagulant blood collection tubes, the samples should be processed within 2 hours at room temperature. The processing procedure includes incubation with red blood cell lysis buffer for 5–8 minutes, followed by centrifugation to remove lysis residue, resuspending the cell pellet in PBS buffer and adjusting to the required concentration (approximately 1 × 10⁻⁶). 6 (cells / mL). After resuspending, the sample is placed into the sample reagent bottle in the reagent carrier module and connected to the injection module via a flexible tubing, awaiting the subsequent injection process.

[0078] S2, Barcode Recognition:

[0079] Before the injection, the barcode scanning module is activated to identify the external label of the reagent bottle, and the system automatically reads and parses the sample number. The software system compares this number with the sample scheduling database to extract personalized injection parameters such as the channel selection strategy, preset injection volume range, and channel volume parameters corresponding to the sample. This data structure is then used for the initialization of subsequent injection control tasks in the switching valve module.

[0080] After the chip is loaded, the coupler temperature control module is activated and enters the initialization phase. The coupler temperature control module distributes a graphene heating film on the bottom of the chip and presets an independent thermistor sensing point directly below each enrichment cavity. The thermal control program sets the temperature zone of the enrichment cavity to 36–38°C and the temperature zone of the branch channels to 26–28°C, maintaining a temperature difference of 10 ± 1°C to establish a stable heat flux gradient.

[0081] This gradient generates a thermally induced flow towards the enrichment cavity, aiding in the migration and adhesion of target cells. Temperature regulation is achieved through a feedback closed-loop control logic, with a temperature control accuracy better than ±0.3℃. Sample loading can only proceed after the system temperature has stabilized.

[0082] S3, Fluid Loading:

[0083] The fluid injection unit is configured as an integrated CNC injection pump system, with the pump flow rate and injection volume set via drive control software. The initial flow rate is set to 0.5 μL / min and adjusted in real time based on the channel response.

[0084] To reduce startup shock and fluctuation interference, a flexible pipeline buffer unit is configured at the front end of the injection channel. A spiral compression structure is embedded in the buffer chamber, which can disperse the instantaneous resistance fluctuation of the fluid during high-pressure injection and ensure that the fluid is linearly propelled into the main channel of the chip.

[0085] S4, Enrichment Capture:

[0086] Before entering the injection process, the switching valve module initiates a scoring algorithm to prioritize the pathways. The valve body employs an eight-channel rotor valve structure, with the valve core driven to rotate by a stepper motor, forming pathway connections between different channel outlets. Before each injection, the system acquires the historical injection status of the current eight branch channels and calculates the priority score for each channel. ,satisfy:

[0087]

[0088] in, For branch channel number, For the injection sequence number; For the serial number The time interval between the current time and the last time fluid was injected into the branch channel. and These represent the shortest and longest time intervals recorded for all branch channels, respectively. For the serial number The valve back pressure of a branch channel is the outlet pressure of the valve channel corresponding to that branch channel. and These are the minimum and maximum valve back pressures recorded for all branch channels, respectively. For the serial number The branch channel in the Sample volume injected in the second injection For the serial number The volume of the enrichment cavity corresponding to the branch channel, α represents the total number of injections into this branch channel; α, β, and γ are adjustable weighting coefficients.

[0089] Before entering the enrichment chamber through the branch channels under the influence of fluid pressure and temperature difference, the sample first passes through the interface microstructure control zone. This zone, with a length of 800–1000 μm, contains an array of hexagonally arranged variable-diameter micropillars, integrally molded from PDMS-compatible polyurethane material. The column diameter gradually increases along the flow direction, transitioning from 10 μm to 20 μm, while the height is maintained at 30–60 μm. This structure provides a multi-level turbulent field and a controllable shear region, effectively reducing cell flow velocity and guiding them to adhere to the inner wall of the chamber.

[0090] The enrichment cavity volume is set to 50–150 nL, the inlet is a tapered horn-shaped structure (angle of 20–30°), and the inner surface roughness is controlled to Ra≤0.2 μm.

[0091] The inner wall of the cavity is fixed with a three-dimensional supporting framework structure, formed by confined polymerization of isopropyltriethoxysilane within a polystyrene microsphere template. After pyrolysis to remove the template, a porous, interconnected structure of 50–150 nm is obtained. Its surface is then modified with an amino block polymer and coupled with anti-EpCAM and anti-CK antibodies, with the coupling density controlled at 20–60 μg / cm², providing high-density anchor binding sites for cells.

[0092] Cells gradually settle and adhere to the cavity, completing specific recognition and binding with the antibody. The enrichment process lasts 30–50 minutes, with the flow rate maintained at 0.3–0.8 μL / min throughout the injection process. After enrichment is complete, the system automatically cuts off the flow path and enters the washing process.

[0093] S5, Rinse:

[0094] PBS buffer was injected via a syringe pump to quantitatively flush the branch channels and enrichment chambers (approximately 10 μL flush volume per channel) to remove unbound cells and solution residue.

[0095] S5. Fixation and staining:

[0096] Cells were fixed by injecting 4% formaldehyde solution for no more than 10 minutes. After fixation, a specific staining solution was injected, including:

[0097] Excite the nuclear dye at 405 nm fluorescence wavelength;

[0098] 488 nm was used to label EpCAM;

[0099] 594 nm was used to label CK.

[0100] After incubating the staining reaction at room temperature in the dark for 15 minutes, rinse with PBS three times to prepare for image acquisition.

[0101] S8. Fluorescence Image Acquisition:

[0102] The chip is moved to a fluorescence imaging platform, and a laser confocal scanning system is used for simultaneous three-channel acquisition to obtain the spatial location information and staining signal intensity of the target cells. The system extracts cell morphology parameters, nucleocytoplasmic ratio, and fluorescence distribution ratio through an image recognition module, and determines whether the target cells meet the CTC identification criteria based on a set threshold. The results are output to the main control software terminal, which can automatically generate a detection report and archive the data.

[0103] This embodiment achieves the following in multi-channel enrichment tasks through deep coupling of structural design and dynamic control strategy: precise channel control; asynchronous injection scheduling; migration guided by thermal field difference between channels; multi-point three-dimensional capture; and intelligent scheduling and recognition linkage.

[0104] This method not only optimizes throughput and capture efficiency, but also ensures cell viability and the stability of detection results. It is a feasible and innovative core process in the circulating epithelial cell enrichment device.

[0105] The foregoing has shown and described 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 to the above embodiments. The embodiments and descriptions in the specification are merely illustrative of the principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the present invention as claimed. The scope of protection of the present invention is defined by the appended claims and their equivalents.

Claims

1. A multi-channel circulating epithelial cell separation and enrichment device based on microfluidic immune capture, comprising: Fluid injection unit, switching valve, chip body, reagent carrier module, barcode scanning module, coupler temperature control module, exhaust air cooling module and software control platform; Its features are, The chip body has a three-layer composite structure, consisting of a transparent cover layer, a middle microfluidic structure layer, and a bottom support substrate layer. The intermediate microfluidic structure layer includes a microfluidic multichannel structure, an interface microstructure control region, and an enrichment cavity; The microfluidic multichannel structure has multiple branch channels, and the switching valve has multiple outlets, which are respectively connected to the inlets of the corresponding branch channels, and the outlets of the branch channels are connected to the enrichment chamber. The interface microstructure control area is located at the junction of the branch channel outlet and the enrichment cavity inlet. The cross-section has multiple rows of micropillar arrays arranged in a hexagonal pattern. The column diameter is 10-20 μm, the column spacing is no more than 15 μm, and the height is 30-60 μm. It is used to guide cells to slowly enter the enrichment cavity. The enrichment cavity is located at the end of each branch channel and consists of a closed cavity. A three-dimensional support skeleton is provided on the inner surface of the cavity. The three-dimensional support skeleton is formed by template-confined silane polymerization reaction and the surface is modified with amino block polymer to introduce immune antibody anchors. The three-dimensional support framework is formed by confined polymerization of isopropyltriethoxysilane in a polystyrene microsphere template, and a porous interconnected structure is formed after the template is removed by pyrolysis. The surface capture functional layer is located on the surface of the three-dimensional support framework and includes conjugated anti-EpCAM and anti-CK antibodies. The antibodies are fixed in the form of covalent bonds, and the conjugation density is controlled at 20–60 μg / cm².

2. The multi-channel circulating epithelial cell separation and enrichment device based on microfluidic immune capture according to claim 1, characterized in that, The pore size distribution of the framework is concentrated in the range of 50–150 nm, and the overall framework thickness is 1–3 μm, which provides stable anchor points and enhances the capture efficiency in the liquid flow path. Block polymers use amino terminal groups as linking groups and introduce carboxyl or epoxy terminal groups to enhance the multi-point stability of antibody binding.

3. The multi-channel circulating epithelial cell separation and enrichment device based on microfluidic immune capture according to claim 1, characterized in that, The interface microstructure control area is provided with a micropillar array to form a transition buffer section. The micropillar structure is integrally formed by micro-molding process, and the material used is a polyurethane elastomer compatible with PDMS. The micropillar array has a group of variable-diameter pillars with gradually increasing diameters arranged in the flow direction, forming a deceleration gradient along the flow direction. This decelerates the target cells by intercepting them, thereby prolonging their residence time in the enrichment cavity.

4. The multi-channel circulating epithelial cell separation and enrichment device based on microfluidic immune capture according to claim 1, characterized in that, The switching valve uses a weighted scoring algorithm to select the injection branch channel, and the weighted scoring algorithm includes the following steps: S101. Before each injection, obtain the last injection time, valve back pressure and cumulative injection volume of all branch channels; S102. Based on the time interval, valve end resistance, and time interval since the last injection, calculate the priority score for each channel, satisfying the following: ； in, For branch channel number, For the injection sequence number; For the serial number The time interval between the current time and the last time fluid was injected into the branch channel. and These represent the shortest and longest time intervals recorded for all branch channels, respectively. For the serial number The valve back pressure of a branch channel is the outlet pressure of the valve channel corresponding to that branch channel. and These are the minimum and maximum valve back pressures recorded for all branch channels, respectively. For the serial number The branch channel in the Sample volume injected in the second injection For the serial number The volume of the enrichment cavity corresponding to the branch channel, This represents the total number of injections into this branch channel; α, β, and γ are adjustable weighting coefficients. S103. Inject the samples sequentially into the high-priority branch channels, and recalculate the scores after each round of injection to achieve dynamic channel switching based on load balancing and fluid resistance coordinated control.

5. The multi-channel circulating epithelial cell separation and enrichment device based on microfluidic immune capture according to claim 1, characterized in that, The width of the branch channel is 80–100 μm, the volume of the enrichment cavity is 50–150 nL, the cavity inlet is designed as a 20–30° tapered flared transition structure, and the inner wall roughness is controlled to Ra≤0.2 μm.

6. The multi-channel circulating epithelial cell separation and enrichment device based on microfluidic immune capture according to claim 1, characterized in that, The switching valve is an eight-channel rotor valve, with the rotation angle controlled by a stepper motor. Different angles connect to branch channels, and the valve body is made of polytetrafluoroethylene material.

7. The multi-channel circulating epithelial cell separation and enrichment device based on microfluidic immune capture according to claim 1, characterized in that, The fluid injection unit consists of a CNC injection pump, a flexible pipe, and a buffer connector. The maximum flow rate of the injection pump is no more than 5 μL / min, and the accuracy is controlled within the range of ±0.1 μL. A pressure buffer chamber is provided before the flexible pipe is connected to the chip injection port. The interior of the buffer chamber is a spiral compression tube structure.

8. The multi-channel circulating epithelial cell separation and enrichment device based on microfluidic immune capture according to claim 1, characterized in that, The coupler temperature control module uses a graphene heating film distributed at the bottom of the chip, and thermistors are arranged according to the location of the enrichment cavity. The thermistors adjust the power supply through the temperature controller to achieve independent temperature zone adjustment for each enrichment cavity. The enrichment cavity is set in a constant temperature zone of 36-38℃, and the branch channels are controlled between 26-28℃. The temperature difference forms a stable heat flow field, which helps cells to move towards the enrichment cavity.

Images