CN-121472016-B - Multichannel circulating epithelial cell separation and enrichment device based on microfluidic immunocapture and application

CN121472016BCN 121472016 BCN121472016 BCN 121472016BCN-121472016-B

Abstract

The invention belongs to the technical field of biological detection, and particularly relates to a multichannel circulating epithelial cell separation and enrichment device based on microfluidic immunocapture and application thereof; the device comprises a fluid injection unit, a switching valve, a three-layer structure chip body, a reagent bearing module, a bar code scanning module, a temperature control module and an exhaust air cooling system, wherein a multichannel micro-fluidic structure, an interface micro-column regulation area and a three-dimensional structure enrichment cavity are integrated in the chip body. The switching valve is controlled by a dynamic scoring algorithm to automatically switch the sample injection channel, and the accurate capture of target cells in the multi-channel enrichment cavity is realized by combining microstructure regulation and control and a temperature difference induction mechanism. The device has the advantages of high capture efficiency, low cell damage, large flux and high intelligent degree, and is suitable for enrichment detection of rare cells such as circulating tumor cells.

Inventors

CUI WEI
HUANG JIAN
ZHANG KAISHAN
GUO ZHIMIN
ZHANG YUJUAN
WU YUANJI

Assignees

杭州华得森生物技术有限公司
中国医学科学院肿瘤医院

Dates

Publication Date: 20260508
Application Date: 20260109

Claims (8)

1. A multichannel circulating epithelial cell separation and enrichment device based on microfluidic immunocapture comprises a fluid injection unit, a switching valve, a chip body, a reagent bearing module, a bar code scanning module, a coupler temperature control module, an exhaust air cooling module and a software control platform; It is characterized in that the method comprises the steps of, The chip body is of a three-layer composite structure and sequentially comprises a transparent cover layer, a middle microfluidic structure layer and a bottom supporting basal layer; the middle microfluidic structure layer comprises a microfluidic multichannel structure, an interface microstructure regulation region and an enrichment cavity; the microfluidic multichannel structure is provided with a plurality of branch channels, the switching valve is provided with a plurality of outlets, the switching valve is respectively connected with the inlets of the corresponding branch channels, and the outlets of the branch channels are connected with the enrichment cavity; the interface microstructure regulation and control region is arranged in the junction region of the branch channel outlet and the enrichment cavity inlet, a plurality of rows of micro-column arrays are arranged on the cross section, the arrangement structure is in hexagonal distribution, the column diameter is 10-20 mu m, the column distance is not more than 15 mu m, and the height is 30-60 mu m, and the interface microstructure regulation and control region is used for guiding cells to slowly enter the enrichment cavity; The enrichment cavity is arranged at the tail end of each branch channel and consists of a cavity body with a closed structure, the inner surface of the cavity body is provided with a three-dimensional support framework, the three-dimensional support framework is formed by template limited-domain silane polymerization reaction, and the surface of the three-dimensional support framework is modified by amino block polymers and then is introduced into immune antibody anchor points; the three-dimensional supporting framework is formed by carrying out limited-domain polymerization on isopropyl triethoxysilane in a polystyrene microsphere template, and a porous intercommunication structure is formed after the template is removed by pyrolysis; the surface capturing functional layer is positioned on the surface of the three-dimensional supporting framework and comprises a coupled anti-EpCAM antibody and an anti-CK antibody, the antibodies are fixed in a covalent bond mode, and the coupling density is controlled to be 20-60 mug/cm < 2 >.
2. The microfluidic immunocapture-based multichannel circulating epithelial cell separation and enrichment device according to claim 1, wherein the device comprises a plurality of microfluidic channels, The pore size distribution of the framework is concentrated in the range of 50-150 nm, the thickness of the whole framework is 1-3 mu m, stable anchor points are provided, and the capturing efficiency in a liquid flow path is enhanced; The block polymer takes amino end groups as connecting groups, and carboxyl or epoxy end groups are introduced to enhance the multi-point stability of antibody binding.
3. The microfluidic immunocapture-based multichannel circulating epithelial cell separation and enrichment device according to claim 1, wherein the device comprises a plurality of microfluidic channels, The interface microstructure regulation and control area is provided with a micro-column array to form a transition buffer section, the micro-column structure is integrally formed through a micro-mould pressing process, and the adopted material is polyurethane elastomer compatible with PDMS; The micro-column array is provided with a variable-diameter column group with gradually increased diameter along the flow direction, a deceleration gradient is formed along the flow direction, and the residence time of the target cells in the enrichment cavity is prolonged by intercepting the target cells to decelerate the target cells.
4. The microfluidic immunocapture-based multichannel circulating epithelial cell separation and enrichment device according to claim 1, wherein the switching valve selects the injected branch channel by using a weighted scoring algorithm, and the weighted scoring algorithm comprises the following steps: s101, before each sample injection, acquiring the last sample injection time, valve back pressure and accumulated injection volume of all branch channels; s102, calculating the priority score of each channel according to the time interval, the valve end resistance and the time interval from the last sample injection, and meeting the following conditions: ; Wherein, the For the number of the branch channel, Is the injection sequence number; is of sequence number At the time interval between the current time and the time when the last liquid injection of the channel is completed, And The shortest time interval and the longest time interval that all branch channels have recorded are respectively; is of sequence number The valve back pressure of the branch channel is the outlet pressure of the valve channel corresponding to the branch channel, And The minimum and maximum valve back pressures that have been recorded for all branch channels, respectively; is of sequence number Branch channel of (1) is at the first The volume of the sample to be injected a time, Is of sequence number The volume of the enrichment cavity corresponding to the branch channel, The total number of injections for the branch channel; alpha, beta and gamma are adjustable weight coefficients; S103, sequentially injecting samples into branch channels with high priority, and recalculating the scores after each round of sample injection, so that dynamic channel switching based on load balancing and fluid resistance cooperative control is realized.
5. The multichannel circulating epithelial cell separation and enrichment device based on microfluidic immunocapture according to claim 1, wherein the width of the branch channel is 80-100 microns, the volume of the enrichment cavity is 50-150 nL, the cavity inlet is designed into a 20-30-degree tapered bell mouth transition structure, and the roughness of the inner wall is controlled to be less than or equal to 0.2 microns.
6. The multi-channel circulating epithelial cell separation and enrichment device based on microfluidic immunocapture according to claim 1, wherein the switching valve is an eight-channel rotor valve, the rotating angle is controlled by a stepping motor, the different angles are connected with branch channels, and the valve body is made of polytetrafluoroethylene materials.
7. The multichannel circulating epithelial cell separation and enrichment device based on microfluidic immunocapture of claim 1, wherein the fluid injection unit consists of a numerical control injection pump, a flexible pipeline and a buffer joint, the maximum flow rate of the injection pump is not more than 5 mu L/min, the accuracy is controlled within a range of +/-0.1 mu L, a pressure buffer cavity is arranged in front of the flexible pipeline connected to a chip sample inlet, and a spiral compression tube structure is arranged in the buffer cavity.
8. The multichannel circulating epithelial cell separation and enrichment device based on microfluidic immunocapture according to claim 1 is characterized in that the coupler temperature control module adopts a graphene heating film, is distributed at the bottom of a chip, and is correspondingly provided with thermistors according to the positions of enrichment cavities, the thermistors feed back and adjust the power through a temperature control controller, so that the independent temperature area adjustment of each enrichment cavity is realized, the enrichment cavity is set to be in a 36-38 ℃ constant temperature area, the branch channels are controlled to be in a 26-28 ℃ constant temperature area, the temperature difference forms a stable thermal flow field, and auxiliary cells tend to the enrichment cavity.

Description

Multichannel circulating epithelial cell separation and enrichment device based on microfluidic immunocapture and application Technical Field The invention belongs to the technical field of biological detection, and particularly relates to a multichannel circulating epithelial cell separation and enrichment device based on microfluidic immunocapture and application thereof. Background In the context of rapid development of tumor diagnosis and liquid biopsy techniques, circulating Tumor Cells (CTCs) have received attention for their important role in early diagnosis, treatment monitoring and prognosis evaluation. The number of CTCs is very small, the heterogeneity is strong, and the CTCs are required to be extracted by an efficient and stable separation and enrichment technology. The traditional CTC separation technology mainly comprises a density gradient centrifugation method, an immunomagnetic bead method, a membrane filtration method and the like, but the methods have the defects of complex operation, high nonspecific capture rate, impaired cell activity and the like, and are difficult to meet the comprehensive requirements of clinic on sensitivity, selectivity and flux. The micro-fluidic technology is applied to the field of CTC separation due to the micro-scale control capability and the multifunctional integration characteristic, and can realize high-specificity cell identification and fixation by utilizing CTC surface specific markers (such as EpCAM, CK and the like) and a functional interface, and meanwhile, the cell integrity and activity are maintained. However, the current mainstream microfluidic CTC enrichment device is mostly of a single-channel structure, has low flux, is easy to block, has unstable efficiency and the like, is difficult to adapt to diversified sample processing requirements, and limits popularization of the device in high-flux clinical application. Therefore, the design of introducing multiple channels into a chip structure is considered, so that the high-selectivity enrichment and separation of circulating epithelial cells are realized, a more reliable and efficient sample source is provided for downstream molecular analysis and accurate diagnosis and treatment, and the technology is becoming an important direction of the development of a liquid biopsy platform. Disclosure of Invention Aiming at the problems, the invention aims to provide a multichannel circulating epithelial cell separation and enrichment device based on microfluidic immunocapture, which comprises a fluid injection unit, a switching valve, a chip body, a reagent bearing module, a bar code scanning module, a coupler temperature control module, a temperature digital display module and an exhaust air cooling module; The chip body is of a three-layer composite structure and sequentially comprises a transparent cover layer, a middle microfluidic structure layer and a bottom supporting basal layer; the middle microfluidic structure layer comprises a microfluidic multichannel structure, an interface microstructure regulation region and an enrichment cavity; the microfluidic multichannel structure is provided with a plurality of branch channels, the switching valve is provided with a plurality of outlets, the switching valve is respectively connected with the inlets of the corresponding branch channels, and the outlets of the branch channels are connected with the enrichment cavity; the interface microstructure regulation and control region is arranged in the junction region of the branch channel outlet and the enrichment cavity inlet, a plurality of rows of micro-column arrays are arranged on the cross section, the arrangement structure is in hexagonal distribution, the column diameter is 10-20 mu m, the column distance is not more than 15 mu m, and the height is 30-60 mu m, and the interface microstructure regulation and control region is used for guiding cells to slowly enter the enrichment cavity; The enrichment cavity is arranged at the tail end of each branch channel and consists of a cavity body with a closed structure, the inner surface of the cavity body is provided with a three-dimensional support framework, the three-dimensional support framework is formed by template limited-domain silane polymerization reaction, and the surface of the three-dimensional support framework is modified by amino block polymers and then is introduced into immune antibody anchor points; The surface capture functional layer is positioned on the surface of the three-dimensional support framework and comprises a coupled anti-EpCAM antibody and an anti-CK antibody, the antibodies are fixed in a covalent bond mode, and the coupling density is controlled to be 20-60 mug/cm < 2 >. The three-dimensional support skeleton is formed by polymerizing isopropyl triethoxysilane in a polystyrene microsphere template in a limited domain, a porous intercommunication structure is formed after the template is removed through pyrolysis, the skelet