Microfluidic Chip for Capturing Circulating Tumor Cells and Its Preparation Method

Abstract

The present invention relates to a microfluidic chip for capturing circulating tumor cells and a preparation method thereof, which includes a PDMS substrate and a glass slide bonded to each other. Its microchannels include an inlet channel, an array of swimming lanes, and an outlet channel. Several microfluidic groove array units are equally spaced along the axial direction of the swimming lane. The microfluidic groove array unit includes a first microfluidic groove array to a sixth microfluidic groove array equally spaced in sequence along the axial direction of the swimming lane. The first microfluidic groove array is formed by arranging L W-shaped microfluidic grooves equally spaced along the axial direction of the swimming lane, and the lateral dimension of the W-shaped microfluidic groove is equal to the lateral dimension of the swimming lane. The structure of the i-th microfluidic groove array is based on the tandem double W-shaped microfluidic groove as the offset main body, obtained by laterally offsetting the first microfluidic groove array by the i-th preset dimension along the lateral direction of the swimming lane, and the offset directions are the same. The inner surface of the glass slide is loaded with capture probes. The CTC cell capture rate of the microfluidic chip of the present invention is ≥95%, and the enrichment purity of CTC cells is ≥80%.

Description

Technical Field

[0001] The present invention belongs to the technical field of biological detection, and particularly relates to a microfluidic chip for capturing circulating tumor cells and a preparation method thereof. Background Art

[0002] Tumor detection runs through the entire onset and treatment cycle, from early benign and malignant screening, surgical treatment, various conventional radiotherapy, chemotherapy, targeted and immunotherapy. Conventional imaging techniques, such as PET-CT and MRI, etc., can be used to initially confirm the occurrence of malignant tumors. However, for the accurate clinical diagnosis and the formulation of clinical treatment plans, IHC detection of patient tissues needs to be carried out simultaneously, that is, invasive tissue biopsy. However, in terms of current detection methods, IHC detection has high requirements for sampling, and there are certain subjectivity and heterogeneity, which are affected by factors such as the quality of detection antibodies, detection processes (such as fixation, staining), etc. At the same time, it is difficult to obtain tumor tissues in the middle and late stages of some tumor patients. Therefore, although IHC detection is the current gold standard for the detection of clinical tumor patients, there are still certain limitations due to the above reasons.

[0003] In recent years, liquid biopsies have become increasingly attractive for cancer screening and auxiliary diagnosis due to their non-invasiveness and simple sampling process. Liquid biopsy is a revolutionary technology compared with traditional tissue biopsy. It can present a static snapshot of the tumor and has unique and great advantages; it provides information about the "real-time" cancer burden and reveals the evolution and heterogeneity of the disease over time. The main targets of liquid biopsy include circulating tumor cells (CTC), circulating tumor DNA (ctDNA), and extracellular vesicles. Tumor cells that enter the blood from primary or metastatic cancers are called CTC.

[0004] CTC separation techniques can be divided into two categories according to the physical properties of the cells themselves or the specific antigens expressed on the surface. Each category has derived different methods, among which membrane filtration, density gradient centrifugation, and immunological affinity are relatively commonly used methods, but a single method has its unique advantages and disadvantages. Membrane filtration is a method of separating CTC based on cell size, and a part of CTC will be correspondingly lost in the selection of the filtration pore size; density gradient centrifugation is a method of enriching CTC in the monocyte layer based on the principle of density difference between cells. The disadvantage is that white blood cells cannot be easily removed. Immunological affinity is a method of enriching CTC based on the specific binding of specific antigens on the surface of CTC to capture antibodies. The disadvantage is that there are various interferences such as red blood cells and white blood cells in the whole blood sample, which will affect the specific binding of CTC to capture antibodies and the separation after binding. The separated and enriched CTC cells are further identified by immunocytochemistry, immunofluorescence or other techniques.

[0005] With the continuous update and iteration of CTC separation and capture technologies, the enrichment efficiency and purity have also been continuously improved. Among them, the development of a CTC enrichment and capture platform based on microfluidic chips has been a research hotspot in the field of CTC liquid biopsy in recent years; it can not only separate CTC cells by using differences in cell physical properties (such as size and charge differences) and combined microfluidics (such as shear force combination modes), but also capture antibodies for immunological affinity combination separation of CTC by using cell surface antigens and functionalized modifications of the microfluidic chip substrate layer. Since the chamber geometry of the microfluidic chip is designed according to the specific surface area of CTC cells in contact with the inner cavity of the chip, the reaction volume in the inner cavity of the chip is generally controlled at 10-100 microliters, and through the design and adjustment of the microfluidics and angles in the chip chamber, the morphology of the enriched CTC cells can be more completely maintained. Therefore, compared with the conventional negative immunomagnetic beads or positive immunomagnetic beads for CTC enrichment, it has the characteristics of less blood sample consumption, low cost, high sensitivity and specificity for CTC enrichment and capture.

[0006] Currently, CTC-chip, HB-Chip, and CTC-iChip on the market are the most representative CTC capture and identification platforms based on microfluidic chip enrichment. The design of the microfluidic chips of these platforms captures and enriches CTC cells by using immunological affinity through specific antigens on the surface of CTC target cells and bioactive molecular protein antibodies and nucleic acid ligands modified on the surface structure of the microfluidic channels, or enriches and separates CTC cells by combining the physical properties and charge distribution of CTC cells with the microfluidics design in the inner cavity of the microfluidic chip and adjusting the direction and magnitude of the shear force.

[0007] With the development of micro-nano chip manufacturing technology, it has become possible to enhance the CTC enrichment efficiency by designing and modifying the structure of microfluidic chips. A typical microfluidic device is designed in a grooved herringbone (HB) structure, which has been proven to be able to separate and enrich CTC. By stretching the HB structure and changing the streamline pattern of the microfluidics, the interaction on the cell surface is changed, so that the CTC capture rate is as high as 93%, and the purity is close to 14%. Subsequently, various derivative microfluidic chip products with HB microfluidic design have been developed. For example, nanostructure designs (including nanocolumns and nanoloops) are incorporated into the microfluidic chip to increase the total surface area and enhance the cell surface interaction; these microfluidic structure designs show better CTC capture efficiency. However, a major limitation of the grooved HB microfluidic chip structure is the uneven distribution of shear stress in the microchannel, resulting in the capture of white blood cells (WBC) in areas with extremely low shear stress, sacrificing the purity of CTC enrichment and capture on the microfluidic chip. Summary of the Invention

[0008] Based on the above-mentioned drawbacks and deficiencies existing in the prior art, one of the objectives of the present invention is to at least solve one or more of the above-mentioned problems existing in the prior art. In other words, one of the objectives of the present invention is to provide a microfluidic chip for capturing circulating tumor cells and a preparation method thereof that meet one or more of the foregoing requirements.

[0009] In order to achieve the above-mentioned invention objective, the present invention adopts the following technical solutions:

[0010] A microfluidic chip for capturing circulating tumor cells, comprising a PDMS substrate and a glass slide bonded to each other; the inner surface of the PDMS substrate has a microchannel, and the microchannel and the inner surface of the glass slide form a microfluidic channel. The microchannel includes an inlet channel, an array of swimming lanes, and an outlet channel. The array of swimming lanes includes N swimming lanes arranged in parallel at equal intervals. The inlet and outlet of each swimming lane are respectively connected to the inlet channel and the outlet channel; where N is an integer.

[0011] Several microfluidic groove array units are distributed at equal intervals along the axial direction of the swimming lane. The microfluidic groove array unit includes a first microfluidic groove array to a sixth microfluidic groove array arranged at equal intervals along the axial direction of the swimming lane. The first microfluidic groove array is composed of L W-shaped microfluidic grooves arranged at equal intervals along the axial direction of the swimming lane. The axis of symmetry of the W-shaped microfluidic groove coincides with the axis of symmetry of the axial direction of the swimming lane. The W opening of the W-shaped microfluidic groove faces the inlet of the swimming lane, and the transverse dimension of the W-shaped microfluidic groove is equal to the transverse dimension of the swimming lane.

[0012] The structure of the i-th microfluidic groove array is based on the tandem double W-shaped microfluidic groove as the offset main body, and is obtained by offsetting the first microfluidic groove array by the i-th preset dimension along the transverse direction of the swimming lane, and the offset directions are the same; where the i-th preset dimension is i*D / 6, and i takes an integer value from 2 to 6, and D is the transverse dimension of the swimming lane.

[0013] The inner surface of the glass slide is loaded with capture probes corresponding to the microfluidic channel.

[0014] As a preferred solution, the inlet channel is in a tree root-like structure, including a main branch channel, a secondary branch channel, and a swimming lane connection channel that are sequentially connected along the inlet flow direction. The inlet of the main branch channel is connected to the inlet of the PDMS substrate.

[0015] As a preferred solution, the main branch channel, the secondary branch channel, and the swimming lane connection channel are all in an arc-shaped channel structure.

[0016] As a preferred solution, the axis of symmetry of the inlet channel coincides with the axis of symmetry of the axial direction of the array of swimming lanes.

[0017] As a preferred solution, the capture probe is sequentially conjugated with a bifunctional cross-linking agent, streptavidin, and a biotinylated monoclonal antibody.

[0018] As a preferred embodiment, the capture probe further comprises a biotinylated antibody against a key tumor marker.

[0019] As a preferred embodiment, the biotinylated antibody against a key tumor marker comprises at least one of a biotinylated EpCAM antibody, a biotinylated CSV antibody, and a biotinylated VIM (vimentin) antibody.

[0020] As a preferred embodiment, N takes an even number from 4 to 10, and L takes a value from 8 to 15.

[0021] As a preferred embodiment, for the microfluidic chip for capturing circulating tumor cells, the capture rate of circulating tumor cells is ≥95%, and the enrichment purity of circulating tumor cells is ≥80%.

[0022] The present invention also provides a method for preparing the microfluidic chip according to any one of the above embodiments, comprising the following steps:

[0023] S1. Spin-coat a photoresist on the silicon wafer after plasma cleaning, heat and dry it, and then perform exposure, heating, and development in sequence to obtain a photoresist nanoarray; then pour PDMS glue based on the photoresist nanoarray, punch and cut the glue, perform plasma cleaning, and bond to obtain a PDMS substrate;

[0024] S2. Perform sequential conjugate loading of the capture probe components - bifunctional crosslinker, streptavidin, and biotinylated monoclonal antibody on the glass slide after oxygen plasma treatment.

[0025] S3. Bond the PDMS microfluidic channel substrate after oxygen plasma treatment and the glass slide with conjugate loading of the capture probe.

[0026] Compared with the prior art, the present invention has the following beneficial effects:

[0027] (1) The present invention has made an original improvement to the channel structure, changing the problem of high interception rate of white blood cell microfluidic chips caused by uneven shear force distribution in conventional microfluidic chips. The CTC cell capture rate is ≥95% and the enrichment purity of CTC cells is ≥80%;

[0028] (2) The structural design of the arc-shaped channel of the inlet channel in the present invention is beneficial to ensuring the integrity of the morphological form of CTC live cells and preventing rupture;

[0029] (3) The present invention can achieve higher CTC capture efficiency even with a very small blood sample volume (0.2 - 1 mL), and its clinical practicability has been proven in the detection of CTCs in patients with different cancer phenotypes such as breast cancer, lung cancer, prostate cancer, and colorectal cancer;

[0030] (4) The present invention designs the functionalization modification of specific bifunctional groups in the microfluidic channels of the microfluidic chip and couples streptavidin, realizing a cascade amplification reaction of CTC separation and enrichment signals, and enabling more efficient CTC enrichment;

[0031] (5) The present invention further couples biotinylated epithelial marker EPCAM and mesenchymal tumor marker CSV in the microfluidic channels of the microfluidic chip, thereby realizing the efficient enrichment of three subtypes of epithelial CTCs, mesenchymal CTCs, and epithelial-mesenchymal transition CTCs at one time; compared with the existing CTC systems that simply enrich based on epithelial or mesenchymal types or a single tumor marker, it can more comprehensively and sensitively separate and enrich CTCs in the peripheral blood of various tumor patients;

[0032] (6) The microfluidic chip of the present invention is based on the combined immunoassay of various markers of circulating rare cells such as CTCs in various body fluids for different tumor classifications, which can avoid the limitations of high sampling requirements, certain subjectivity and heterogeneity in the IHC detection of tumor patient tissues, and the uneven detection accuracy of each center affected by factors such as the quality of detection antibodies and the detection process (fixation, staining);

[0033] (7) The immunological enrichment of circulating rare cells CTCs by the microfluidic chip of the present invention can detect and diagnose the benign and malignant nature of early masses and the expression of different proteins of tumor markers earlier than morphological imaging FDG-PET / CT, PET / MRI, and nuclear-labeled imaging PET / CT, and can provide early intervention for the clinical immunotherapy and protein-targeted therapy guidance of tumor patients;

[0034] (8) For tumor patients in the advanced and metastatic stages after surgery without available tissue, compared with the IHC pathological detection and identification that must be based on tumor tissue, the immunological enrichment of circulating rare cells CTCs by the microfluidic chip of the present invention can non-invasively, early, and sensitively detect and analyze the number and subtype changes of CTCs in the peripheral blood of patients, so as to accurately perform non-invasive clinical auxiliary dynamic detection on tumor patients in the postoperative recurrence and metastasis stage, and be used for immunotherapy guidance of pan-cancer, immunotherapy efficacy monitoring, and prognosis evaluation. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 is a schematic cross-sectional structure design diagram of the microfluidic chip of Embodiment 1 of the present invention;

[0036] Figure 2 is a schematic diagram of the structure of the inlet or outlet part of the microchannel of the microfluidic chip of Embodiment 1 of the present invention;

[0037] Figure 3 is a schematic diagram of the structure of the microfluidic groove array unit of Embodiment 1 of the present invention;

[0038] Figure 4 It is a schematic structural diagram of the W-shaped microfluidic groove in Embodiment 1 of the present invention;

[0039] Figure 5 It is a bar chart of the capture rate of the microfluidic chip in Embodiment 1 of the present invention for MCF7 and PC9 cell lines;

[0040] Figure 6 It is a bar chart of the capture rate and enrichment purity of the microfluidic chip in Embodiment 1 of the present invention for PC-9 and SK-BR-3 quality control cell lines respectively. Detailed implementation manners

[0041] To more clearly illustrate the embodiments of the present invention, the following will describe the specific implementation manners of the present invention with reference to the accompanying drawings. Obviously, the accompanying drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can be obtained according to these drawings, and other implementation manners can be obtained.

[0042] Embodiment 1:

[0043] As Figure 1 shown, the microfluidic chip for capturing circulating tumor cells in this embodiment includes a PDMS substrate 1 and a glass slide 2 loaded with probes that are bonded to each other.

[0044] Specifically, the inner surface of the PDMS substrate 1 has a microchannel, and the microchannel and the inner surface of the glass slide form a microfluidic channel. As Figure 2 shown, the microchannel includes an inlet channel, an array of lanes, and an outlet channel. The inlet channel is a tree-like structure, including a main branch channel I, a secondary branch channel II, and a lane connection channel III that are sequentially connected along the inlet flow direction. The inlet of the main branch channel I is connected to the inlet IV of the PDMS substrate, and the axis of symmetry of the inlet channel coincides with the axial axis of symmetry of the array of lanes. Among them, the main branch channel I, the secondary branch channel II, and the lane connection channel III are all arc-shaped channel structures, which are beneficial to ensuring the integrity of the morphology of CTC live cells and preventing cell rupture.

[0045] The array of lanes in this embodiment includes eight lanes V arranged in parallel at equal intervals, and the inlets and outlets of each lane are respectively connected to the inlet channel and the outlet channel.

[0046] The following will describe the structure of the lanes in detail:

[0047] In this embodiment, several microfluidic groove array units are arranged at equal intervals along the axial direction of the lane. As Figure 3As shown, the microfluidic groove array unit includes a first microfluidic groove array a, a second microfluidic groove array b, a third microfluidic groove array c, a fourth microfluidic groove array d, a fifth microfluidic groove array e, and a sixth microfluidic groove array f that are sequentially distributed at equal intervals along the axial direction of the flow channel.

[0048] As Figure 4 shown, the first microfluidic groove array is formed by arranging L W-shaped microfluidic grooves at equal intervals along the axial direction of the flow channel. The value of L is 10, which is specifically determined according to actual application requirements. The symmetry axis of the W-shaped microfluidic groove coincides with the symmetry axis of the axial direction of the flow channel. The W opening of the W-shaped microfluidic groove faces the liquid inlet of the flow channel, and the transverse dimension of the W-shaped microfluidic groove is equal to the transverse dimension D of the flow channel;

[0049] The structure of the second microfluidic groove array b takes the series-connected double W-shaped microfluidic groove as the offset main body and is obtained by offsetting the first microfluidic groove array a by D / 6 in the transverse direction of the flow channel, that is, only retaining the part of the double W-shaped microfluidic groove covered by the transverse dimension of the flow channel.

[0050] And so on, the structure of the third microfluidic groove array c takes the series-connected double W-shaped microfluidic groove as the offset main body and is obtained by offsetting the first microfluidic groove array a by D / 3 in the transverse direction of the flow channel; the structure of the fourth microfluidic groove array d takes the series-connected double W-shaped microfluidic groove as the offset main body and is obtained by offsetting the first microfluidic groove array a by D / 2 in the transverse direction of the flow channel; the structure of the fifth microfluidic groove array e takes the series-connected double W-shaped microfluidic groove as the offset main body and is obtained by offsetting the first microfluidic groove array a by 2D / 3 in the transverse direction of the flow channel; the structure of the sixth microfluidic groove array f takes the series-connected double W-shaped microfluidic groove as the offset main body and is obtained by offsetting the first microfluidic groove array a by 5D / 6 in the transverse direction of the flow channel. Among them, the above offsets are all offset in the same direction.

[0051] In addition, the spacing between adjacent microfluidic groove array units is the same as the spacing between adjacent microfluidic grooves within the same microfluidic groove array.

[0052] The micro-hydrodynamics adjustment formula for the microfluidic chip design of this embodiment is:

[0053] Tc = UEμ / △P 2 d

[0054] where U is the cell flow rate, E is the cell elastic modulus, μ is the suspension flow rate, △P 2 is the capillary driving pressure (i.e., the driving force for driving the cell suspension to flow), and d is the outer diameter of the necking (i.e., the outer diameter of the main branch flow channel I). Adjust the structural design of the flow channel according to the above micro-hydrodynamics adjustment formula to improve the cell capture rate and enrichment purity.

[0055] The inner surface of the slide 2 in this embodiment is loaded with capture probes corresponding to the microfluidic channels for capturing CTC cells. Specifically, the capture probes include silane agents, bifunctional crosslinkers, streptavidin, and also include other biotinylated tumor key marker antibodies, such as biotinylated EpCAM antibody, biotinylated CSV antibody, and VIM antibody.

[0056] The microfluidic chip of this embodiment, with the design of smooth liquid inlet and outlet channels and well - arranged microfluidic grooves in the swimming lanes, realizes the elimination of regions with extremely low shear force of microfluidics by adjusting microfluidics; at the same time, through the design of cascade signal amplification reaction modified by specific bifunctional groups, the microfluidic chip shows high capture efficiency and CTC enrichment purity in the separation and enrichment of CTCs in breast cancer, lung cancer, prostate cancer, and colorectal cancer. Moreover, through the coupling design of bifunctional groups, the controllable release of enriched circulating rare cells can be realized simultaneously, so as to flexibly dock with the detection of targeted driving genes of downstream CTC single cells. In addition, biotinylated epithelial marker EPCAM is coupled with mesenchymal tumor marker CSV, thus realizing the efficient enrichment of three subtypes of epithelial CTCs, mesenchymal CTCs, and epithelial - mesenchymal transition CTCs at one time.

[0057] This embodiment performs functional modification on the basis of a microfluidic chip, combines biotinylated tumor key marker antibodies to form a positive microfluidic separation and enrichment system for separating and capturing CTCs from peripheral blood circulating tumor cells; uses detection antibodies labeled with different fluorescent groups for in - situ immunofluorescence hybridization detection of CTC cells enriched by the microfluidic chip, and uses an automated fluorescence system for scanning and interpretation analysis of the captured and detected CTC cells.

[0058] In addition, the preparation method of the microfluidic chip for capturing circulating tumor cells in this embodiment includes the following steps:

[0059] S1. Spin - coat photoresist on the silicon wafer after plasma cleaning, and after heating and drying, perform exposure, heating, and development in sequence to obtain a photoresist nano - array; then pour PDMS glue based on the photoresist nano - array, punch holes and cut the glue, and then perform plasma cleaning and bonding to obtain a PDMS substrate; the specific production process includes:

[0060] 1. Plasma: Place a clean silicon wafer in a plasma cleaner for treatment;

[0061] 2. Spin - coating: Place the treated silicon wafer in the center of the spin - coater platform, pour SU - 8 photoresist for spin - coating treatment;

[0062] 3. Heating: Place it on a heater for heating treatment according to the required thickness of the glue;

[0063] 4. Exposure: Place the heated film on an ultraviolet lithography machine for exposure processing;

[0064] 5. Heating (hard baking): Place the lithography film on a heating machine for heating treatment;

[0065] 6. Cleaning (development, degluing): Place the film in a cleaning agent, shake it, quickly dry it with compressed air, and then heat it on a heating machine;

[0066] 7. Silanization treatment: Treat the silica gel sheet with a plasma cleaner, then place it in a vacuum dryer, drop a few drops of silanization reagent, evacuate to the maximum scale of the pressure gauge, tighten the air extraction valve, and turn off the air extraction pump. After silanization treatment, place the lithography film in a petri dish of appropriate size;

[0067] 8. First pouring of PDMS: Mix the prepared PDMS glue evenly, remove air bubbles by pumping, pour a small amount onto the silicon wafer, place it flat, remove bubbles with a blowing device, heat it, and then carefully lift it with flat-tip tweezers;

[0068] 9. Second pouring of PDMS: Pour the prepared PDMS glue onto the lithography resist silicon wafer to an appropriate thickness, place it flat, defoam, and heat;

[0069] 10. Cutting the glue: Punch holes;

[0070] 11. Plasma: Perform plasma cleaning treatment on the clean channel film and non-channel film;

[0071] 12. Bonding: Press it tightly to avoid air bubbles, and bake for bonding.

[0072] S2. Functional modification of the microfluidic chip.

[0073] Specifically, perform silanization treatment on the surface of the glass slide after oxygen plasma treatment with 3-aminopropyltriethoxysilane, and then sequentially combine a bifunctional amine-thiol crosslinker and streptavidin (SA), and finally combine a biotin-labeled antibody; the specific modification process is as follows:

[0074] 1. Oxygen plasma treatment of the glass slide,

[0075] 2. Then bond the glass slide with the PDMS substrate to obtain a microfluidic chip;

[0076] 2. Silane surface treatment: React 3-aminopropyltriethoxysilane (ATPS) in acetone at room temperature, and rinse with acetone to remove the unreacted ATPS;

[0077] 3. Combine the bifunctional amine-thiol crosslinker (SMCC) with 3-aminopropyltriethoxysilane: Incubate the bifunctional amine-thiol crosslinker in a physiological solution at room temperature, and then rinse with the physiological solution;

[0078] 4. Combine streptavidin (SA) with a bifunctional amine-thiol crosslinker: Coat with streptavidin in phosphate buffer to bind SA to the bifunctional amine-thiol crosslinker;

[0079] 5. The functionalized glass slide is dehydrated with absolute ethanol and then air-dried naturally;

[0080] 6. Bonding: After oxygen plasma treatment of the PDMS substrate of the microfluidic channel and the functionalized glass slide loaded with probes, bond the non-microfluidic channel edge regions around the perimeter.

[0081] 7. Binding of biotin-labeled monoclonal antibody (i.e., biotinylated monoclonal antibody): Introduce the biotinylated monoclonal antibody into the microfluidic channel to obtain a microfluidic chip modified with biotin-capturing antibody. Additionally, block the regions covered by the non-biotinylated monoclonal antibody with 5% bovine serum albumin to reduce non-specific cell capture.

[0082] S3. Combine tumor key marker antibodies on the basis of the microfluidic chip modified with functional groups.

[0083] Specifically, on the basis of the functionalized microfluidic chip, combine biotinylated tumor key marker antibodies to form a forward microfluidic separation and enrichment system for separating and capturing circulating tumor cells (CTCs) from peripheral blood. The specific process is as follows:

[0084] 1. Coating and blocking of biotinylated capture antibody and tumor key markers on the microfluidic chip;

[0085] 2. Isolation of peripheral blood mononuclear cells (PBMCs) from patients;

[0086] 3. Enrichment and capture of CTCs in the microfluidic nanochip;

[0087] 4. Fluorescence in situ hybridization with detection antibodies labeled with different fluorophores for the CTCs enriched and captured on the microfluidic chip;

[0088] 5. Hoechst33342 active nuclear staining of the CTCs enriched and captured;

[0089] 6. Scanning and interpretation analysis of the detected CTCs with an automated fluorescence system.

[0090] The following tests the capture rates of the MCF7 and PC9 cell lines for the microfluidic chip of this example, as Figure 5 shown, the capture rate of the MCF7 cell line reaches 96.59%, and the capture rate of the PC9 cell line reaches 98.26%.

[0091] As Figure 6As shown, the capture rates of the microfluidic chip in this embodiment for the PC-9 and SK-BR-3 quality control cell lines reach 100% and 99.82% respectively, and the enrichment purities reach 81.59% and 83.26% respectively.

[0092] Example 2:

[0093] The difference between the microfluidic chip in this embodiment and that in Example 1 lies in:

[0094] There are six lanes; correspondingly, the structure of the liquid inlet channel can be adjusted adaptively;

[0095] Other structures are the same as those in Example 1.

[0096] Example 3:

[0097] The difference between the microfluidic chip in this embodiment and that in Example 1 lies in:

[0098] The value of L is 20;

[0099] Other structures are the same as those in Example 1.

[0100] Example 4:

[0101] The difference between the microfluidic chip in this embodiment and that in Example 1 lies in:

[0102] The value of L is 3;

[0103] Other structures are the same as those in Example 1.

[0104] Comparative Example 1:

[0105] The difference between the microfluidic chip in this comparative example and that in Example 1 lies in:

[0106] The microfluidic groove array unit in the lane is only a single first microfluidic groove array;

[0107] Other structures are the same as those in Example 1.

[0108] Comparative Example 2:

[0109] The difference between the microfluidic chip in this comparative example and that in Example 1 lies in:

[0110] The microfluidic groove array unit in the lane is only a single second microfluidic groove array;

[0111] Other structures are the same as those in Example 1.

[0112] Comparative Example 3:

[0113] The difference between the microfluidic chip in this comparative example and that in Example 1 lies in:

[0114] The microfluidic groove array unit of the lane is only a single third microfluidic groove array;

[0115] Other structures are the same as those in Embodiment 1.

[0116] Comparative Example 4:

[0117] The difference between the microfluidic chip of this comparative example and that of Embodiment 1 lies in:

[0118] The microfluidic groove array unit of the lane is only a single fourth microfluidic groove array;

[0119] Other structures are the same as those in Embodiment 1.

[0120] Comparative Example 5:

[0121] The difference between the microfluidic chip of this comparative example and that of Embodiment 1 lies in:

[0122] The microfluidic groove array unit of the lane is only a single fifth microfluidic groove array;

[0123] Other structures are the same as those in Embodiment 1.

[0124] Comparative Example 6:

[0125] The difference between the microfluidic chip of this comparative example and that of Embodiment 1 lies in:

[0126] The microfluidic groove array unit of the lane is only a single sixth microfluidic groove array;

[0127] Other structures are the same as those in Embodiment 1.

[0128] Comparative Example 7:

[0129] The difference between the microfluidic chip of this comparative example and that of Embodiment 1 lies in:

[0130] The microfluidic groove array unit of the lane is only a combination of the first microfluidic groove array to the third microfluidic groove array;

[0131] Other structures are the same as those in Embodiment 1.

[0132] Comparative Example 8:

[0133] The difference between the microfluidic chip of this comparative example and that of Comparative Example 7 lies in:

[0134] The microfluidic groove array unit of the lane is only a combination of the first microfluidic groove array to the third microfluidic groove array, and the offset of the second microfluidic groove array is D / 3, and the offset of the third microfluidic groove array is 2D / 3;

[0135] Other structures are the same as those in Embodiment 1.

[0136] Comparative Example 9:

[0137] The microfluidic chip of this comparative example is different from that of Comparative Example 7 in that:

[0138] The microfluidic groove array unit of the lane is only a combination of the first microfluidic groove array to the second microfluidic groove array, and the offset of the second microfluidic groove array is D / 2;

[0139] Other structures are the same as those in Example 1.

[0140] The capture rate and enrichment purity of PC9 cells of the microfluidic chips of the above-mentioned Example 1 and Comparative Examples 1-9 were tested, and the results are shown in Table 1.

[0141] Table 1 Capture rate and enrichment purity of PC9 cells by the microfluidic chips of Example 1 and Comparative Examples 1-9

[0142] Test group Capture rate (%) Enrichment purity (%) Example 1 100％ 81.59 Example 2 97.13 81.25 Example 3 97.56 72.36 Example 4 97.39 68.92 Comparative example 1 92.49 36.75 Comparative example 2 91.92 38.56 Comparative example 3 93.67 39.61 Comparative example 4 90.73 32.54 Comparative example 5 92.13 33.69 Comparative example 6 94.39 36.25 Comparative example 7 96.27 51.32 Comparative example 8 96.13 46.34 Comparative example 9 95.91 42.35

[0143] As can be seen from the above, the lane structure of the present invention changes the problem of high interception rate of white blood cell microfluidic chips caused by uneven shear force distribution in conventional typical microfluidic chips. The capture rate of CTC cells ≥ 95% and the enrichment purity of CTC cells ≥ 80%.

[0144] In view of the large number of embodiments of the present invention and the huge amount of experimental data for each embodiment, it is not suitable to list them one by one here. Only Examples 1 and 2 are used for illustration, but the content to be verified and the final conclusions obtained for each embodiment all meet the capture rate of CTC cells ≥ 95% and the enrichment purity of CTC cells ≥ 80%.

[0145] The above is only a detailed description of the preferred embodiments and principles of the present invention. For those of ordinary skill in the art, according to the idea provided by the present invention, there will be changes in the specific implementation manners, and these changes should also be regarded as the protection scope of the present invention.

Claims

1. A microfluidic chip for capturing circulating tumor cells, comprising a PDMS substrate and a glass slide bonded to each other; the inner surface of the PDMS substrate has microchannels, and the microchannels and the inner surface of the glass slide form a microfluidic channel, characterized in that, The microchannel includes a liquid inlet channel, an array of swimming lanes, and a liquid outlet channel. The array of swimming lanes includes N swimming lanes arranged in parallel at equal intervals. The inlets and outlets of each swimming lane are respectively connected to the liquid inlet channel and the liquid outlet channel. Herein, N is an integer; Several microfluidic groove array units are equally spaced along the axial direction of the swimming lane. The microfluidic groove array unit includes a first microfluidic groove array to a sixth microfluidic groove array arranged at equal intervals along the axial direction of the swimming lane. The first microfluidic groove array is formed by arranging L W-shaped microfluidic grooves at equal intervals along the axial direction of the swimming lane. The axis of symmetry of the W-shaped microfluidic groove coincides with the axis of symmetry of the axial direction of the swimming lane. The W opening of the W-shaped microfluidic groove faces the liquid inlet of the swimming lane. The lateral dimension of the W-shaped microfluidic groove is equal to the lateral dimension of the swimming lane; The structure of the i-th microfluidic groove array is based on the tandem double W-shaped microfluidic groove as the offset main body, and is obtained by offsetting the first microfluidic groove array by the i-th preset dimension along the lateral direction of the swimming lane, and the offset directions are the same. Herein, the i-th preset dimension is i*D / 6, where i is an integer from 2 to 6, and D is the lateral dimension of the swimming lane; The inner surface of the glass slide is loaded with capture probes corresponding to the microfluidic channel; The liquid inlet channel has a tree root-like structure, including a main branch channel, a secondary branch channel, and a swimming lane connection channel connected in sequence along the liquid inlet direction. The inlet of the main branch channel is connected to the liquid inlet of the PDMS substrate; The main branch channel, the secondary branch channel, and the swimming lane connection channel are all arc-shaped channel structures; The capture probe includes a bifunctional crosslinking agent, streptavidin, and a biotinylated monoclonal antibody, which are coupled in sequence.

2. The microfluidic chip for capturing circulating tumor cells according to claim 1, wherein, The axis of symmetry of the liquid inlet channel coincides with the axis of symmetry of the axial direction of the array of swimming lanes.

3. The microfluidic chip for capturing circulating tumor cells according to claim 1, wherein The capture probe further includes a biotinylated tumor key marker antibody.

4. The microfluidic chip for capturing circulating tumor cells according to claim 3, wherein The biotinylated tumor key marker antibody includes at least one of a biotinylated EpCAM antibody, a biotinylated CSV antibody, or a biotinylated VIM antibody.

5. The microfluidic chip for capturing circulating tumor cells according to any one of claims 1-2, characterized in that The value of N is an even number from 4 to 10, and the value of L is from 8 to 15.

6. The microfluidic chip for capturing circulating tumor cells according to any one of claims 1-2, characterized in that, The capture rate of circulating tumor cells ≥ 95%, and the enrichment purity of circulating tumor cells ≥ 80%.

7. The preparation method of the microfluidic chip according to any one of claims 1-6, characterized in that, It includes the following steps: S1. Spin-coat photoresist on the silicon wafer after plasma cleaning. After heating and drying, perform exposure, heating, and development in sequence to obtain a photoresist nanoarray; Then, pour PDMS glue based on the photoresist nanoarray, punch holes and cut the glue, perform plasma cleaning, and bond to obtain a PDMS substrate; S2. Perform sequential coupling and loading of the capture probe components - bifunctional crosslinking agent, streptavidin, and biotinylated monoclonal antibody on the glass slide after oxygen plasma treatment; S3. Bond the PDMS microfluidic substrate fabricated in S1 with the probe-functionalized glass slide in S2.

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