Microfluidic chip, system, and method for single-cell extracellular vesicle isolation and analysis

Abstract

A microfluidic control chip, system, and method for single-cell extracellular vesicle isolation and analysis are disclosed. The microfluidic control chip includes a flow layer and a control layer. The flow layer includes a sample / air inlet, a waste liquid outlet, a micron-sized trapping channel containing contraction and expansion structures, a micron-sized gas flow channel, a micron-sized single-cell trapping structure, and a micron-sized single microsphere trapping structure. The control layer includes an air inlet, a micron-sized gas flow channel, and a micron-sized protrusion structure. A microfluidic control chip imaging system for single-cell extracellular vesicle isolation and analysis is also disclosed, comprising the microfluidic control chip, a micro-injection pump, a pressure controller, a capillary tube, a PFA tube, and a fluorescence microscope. The microfluidic control chip of this invention can simultaneously analyze biomarkers expressed on the surface of single cells and single-cell secretory extracellular vesicles. Correlation analysis of cancer cell drug resistance with the expression levels of biomarker proteins on the surface of cells and cell secretory extracellular vesicles is achieved at single-cell resolution.

Description

Technical Field

[0001] This invention belongs to the fields of microfluidic chips, cell analysis and analysis technology, and particularly relates to a microfluidic control chip, system and method for the separation and analysis of single-cell extracellular vesicles. Background Technology

[0002] Extracellular vesicles are nanoscale (30-200 nm) vesicles secreted by cells into the extracellular environment. Almost all cell types, including normal and cancer cells, can secrete extracellular vesicles, and the number of extracellular vesicles secreted by cancer cells is significantly increased compared to normal cells in various types of cancer. Extracellular vesicles are potential biomarkers with advantages such as high abundance in body fluids, carrying multiple biomarkers reflecting the state of their source cells, and their liposome-like membrane structure maintaining vesicle integrity. Currently, extracellular vesicles have been shown to play a crucial role in intercellular communication, disease diagnosis, treatment, and prognosis. In particular, the differential expression of multiple biomarker proteins carried by extracellular vesicles secreted by cancer cells has been shown to be associated with cancer occurrence and metastasis. However, due to the small size and high heterogeneity of extracellular vesicles, their separation and detection face significant challenges. Microfluidic chips, as a technology capable of precisely manipulating micron- or even nanometer-sized particles, offer advantages such as efficient single-particle separation, high-throughput analysis, integration of multiple analytical steps, and analysis of small / micro-volume samples. Therefore, it plays a crucial role in single-cell analysis, extracellular vesicle sorting, drug screening, and evaluation. Single-cell analysis can reveal heterogeneous information that cannot be obtained from population analysis, further advancing our understanding of cancer occurrence, development, and prognosis. Furthermore, analyzing extracellular vesicles secreted by different individual cells from the same / different cancer types is of great significance for studying cellular drug resistance, phenotypic analysis, subgroup analysis, and personalized treatment.

[0003] Breast cancer is the most common malignant tumor (cancer) in women. With the widespread adoption of screening technologies, the incidence of breast cancer continues to rise. Breast cancer is generally classified into triple-negative breast cancer, human epidermal growth factor (+, HER2) breast cancer, and luminal breast cancer. Triple-negative breast cancer refers to a subtype of breast cancer that is negative for estrogen receptor (ER), progesterone receptor (PR), and HER2. HER2-positive breast cancer refers to a subtype of breast cancer with high expression of the HER2 gene. Luminal breast cancer can be further classified into luminal A and luminal B types based on differences in ER, PR, and HER2 expression levels. Therefore, HER2 protein, as an important biomarker, plays a crucial role in the diagnosis and treatment of breast cancer. Studies have shown that overexpression of HER2 protein leads to uncontrolled growth and increased invasiveness of cancer cells, thereby increasing the likelihood of recurrence and metastasis. Therefore, by detecting the expression level of HER2 protein, the specific breast cancer subtype can be identified, ultimately allowing for personalized treatment for patients. Especially for HER2-positive patients, the primary treatment method is targeted drug therapy. Trastuzumab, a humanized monoclonal antibody, is one of the commonly used targeted drugs in clinical practice. It can inhibit further tumor growth and reduce the possibility of cancer cell metastasis by controlling the expression of the HER2 gene in cells. Therefore, analyzing the expression level of HER2 in breast cancer cells is particularly important. However, most of the reported methods currently available study the HER2 expression level of breast cancer cells in populations and their resistance to trastuzumab. Studies on the drug resistance of individual breast cancer cells are scarce, especially those based on the analysis of exovesicles secreted by single cells to study the drug resistance of the cells of origin. Therefore, designing a microfluidic chip that can realize the separation and analysis of exovesicles in single cells and then evaluating the drug resistance of the cells of origin through exovesicle analysis is of great significance for the subtype classification and treatment prognosis of breast cancer.

[0004] Currently, methods for separating and analyzing single-cell secretory extracellular vesicles based on microfluidic chips mainly include micropore array microfluidic chips, pneumatic valve array microfluidic chips, and antibody barcode microfluidic chips. However, all of these detection methods have some problems in practical applications:

[0005] The microfluidic control chip with a microporous array involves dropwise addition of a tumor cell suspension onto the surface of a polydimethylsiloxane (PDMS) filter containing a microporous array, followed by centrifugation to capture multiple single cells. The PDMS filter is then removed, resulting in an ordered arrangement of the single cells. Single-cell extracellular vesicles are captured and analyzed by attaching and immobilizing a slide with modified capture antibodies to a culture dish containing the single-cell array. However, this method requires centrifugation and filter removal to achieve effective single-cell arrangement. The entire process demands high precision in controlling cell density, size, and viscosity, and the removal process may damage the already arranged cells, affecting subsequent single-cell analysis. Furthermore, the method requires close contact between the slide and the culture dish containing the arranged cells for cell culture and extracellular vesicle capture / analysis, placing high demands on the operator's skill. While this method can capture extracellular vesicles secreted by multiple single cells, it cannot guarantee against cross-contamination between vesicles secreted by individual cells.

[0006] The pneumatic valve array microfluidic control chip consists of a flow layer and a control layer. Tumor cells are introduced into the flow layer using an injection pump. Because a single-cell capture array structure is constructed within the flow layer, multiple single cells can be captured simultaneously. Subsequently, air is introduced into the control layer via a pressure controller to cause the PDMS film to bulge, effectively isolating single cells and preventing cross-contamination between adjacent cells. However, this method requires prolonged pressure control, placing high demands on the equipment. Furthermore, the control layer needs to remain in a bulging state for an extended period, posing significant challenges to the fabrication process of the microfluidic control chip. In addition, this method achieves single-cell extravesicular vesicle detection by modifying the control layer surface with antibodies that capture extravesicles, combined with immunolabeling. Therefore, there is a risk of loss of captured extravesicles after rinsing with phosphate-buffered saline (PBS), requiring precise experimental techniques. The high equipment and microfluidic control chip fabrication requirements limit the application of this method in single-cell extravesicular vesicle detection.

[0007] Antibody barcode microfluidic control chips typically combine single-cell chips with capture arrays and spatially encoded antibody barcode chips to achieve the separation and analysis of single-cell extracellular vesicles. Tumor cells are loaded onto the single-cell capture chip, ensuring that each microcavity contains only a single cell. Each microcavity is covered by the entire antibody barcode array, thus capturing extracellular vesicles secreted by single cells with different biomarker proteins from different antibodies. Finally, immunolabeling is used to obtain the secretion data of single-cell extracellular vesicles. This method has the advantage of simultaneously detecting multiple biomarkers; however, the high cost of antibodies leads to a high overall cost. Furthermore, the difficulty in modifying the antibody barcode array and the requirements for its integration with single-cell capture chips limit the universality of antibody barcode microfluidic control chips.

[0008] Most importantly, the aforementioned methods for separating and analyzing single-cell extravesicles based on microfluidic chips all require complex and challenging experimental procedures, which greatly limits their application in extravesicle analysis at single-cell resolution. Furthermore, they pose a significant challenge to achieving simultaneous analysis of single cells and extravesicles. Summary of the Invention

[0009] To address the problems existing in current microfluidic control chip-based single-cell extracellular vesicle separation and analysis techniques, this invention provides a simple-to-operate microfluidic control chip for single-cell extracellular vesicle separation. Combined with fabricated aptamer nanoprobes, it proposes a system capable of zero-cross-contamination analysis of single-cell extracellular vesicles and simultaneous analysis of single cells and extracellular vesicles. The core of this system is a bilayer microfluidic control chip consisting of a flow layer and a control layer. This microfluidic control chip is fabricated from easily manufactured and cost-effective PDMS material. A cell suspension obtained through trypsin digestion is introduced into the microfluidic control chip through the inlet of the flow layer using a syringe pump. Highly efficient single-cell capture is achieved based on a specially designed single-cell capture array structure. Subsequently, a solution of microspheres modified with capture aptamers is introduced into the microfluidic control chip through the inlet of the flow layer using the syringe pump. Individual microspheres are then captured by the specially constructed capture structure. Throughout these processes, air is introduced into the channels of the control layer via a pressure controller to cause the PDMS film to bulge, thereby achieving efficient capture of single cells and single microspheres. Since each capture unit contains only one capture structure for a single cell and one capture structure for a single microsphere, the matching process between the single cell and the single microsphere can be quickly completed in the two steps described above, without the need for additional complex steps. Subsequently, based on the differential flow resistance effect, air is introduced in reverse into the flow layer to form a single droplet within each capture unit, thereby achieving effective barrier between adjacent single cells. In-situ culture of single cells is achieved by constructing a cell culture method within the microfluidic chip. Effective capture of single-cell extracellular vesicles is achieved through microspheres modified with aptamers. Using aptamer nanoprobes, not only can the analysis of secreted extracellular vesicles by single cells be achieved, but also the simultaneous analysis of single-cell extracellular vesicles and single cells can be further realized. Using the microfluidic chip for capturing single-cell extracellular vesicles in conjunction with a fluorescence microscope enables analysis of single-cell extracellular vesicles with zero cross-contamination, simultaneous analysis of single cells and single-cell extracellular vesicles, and drug resistance analysis of single cells.

[0010] The specific technical solution adopted in this invention is as follows:

[0011] A microfluidic control chip for the separation and analysis of single-cell extracellular vesicles comprises an upper flow layer and a lower control layer. The flow layer includes three inlets (inlet I, inlet II, and inlet III) and a waste liquid outlet. The flow layer also includes a micrometer-scale trapping channel, a micrometer-scale single-cell trapping structure, a micrometer-scale single-microsphere trapping structure, and a micrometer-scale gas flow channel. The control layer includes one inlet (inlet IV) and a micrometer-scale gas flow channel.

[0012] In the flow layer, both inlet I and inlet II are connected to the inlet of the micron-level capture channel. The micron-level single-cell capture structure and the micron-level single microsphere capture structure are located within the micron-level capture channel. The micron-level capture channel and the micron-level gas flow channel are connected at the front and rear ends of each unit. The micron-level gas flow channel is connected to inlet III. In the control layer, inlet IV is connected to the inlet of the micron-level gas flow channel.

[0013] A microfluidic control chip for single-cell extracellular vesicle separation and analysis consists of 54 capture units. Each capture unit includes a micrometer-scale single-cell capture structure in the flow layer, a micrometer-scale single microsphere capture structure, a micrometer-scale capture channel, a micrometer-scale gas flow channel (the micrometer-scale capture channel in each unit is connected to the micrometer-scale gas flow channel before and after it), and a micrometer-scale gas flow channel in the control layer.

[0014] Inlet I is used to introduce cell suspension, and inlet II is used to introduce microsphere suspension. After entering the micron-scale capture channel, cells and microspheres are captured by single-cell capture structures and single-microsphere capture structures, respectively. When the cell suspension is introduced at a flow rate of 80 μL / min, the probability of a single-cell capture event is 93.1%. When the microsphere suspension is introduced at a flow rate of 50 μL / min, the probability of simultaneous capture of single cells and single microspheres is 75.5%. Figure 6 After air is introduced from inlet III at a pressure of 40 mbar, a single droplet is formed in each capture unit, effectively isolating adjacent single cells and providing a basis for subsequent separation of single-cell extracellular vesicles. Throughout the process, the fluid shear forces experienced by single cells and individual microspheres are low, and the fluid conditions are ultra-mild, which does not affect the normal growth of single cells or the aptamers modified on the surface of microspheres.

[0015] To avoid fluid diversion effects that could affect capture efficiency, air is introduced into the control layer at a pressure of 1000 mbar from inlet IV during single-cell and single-microsphere capture operations to reduce the impact of fluid diversion. During single-cell culture, the different channel widths at both ends of each unit cause fluid to flow from the narrower channel to the wider channel. That is, fluid carrying single-cell extracellular vesicles flows towards the location of the individual microspheres, where it is recognized and captured by the aptamers modified on the microsphere surface. Figure 3 The gray arrow drawn within 27 indicates the direction of fluid flow.

[0016] The reference dimensions of each part of the microfluidic control chip are as follows:

[0017] The normal width of the micron-scale capture channel (normal width refers to the width when there are no contraction or expansion structures in the channel) is 100–200 μm, and the total length is 10.5–25.5 cm. The micron-scale single microsphere capture structure and the micron-scale single cell capture structure are designed according to the size of the microsphere and the cell, and both are composed of two mirror-arranged PDMS micropillars. Because the slit width between the micropillars is smaller than the size of the microspheres / cells, while the diameter of the built-in grooves is similar to the size of a single microsphere / cell, a single microsphere / cell can be captured by the trapping structure. The length of the micron-scale single microsphere trapping structure is 50–100 μm, the width is 20–55 μm, the radius of the built-in groove is 20–30 μm, and the width of the built-in slit is 20–30 μm. The length of the micron-scale single-cell trapping structure is 15–36 μm, the width is 10–20 μm, the radius of the built-in groove is 5–10 μm, and the width of the built-in slit is 4–6 μm. The distance between the micron-scale single-cell trapping structure and the micron-scale single microsphere trapping structure is 150–200 μm. Figure 5 ).

[0018] An expansion and contraction structure is incorporated within the micrometer-scale capture channel. Expansion structure I has a length of 150–400 μm and a width of 220–280 μm; expansion structure II has a length of 180–650 μm and a width of 500–600 μm; contraction structure I has a length of 100–250 μm and a width of 150–200 μm; contraction structure II has a length of 550–750 μm and a width of 50–250 μm. The width of the connection between contraction structure II and the micrometer-scale gas flow channel is 25–75 μm. The distance between expansion structure I and contraction structure I is 350–400 μm; the distance between contraction structure I and expansion structure II is 100–120 μm; expansion structure II and contraction structure II are connected. The arrangement order of these four expansion and contraction structures is expansion structure I, contraction structure I, expansion structure II, and contraction structure II, respectively. In each capture unit, there are a total of 54 expansion structures I, contraction structures I, expansion structures II, and contraction structures II within the micron-scale capture channel.

[0019] The length of the micron-level gas flow channels in the flow layer is 26–35 cm, and the width is 100–200 μm. The normal width of the micron-level gas flow channels in the control layer is 350–400 μm, and the length is 8–13 cm, arranged in three rows. The control layer gas flow channels also include 108 protrusion structures, each with a length of 450–900 μm and a width of 150–300 μm. The entire microfluidic control chip consists of 54 structural units arranged in 6 rows. Each unit comprises an expansion structure I, a contraction structure I, an expansion structure II, a contraction structure II, a micron-level single-microsphere trapping structure, a micron-level single-cell trapping structure, two protrusion structures in the control layer, a 0.25–0.50 cm long micron-level trapping channel a, a 0.35–0.75 cm long flow layer micron-level gas flow channel b, and a 0.25–0.50 cm long control layer micron-level gas flow channel c. Figure 3 ).

[0020] In the aforementioned microfluidic control chip, the height of each channel in the flow layer, such as the micron-level capture channel, the micron-level single-cell capture structure, and the micron-level gas flow channel, is 30–70 μm; the height of all channels in the control layer is 30–70 μm.

[0021] The microfluidic control chip has an area of ​​1.27 cm². 2 ~10.50cm 2 , to support the above structure.

[0022] The present invention also includes a microfluidic control chip imaging system for single-cell extracellular vesicle separation and analysis, which, in addition to the horizontally placed microfluidic control chip mentioned above, also includes a microinjection pump a, a microinjection pump b, a pressure controller a, a pressure controller b, a capillary tube a, a capillary tube b, a capillary tube c, a PFA tube a, a PFA tube b, and a fluorescence microscope.

[0023] The microinjection pump a is connected to the inlet I of the microfluidic control chip via capillary tube a, which serves as the inlet channel for cell suspension and matrix gel; the microinjection pump b is connected to the inlet II of the microfluidic control chip via capillary tube b, which serves as the inlet channel for microsphere solution; the pressure controller a is connected to the inlet III of the microfluidic control chip via PFA tube a, which serves as the air inlet channel; the pressure controller b is connected to the inlet IV of the microfluidic control chip via PFA tube b, which serves as the air inlet channel.

[0024] The capillary tube c serves as a waste liquid channel, with one end connected to the waste liquid outlet of the microfluidic control chip and the other end used for waste liquid discharge.

[0025] Microfluidic control chips, combined with aptamer probes, enable the detection of biomarkers on the surface of single-cell extracellular vesicles using fluorescence microscopy.

[0026] This invention provides a method for zero-cross-contamination analysis of single-cell extracellular vesicles using the aforementioned system, specifically comprising the following steps:

[0027] Step 1, Preparation of Matrix Gel: A matrix gel for cell adhesion and growth was prepared using Collagen, Matrigel, and Fibrican. The matrix gel was supplied by a microinjection pump a and connected to the microfluidic control chip inlet I via capillary tubing a.

[0028] Step 2, cell sample preparation: The cells to be tested are prepared into a cell suspension by means of trypsin digestion, and then filtered through a cell filter sieve.

[0029] Step 3, Preparation of microsphere samples: The aptamer used to capture the outer vesicles is attached to the surface of the microspheres through methods such as EDC-NHS reaction, and a microsphere suspension is prepared.

[0030] Step 4, Sample preparation: Cell suspension is supplied by microinjection pump a and connected to inlet I of the microfluidic control chip through capillary tube a; microsphere suspension is supplied by microinjection pump b and connected to inlet II of the microfluidic control chip through capillary tube b; air is supplied by pressure controller b and connected to inlet IV of the microfluidic control chip through PFA tube b.

[0031] Step 5, Capture of Single Cells and Microspheres: Air is introduced into the micron-level gas flow channels of the control layer via pressure controller b and PFA tube b, causing the PDMS film to bulge. Cell suspensions are introduced into the micron-level capture channels of the microfluidic control chip via microinjection pump a and capillary tube a, and microsphere suspensions are introduced into the micron-level capture channels of the microfluidic control chip via microinjection pump b and capillary tube b. Cells and microspheres will be captured by the micron-level single-cell capture structures and micron-level single-microsphere capture structures within the micron-level capture channels.

[0032] Step 6, Droplet Generation: Air is supplied by pressure controller a and connected to the microfluidic control chip inlet III via PFA tube a; air is introduced into the micron-level gas flow channel of the flow layer through pressure controller a and PFA tube a. Since the width of the micron-level gas flow channel is wider than the width of the contraction structure II, the gas will preferentially flow into the micron-level gas flow channel, thus forming droplets.

[0033] Step 7, Single-cell culture and capture of external vesicles: The droplet microfluidic control chip containing a single cell and a single microsphere is placed in an incubator for culture, and the external vesicles secreted by the single cell are captured by an aptamer modified on the surface of the microsphere.

[0034] Step 8, Labeling of single cells and single-cell extracellular vesicles: The prepared fluorescent nanoprobe solution is introduced into the microfluidic control chip to label single cells and single-cell secretory extracellular vesicles.

[0035] Step 9, Fluorescence Imaging Analysis of Single Cells and Single-Cell Extracellular Vesicles: Microfluidic control chips containing fluorescently labeled single cells and single microspheres are placed on the stage of a fluorescence microscope for observation, thereby obtaining the expression information of cancer-related biomarker proteins on the surface of single cells and single-cell extracellular vesicles.

[0036] If acridine orange (AO) is used to stain cells in the above analytical detection method, it can also be used to analyze cell drug resistance. After completing step 9 above, cells can be cultured in a specific anticancer drug for a period of time; then, single cells are stained and fluorescently imaged using AO, and the strength of drug resistance of single cells is analyzed by the color and brightness of the fluorescence.

[0037] In step 4, the injection flow rate of the cell suspension into the microfluidic control chip is 10–100 μL / min, and the injection flow rate of the microsphere solution into the microfluidic control chip is 20–120 μL / min; the pressure of air entering the control layer is 800–1500 mbar.

[0038] In step 6, the pressure in the micron-level gas flow channel through which air enters the flow layer is 10–150 mbar.

[0039] Preferably, in step 9, the specific detection method is as follows: capture and photograph the fluorescence on the surface of single cells and single microspheres in dark field mode, and extract the fluorescence intensity using ImageJ software; finally, analyze the expression of different biomarkers on the surface of single cells and single-cell secretory vesicles based on the fluorescence intensity values.

[0040] Compared with the prior art, the advantages of the present invention are:

[0041] 1. Compared with microfluidic control chips with micropore arrays, it can achieve effective arrangement of single cells without complex filtration steps, and the whole process causes less damage to cells;

[0042] 2. Compared with pneumatic valve array microfluidic control chips, it can achieve zero cross-contamination capture and analysis of single-cell extracellular vesicles without the need for long-term pressure control operation, and the operation is simple;

[0043] 3. Compared with antibody barcode microfluidic control chips, the microfluidic control chip in this invention is easier to modify to capture aptamers and has better stability, while having a relatively lower cost;

[0044] 4. Compared with the above three types of microfluidic control chips, the microfluidic control chip of the present invention does not require a complicated two-layer alignment step (the microfluidic control chip of the present invention has a larger channel size and is easier to align); and no additional assembly operation is required during the entire operation.

[0045] 5. The microfluidic control chip of the present invention is equipped with a micron-level gas flow channel, and the air is introduced into the inlet Ⅲ by the pressure controller a. It can achieve single-cell extracellular vesicle capture and analysis with zero cross-contamination without long-term pressure control operation.

[0046] 6. This invention uses microspheres modified with aptamers to capture external vesicles, which is more stable than aptamers / antibodies modified on the surface of PDMS;

[0047] 7. The microfluidic control chip of this invention, possessing both micrometer-scale single-cell capture structures and micrometer-scale single-microsphere capture structures, and incorporating fluorescent probes, can simultaneously analyze biomarkers expressed on the surface of single cells and single-cell secretory vesicles. It enables correlation analysis of cancer cell drug resistance with the expression levels of biomarker proteins on the surface of cells and cell secretory vesicles at single-cell resolution. Attached Figure Description

[0048] Figure 1 A schematic diagram of the microfluidic control chip for single-cell extracellular vesicle separation and analysis of the present invention; (a) is a schematic diagram of the flow layer of the microfluidic control chip; (b) is a schematic diagram of the control layer of the microfluidic control chip;

[0049] Figure 2 Schematic diagram of the microfluidic control chip and imaging system for single-cell extracellular vesicle separation and analysis of the present invention;

[0050] Figure 3 , 4 A schematic diagram of one unit in the microfluidic control chip for single-cell extracellular vesicle separation and analysis of the present invention;

[0051] Figure 5 Schematic diagrams of the micron-scale single microsphere capture structure and the micron-scale single cell capture structure of the present invention; a is the micron-scale single microsphere capture structure, and b is the micron-scale single cell capture structure;

[0052] Figure 6 A bar chart showing the probability of single-cell and / or microsphere capture events;

[0053] Among them, 1. Capillary tube a, 2. Capillary tube b, 3. PFA tube a, 4. Capillary tube c, 5. Waste liquid outlet, 6. Micron-level capture channel, 7. PDMS membrane of the flow layer, 8. Micron-level gas flow channel of the flow layer, 9. Inlet I, 10. Inlet II, 11. Inlet III, 12. PFA tube b, 13. Micron-level gas flow channel of the control layer, 14. Micron-level protrusion structure of the control layer, 15. PDMS membrane of the control layer, 16. Inlet IV, 17. Fluorescence microscope, 18. Pressure controller a, 19. Pressure controller b, 20. Micro-injection pump b, 21. Micro-injection pump a, 22. Microfluidic control chip, 23. Micron-level expansion structure I, 24. Micron-level single microsphere capture structure, 25. Micron-level contraction structure I, 26. Micron-level single cell capture structure, 27. Micron-level expansion structure II, 28. Micron-level contraction structure II. Detailed Implementation

[0054] The specific embodiments of the present invention will be described in detail below with reference to the accompanying drawings and examples.

[0055] A microfluidic chip imaging system for single-cell extracellular vesicle isolation and analysis, such as Figure 1 , 2 As shown, this system enables zero-cross-contamination capture and analysis of single-cell extracellular vesicles. The system includes a horizontally placed microfluidic control chip 22, micro-injection pumps a 21 and b 20, pressure controllers a 18 and b 19, capillary tubes a1, b 2, and c 4, PFA tubes a 3 and b 12; the structure of the microfluidic control chip 22 is as follows... Figure 1-5 As shown, it includes the upper flow layer ( Figure 1 a) and the lower control layer ( Figure 1b) Both are made of PDMS material. After the two layers are combined, they are sealed with a glass slide. The flow layer includes three inlets, namely inlet I9, inlet II10 and inlet III11, as well as a waste liquid outlet 5, a micron-level capture channel 6, a micron-level gas flow channel 8, a micron-level expansion structure I 23, a micron-level single microsphere capture structure 24, a micron-level contraction structure I 25, a micron-level single cell capture structure 26, a micron-level expansion structure II 27, and a micron-level contraction structure II 28. Among them, the micron-level single microsphere capture structure 24 and the micron-level single cell capture structure 26 are respectively set in the micron-level capture channel 6 located on the left and right sides of the micron-level contraction structure I 25. The control layer includes an inlet IV 16, a micron-level gas flow channel 13, and a micron-level protrusion structure 14. One end of capillary tubes a1 and b2 is connected to the outlet of micro-injection pumps a21 and b20, respectively, and the other end is connected to inlet I9 and inlet II10 of microfluidic control chip 22, respectively. One end of PFA tubes a3 and b12 is connected to the outlet of pressure controllers a18 and b19, respectively, and the other end is connected to inlet III11 and inlet IV16 of microfluidic control chip 22, respectively. One end of capillary tube c4 is connected to the waste liquid outlet 5 of microfluidic control chip 22, and the other end discharges waste liquid. A fluorescence microscope 17 is located directly above the micron-level capture channel 6 of microfluidic control chip 22. The fluorescence microscope 17 is provided to improve the system functionality of this invention. Depending on actual usage requirements, other types of fluorescence microscopes or other detection methods can be selected. The capillary tubes a1, b2, and c... The outer diameter of each of the four tubes is 1.6 mm and the inner diameter is 160 μm; the outer diameter of the PFA tube a3 and the inner diameter of the PFA tube b12 are both 1.6 mm and 800 μm.

[0056] In the microfluidic control chip 22, the length of the micron-level gas flow channel 8 in the flow layer is 26–35 cm, and the width is 100–200 μm. The normal width of the micron-level gas flow channel 13 in the control layer is 350–400 μm, and the length is 8–13 cm, arranged in three rows. The micron-level gas flow channel 13 in the control layer also includes 108 protrusion structures, each with a length of 450–900 μm and a width of 150–300 μm.

[0057] The microfluidic control chip 22 is a specially designed six-row parallel array structure, with each row including 9 units, for a total of 54 units. Each unit consists of a micrometer-scale capture channel with a length of 0.25–0.50 cm. Figure 3 The middle section (segment a) has a width of 100–200 μm and a length of 0.35–0.75 cm, forming a micron-sized gas flow channel in the flow layer. Figure 3The middle section (b segment) (width 100–200 μm) includes a micron-sized single microsphere trapping structure 24, a micron-sized single-cell trapping structure 26, a micron-sized expansion structure I 23, a micron-sized expansion structure II 27, a micron-sized contraction structure I 25, a micron-sized contraction structure II 28, and two micron-sized protrusions 14 (length 450–900 μm, width 150–300 μm) in the control layer, and a micron-sized gas flow channel in the control layer with a length of 0.25–0.50 cm. Figure 3 It consists of segment c.

[0058] Two micrometer-scale expansion structures and two micrometer-scale contraction structures are arranged within the micrometer-scale capture channel. Micrometer-scale expansion structure I 23 has a length of 150–400 μm and a width of 220–280 μm; micrometer-scale expansion structure II 27 has a length of 180–650 μm and a width of 500–600 μm; micrometer-scale contraction structure I 25 has a length of 100–250 μm and a width of 150–200 μm; micrometer-scale contraction structure II 28 has a length of 550–750 μm and a width of 50–250 μm. The connection between micrometer-scale contraction structure II 28 and the micrometer-scale gas flow channel (…) Figure 4 The width at point b) is 25–75 μm. The distance between the micron-scale expansion structure I 23 and the micron-scale contraction structure I 25 is 350–400 μm; the distance between the micron-scale contraction structure I 25 and the micron-scale expansion structure II 27 is 100–120 μm; the micron-scale expansion structure II 27 and the micron-scale contraction structure II 28 are connected. The arrangement order of the above four expansion and contraction structures is micron-scale expansion structure I 23, micron-scale contraction structure I 25, micron-scale expansion structure II 27, and micron-scale contraction structure II 28. In each capture unit, there are a total of 54 micron-scale expansion structures I 23, micron-scale contraction structures I 25, micron-scale expansion structures II 27, and micron-scale contraction structures II 28.

[0059] The width of the micron-scale expansion structure I 23 is greater than the width of the micron-scale capture channel 6; the micron-scale expansion structure II 27 is used to provide cell culture medium and its size can be adjusted appropriately, but its width must be greater than the width of the micron-scale capture channel 6; the width of the micron-scale contraction structure II 28 needs to be less than at least half the width of the micron-scale capture channel 6 to ensure successful droplet formation, and it also needs to be greater than the size of the microspheres and cells to ensure that the remaining sample can flow into the next unit.

[0060] In this embodiment, such as Figure 1 and Figure 3As shown, the total length of the micron-scale capture channel 6 is 10.5–25.5 cm, and its normal width is 100–200 μm; the length of a micron-scale single-cell capture structure 26 inside it is 15–36 μm, the width is 10–20 μm, the radius of the built-in groove is 5–10 μm, and the width of the built-in slit is 4–6 μm; the length of a micron-scale single microsphere capture structure 24 is 50–100 μm, the width is 20–55 μm, the radius of the built-in groove is 20–30 μm, and the width of the built-in slit is 20–30 μm; the overall height of each channel within the flow layer of the microfluidic control chip 22 is 30–70 μm, and the area of ​​the microfluidic control chip 22 is 1.27 cm². 2 ~10.50cm 2 .

[0061] It should be noted that the dimensions and parameters described above are only specific dimensions and parameters in this embodiment and are not intended to limit the scope of the present invention. Those skilled in the art can select the specific dimensions and parameters of each part of the microfluidic control chip within the scope of the invention, based on specific practical needs, such as the actual size of the cells or the actual size of the microspheres used.

[0062] Example 1

[0063] A method for analyzing single-cell extracellular vesicles with zero cross-contamination employs a microfluidic control system for the separation and analysis of single-cell extracellular vesicles combined with a fluorescence microscope, comprising the following steps:

[0064] Step 1, Preparation of cell samples: Cells are prepared into a cell suspension through trypsin digestion;

[0065] The method for preparing cell suspension is as follows:

[0066] (1) In a cell culture dish, the cells are cultured to the logarithmic growth phase using a culture medium;

[0067] (2) Cells are digested from cell culture dishes using pancreatic enzymes;

[0068] (3) The digested cells were centrifuged and washed, and the centrifuged products were resuspended in serum-free cell culture medium to prepare a density of 10. 5 Cell suspension with cells / mL;

[0069] Step 2: Prepare microsphere samples by modifying the surface of microspheres (micron-sized polystyrene spherical particles) with a capture aptamer (CD63 aptamer) through an EDC-NHS reaction, and obtain a microsphere suspension.

[0070] The preparation method of the microsphere suspension is as follows:

[0071] (1) The modified carboxyl (-COOH) aptamer, EDC and NHS solution (MES buffer, pH 6.0) were thoroughly mixed by oscillation to achieve -COOH activation;

[0072] (2) Add the amino-modified microspheres to the solution of step (1) and shake overnight;

[0073] (3) The microsphere solution successfully modified to capture aptamers was centrifuged and washed, and then the centrifuged product was resuspended in serum-free cell culture medium to prepare a density of 10. 4 Microsphere suspension with particles / mL;

[0074] Step 3: Pre-treat the microfluidic chip substrate to make it suitable for cell adhesion and growth;

[0075] The microfluidic control chip substrate pretreatment steps are as follows:

[0076] (1) A matrix gel for cell adhesion growth was prepared from Collagen, Matrigel and Fibrican;

[0077] (2) Pass the matrix adhesive into the microfluidic control chip and incubate for 2 hours;

[0078] (3) The matrix adhesive in the microfluidic chip channel was washed away with a phosphate buffer solution, wherein the phosphate particle concentration was 0.01 mol / L PO4. 3- pH 7.4;

[0079] Step 4, Sample preparation: Cell suspension is supplied by microinjection pump a 21 and connected to inlet I9 of microfluidic control chip 22 via capillary tube a 1; microsphere suspension modified with aptamer is supplied by microinjection pump b 20 and connected to inlet II 10 of microfluidic control chip via capillary tube b 2; air for droplet formation is supplied by pressure controller a 18 and connected to inlet III 11 of microfluidic control chip via PFA tube a 3; air for bulging the control layer PDMS film upward is supplied by pressure controller b 19 and connected to inlet IV 16 of microfluidic control chip via PFA tube b 12.

[0080] Step 5, Single-cell capture: Air is introduced into the micron-level gas flow channel of the control layer at a pressure of 1000 mbar through pressure controller b19 and PFA tube b12, causing the PDMS film of the control layer to bulge upwards and reducing the diversion effect of the flow layer. The cell suspension is introduced into the micron-level capture channel 6 of the microfluidic control chip at an injection flow rate of 80 μL / min through micro-injection pump a21 and capillary tube a1. The cell suspension is captured by the micron-level single-cell capture structure 26 in the micron-level capture channel 6.

[0081] Step 6, Microsphere Capture: Air is introduced into the micron-level gas flow channel of the control layer at a pressure of 1000 mbar through pressure controller b19 and PFA tube b12, causing the PDMS film of the control layer to bulge upwards and reducing the diversion effect of the flow layer. The microsphere suspension is introduced into the micron-level capture channel 6 of the microfluidic control chip at an injection flow rate of 50 μL / min through micro-injection pump b20 and capillary tube b2. The microsphere suspension is captured by the micron-level single microsphere capture structure 24 in the micron-level capture channel 6.

[0082] Step 7, cleaning excess cells and microspheres in the microfluidic control chip channels: serum-free cell culture medium is introduced into the micron-level capture channel 6 of the microfluidic control chip at an injection flow rate of 50 μL / min using microinjection pump a21 and capillary tube a1; serum-free cell culture medium is introduced into the micron-level capture channel of the microfluidic control chip at an injection flow rate of 50 μL / min using microinjection pump b20 and capillary tube b2, thus completing the effective arrangement of single cells and single microspheres in the microfluidic control chip;

[0083] Step 8, forming droplets to achieve single-cell isolation: Air is introduced into the micron-level gas flow channel of the flow layer at a pressure of 40 mbar through pressure controller a18 and PFA tube a3. Due to the difference in width between the micron-level contraction structure II28 and the micron-level gas flow channel, the introduced gas preferentially flows to the micron-level gas flow channel (differential flow resistance effect), thus forming a single droplet in each unit, achieving effective single-cell isolation and realizing single-cell extracellular vesicle analysis with zero cross-contamination;

[0084] Step 9, Single-cell culture and extracellular vesicle capture: After completing step 8, place the microfluidic chip in a cell culture incubator. During culture, the extracellular vesicles secreted by the cells are captured by the micron-sized capture channels on both sides (...). Figure 4 The pressure difference at points a and b in the structure causes the solution carrying the outer vesicle to flow to the micron-scale single microsphere trapping structure at point 24; and then it is captured by the microsphere modified with the trapping aptamer.

[0085] Step 10, Labeling of single-cell extracellular vesicles: Wash the entire channel of the microfluidic chip after step 9 with phosphate buffer solution, wherein the phosphate particle concentration is 0.01 mol / L PO4. 3- pH = 7.4; then the fluorescent probe was introduced into the microfluidic chip for incubation;

[0086] Step 11, detection of single-cell extravesicles: The microfluidic control chip is placed on the stage of the fluorescence microscope 17. Since the microspheres that capture extravesicles are labeled by fluorescent probes, the fluorescence signals of the fluorescent probes can be collected by the fluorescence microscope, and finally the expression information of biomarkers on the surface of single-cell secretory extravesicles can be obtained.

[0087] Repeat steps 1 to 11 to sample and re-examine the secretory vesicles from single cells.

[0088] It should be noted that in this example, the cell suspension flow rate is 80 μL / min, the microsphere suspension flow rate is 50 μL / min, the gas pressure introduced into the micron-level gas flow channel of the control layer is 1000 mbar, the diameter of the microspheres capturing the outer vesicles is 50 μm, the pressure used to generate droplets is 40 mbar, the total length of the micron-level capture channel 6 is 24.6 cm, and the normal width is 200 μm; within it, a micron-level single-cell capture structure 26 has a length of 36 μm, a width of 20 μm, a slit width of 5 μm, and a radius of 9 μm for the built-in groove; a micron-level single microsphere capture structure 24 has a length of 94 μm, a width of 52 μm, a slit width of 30 μm, and a radius of 30 μm for the built-in groove; the overall height of each channel within the flow layer of the microfluidic control chip 22 is 60 μm, and the area of ​​the microfluidic control chip 22 is 9.6 cm². 2 .

[0089] Example 2

[0090] A method for analyzing single-cell extracellular vesicles with zero cross-contamination employs a microfluidic chip system for the separation and analysis of single-cell extracellular vesicles combined with a fluorescence microscope, comprising the following steps:

[0091] Step 1, Preparation of cell samples: Cells are prepared into a cell suspension through trypsin digestion;

[0092] The method for preparing cell suspension is as follows:

[0093] (1) In a cell culture dish, the cells are cultured to the logarithmic growth phase using a culture medium;

[0094] (2) Cells are digested from cell culture dishes using pancreatic enzymes;

[0095] (3) The digested cells were centrifuged and washed, and the centrifuged products were resuspended in serum-free cell culture medium to prepare a density of 10. 5 Cell suspension with cells / mL;

[0096] Step 2: Prepare microsphere samples by modifying the capture aptamer onto the surface of the microspheres through EDC-NHS reaction, and obtain a microsphere suspension;

[0097] The preparation method of the microsphere suspension is as follows:

[0098] (1) The modified carboxyl (-COOH) aptamer, EDC and NHS solution (MES buffer, pH 6.0) were thoroughly mixed by oscillation to achieve -COOH activation;

[0099] (2) Add the amino-modified microspheres to the solution of step (1) and shake overnight;

[0100] (3) The microsphere solution successfully modified to capture aptamers was centrifuged and washed, and then the centrifuged product was resuspended in serum-free cell culture medium to prepare a density of 10. 4 Microsphere suspension with particles / mL;

[0101] Step 3: Pre-treat the microfluidic chip substrate to make it suitable for cell adhesion and growth;

[0102] The microfluidic control chip substrate pretreatment steps are as follows:

[0103] (1) A matrix gel for cell adhesion growth was prepared from Collagen, Matrigel and Fibrican;

[0104] (2) Pass the matrix adhesive into the microfluidic control chip and incubate for 2 hours;

[0105] (3) The matrix adhesive in the microfluidic chip channel was washed away with a phosphate buffer solution, wherein the phosphate particle concentration was 0.01 mol / L PO4. 3- pH 7.4;

[0106] Step 4, Sample preparation: Cell suspension is supplied by microinjection pump a21 and connected to microfluidic control chip inlet I9 via capillary tube a1; microinjection pump b20 supplies microsphere suspension modified with aptamer capture and connects to microfluidic control chip inlet II10 via capillary tube b2; air for droplet formation is supplied by pressure controller a18 and connected to microfluidic control chip inlet III11 via PFA tube a3; air for bulging the control layer PDMS film upwards is supplied by pressure controller b19 and connected to microfluidic control chip inlet IV16 via PFA tube b12.

[0107] Step 5, Single-cell capture: Air is introduced into the micron-level gas flow channel of the control layer at a pressure of 900 mbar through pressure controller b19 and PFA tube b12, causing the PDMS film of the control layer to bulge upwards and reducing the diversion effect of the flow layer. The cell suspension is introduced into the micron-level capture channel of the microfluidic control chip at an injection flow rate of 60 μL / min through micro-injection pump a21 and capillary tube a1. The cell suspension is captured by the micron-level single-cell capture structure within the micron-level capture channel.

[0108] Step 6, Microsphere Capture: Air is introduced into the micron-level gas flow channel of the control layer at a pressure of 900 mbar through pressure controller b19 and PFA tube b12, causing the PDMS film of the control layer to bulge upwards and reducing the flow layer's diversion effect. The microsphere suspension is introduced into the micron-level capture channel of the microfluidic control chip at an injection flow rate of 60 μL / min through micro-injection pump b20 and capillary tube b2. The microsphere suspension is captured by the micron-level single microsphere capture structure within the micron-level capture channel.

[0109] Step 7, Cleaning excess cells and microspheres in the microfluidic control chip channels: Introduce serum-free cell culture medium into the micron-level capture channels of the microfluidic control chip at an injection flow rate of 50 μL / min using microinjection pump a21 and capillary tube a1; Introduce serum-free cell culture medium into the micron-level capture channels of the microfluidic control chip at an injection flow rate of 50 μL / min using microinjection pump b20 and capillary tube b2, thus completing the effective arrangement of single cells and individual microspheres in the microfluidic control chip;

[0110] Step 8, forming droplets to achieve single-cell isolation: Air is introduced into the micron-level gas flow channel of the flow layer at a pressure of 50 mbar through pressure controller a18 and PFA tube a3. Due to the difference in width between the micron-level contraction structure II 28 and the micron-level gas flow channel, the introduced gas preferentially flows to the micron-level gas flow channel (differential flow resistance effect), thus forming a single droplet in each unit, achieving effective single-cell isolation and realizing single-cell extracellular vesicle analysis with zero cross-contamination;

[0111] Step 9, Single-cell culture and extracellular vesicle capture: After completing Step 8, the microfluidic chip is placed in a cell culture incubator. During culture, the extracellular vesicles secreted by the cells experience pressure differences due to the varying widths on both sides of the micron-sized capture channel. The solution carrying the extracellular vesicles flows towards the micron-sized single-microsphere capture structure and is then captured by the microspheres modified with capture aptamers.

[0112] Step 10, Labeling of single-cell extracellular vesicles: Wash the entire channel of the microfluidic chip after step 9 with phosphate buffer solution, wherein the phosphate particle concentration is 0.01 mol / L PO4. 3- pH = 7.4; then the fluorescent probe was introduced into the microfluidic chip for incubation;

[0113] Step 11, detection of single-cell extracellular vesicles: The microfluidic control chip is placed on the stage of a fluorescence microscope. Since the microspheres that capture extracellular vesicles are labeled by fluorescent probes, the fluorescence signals of the fluorescent probes can be collected by the fluorescence microscope, and finally the expression information of biomarkers on the surface of single-cell secretory extracellular vesicles can be obtained.

[0114] Repeat steps 1 to 11 to sample and re-examine the secretory vesicles from single cells.

[0115] It should be noted that in this example, the cell suspension flow rate is 60 μL / min, the microsphere suspension flow rate is 60 μL / min, the gas pressure introduced into the micron-level gas flow channel of the control layer is 900 mbar, the diameter of the microspheres capturing the outer vesicles is 40 μm, the pressure used to generate droplets is 50 mbar, the total length of the micron-level capture channel is 16.5 cm, and the normal width is 135 μm; within it, a micron-level single-cell capture structure 26 has a length of 24 μm, a width of 13 μm, a slit width of 4 μm, and a radius of 8 μm for the built-in groove; a micron-level single microsphere capture structure 24 has a length of 63 μm, a width of 35 μm, a slit width of 25 μm, and a radius of 25 μm for the built-in groove; the overall height of each channel within the flow layer of the microfluidic control chip 22 is 50 μm, and the area of ​​the microfluidic control chip 22 is 6.5 cm². 2 .

[0116] Example 3

[0117] A method for analyzing single-cell extracellular vesicles with zero cross-contamination employs a microfluidic control system for the separation and analysis of single-cell extracellular vesicles combined with a fluorescence microscope, comprising the following steps:

[0118] Step 1, Preparation of cell samples: Cells are prepared into a cell suspension through trypsin digestion;

[0119] The method for preparing cell suspension is as follows:

[0120] (1) In a cell culture dish, the cells are cultured to the logarithmic growth phase using a culture medium;

[0121] (2) Cells are digested from cell culture dishes using pancreatic enzymes;

[0122] (3) The digested cells were centrifuged and washed, and the centrifuged products were resuspended in serum-free cell culture medium to prepare a density of 10. 5 Cell suspension with cells / mL;

[0123] Step 2: Prepare microsphere samples by modifying the capture aptamer onto the surface of the microspheres through EDC-NHS reaction, and obtain a microsphere suspension;

[0124] The preparation method of the microsphere suspension is as follows:

[0125] (1) The modified carboxyl (-COOH) aptamer, EDC and NHS solution (MES buffer, pH 6.0) were thoroughly mixed by oscillation to achieve -COOH activation;

[0126] (2) Add the amino-modified microspheres to the solution of step (1) and shake overnight;

[0127] (3) The microsphere solution successfully modified to capture aptamers was centrifuged and washed, and then the centrifuged product was resuspended in serum-free cell culture medium to prepare a density of 10. 4 Microsphere suspension with particles / mL;

[0128] Step 3: Pre-treat the microfluidic chip substrate to make it suitable for cell adhesion and growth;

[0129] The microfluidic control chip substrate pretreatment steps are as follows:

[0130] (1) A matrix gel for cell adhesion growth was prepared from Collagen, Matrigel and Fibrican;

[0131] (2) Pass the matrix adhesive into the microfluidic control chip and incubate for 2 hours;

[0132] (3) The matrix adhesive in the microfluidic chip channel was washed away with a phosphate buffer solution, wherein the phosphate particle concentration was 0.01 mol / L PO4. 3- pH 7.4;

[0133] Step 4, Sample preparation: Cell suspension is supplied by microinjection pump a21 and connected to microfluidic control chip inlet I9 via capillary tube a1; microinjection pump b20 supplies microsphere suspension modified with aptamer capture and connects to microfluidic control chip inlet II10 via capillary tube b2; air for droplet formation is supplied by pressure controller a18 and connected to microfluidic control chip inlet III11 via PFA tube a3; air for bulging the control layer PDMS film upwards is supplied by pressure controller b19 and connected to microfluidic control chip inlet IV16 via PFA tube b12.

[0134] Step 5, Single-cell capture: Air is introduced into the micron-level gas flow channel of the control layer at a pressure of 1200 mbar through pressure controller b19 and PFA tube b12, causing the PDMS film of the control layer to bulge upwards and reducing the diversion effect of the flow layer. The cell suspension is introduced into the micron-level capture channel of the microfluidic control chip at an injection flow rate of 100 μL / min through micro-injection pump a21 and capillary tube a1. The cell suspension is captured by the micron-level single-cell capture structure in the micron-level capture channel.

[0135] Step 6, Microsphere Capture: Air is introduced into the micron-level gas flow channel of the control layer at a pressure of 1000 mbar via pressure controller b19 and PFA tube b12, causing the PDMS film of the control layer to bulge upwards and reducing the flow layer's diversion effect. The microsphere suspension is introduced into the micron-level capture channel of the microfluidic control chip at an injection flow rate of 70 μL / min via micro-injection pump b20 and capillary tube b2. The microsphere suspension is captured by the micron-level single microsphere capture structure within the micron-level capture channel.

[0136] Step 7, Cleaning excess cells and microspheres in the microfluidic control chip channels: Introduce serum-free cell culture medium into the micron-level capture channels of the microfluidic control chip at an injection flow rate of 50 μL / min using microinjection pump a21 and capillary tube a1; Introduce serum-free cell culture medium into the micron-level capture channels of the microfluidic control chip at an injection flow rate of 50 μL / min using microinjection pump b20 and capillary tube b2, thus completing the effective arrangement of single cells and individual microspheres in the microfluidic control chip;

[0137] Step 8, forming droplets to achieve single-cell isolation: Air is introduced into the micron-level gas flow channel of the flow layer at a pressure of 60 mbar through pressure controller a18 and PFA tube a3. Due to the difference in width between the micron-level contraction structure II28 and the micron-level gas flow channel, the introduced gas preferentially flows to the micron-level gas flow channel (differential flow resistance effect), thus forming a single droplet in each unit, achieving effective single-cell isolation and realizing single-cell extracellular vesicle analysis with zero cross-contamination;

[0138] Step 9, Single-cell culture and extracellular vesicle capture: After completing Step 8, the microfluidic chip is placed in a cell culture incubator. During culture, the extracellular vesicles secreted by the cells experience pressure differences due to the varying widths on both sides of the micron-sized capture channel. The solution carrying the extracellular vesicles flows towards the micron-sized single-microsphere capture structure and is then captured by the microspheres modified with capture aptamers.

[0139] Step 10, Labeling of single-cell extracellular vesicles: Wash the entire channel of the microfluidic chip after step 9 with phosphate buffer solution, wherein the phosphate particle concentration is 0.01 mol / L PO4. 3- pH = 7.4; then the fluorescent probe was introduced into the microfluidic chip for incubation;

[0140] Step 11, detection of single-cell extracellular vesicles: The microfluidic control chip is placed on the stage of a fluorescence microscope. Since the microspheres that capture extracellular vesicles are labeled by fluorescent probes, the fluorescence signals of the fluorescent probes can be collected by the fluorescence microscope, and finally the expression information of biomarkers on the surface of single-cell secretory extracellular vesicles can be obtained.

[0141] Repeat steps 1 to 11 to sample and re-examine the secretory vesicles from single cells.

[0142] It should be noted that in this example, the cell suspension flow rate is 100 μL / min, the microsphere suspension flow rate is 70 μL / min, the gas pressure introduced into the micron-level gas flow channel of the control layer is 1200 mbar, the diameter of the microspheres capturing the outer vesicles is 30 μm, the pressure used to generate droplets is 60 mbar, the total length of the micron-level capture channel is 12.6 cm, and the normal width is 100 μm; within it, a micron-level single-cell capture structure 26 has a length of 18 μm, a width of 10 μm, a slit width of 4 μm, and a radius of 7 μm for the built-in groove; a micron-level single microsphere capture structure 24 has a length of 50 μm, a width of 25 μm, a slit width of 20 μm, and a radius of 20 μm for the built-in groove; the overall height of each channel within the flow layer of the microfluidic control chip 22 is 40 μm, and the area of ​​the microfluidic control chip 22 is 4.8 cm². 2 .

Claims

1. A microfluidic control chip for single-cell extracellular vesicle separation and analysis, characterized in that, It includes an upper flow layer and a lower control layer; the flow layer includes three inlets, namely inlet I (9), inlet II (10) and inlet III (11), as well as a waste liquid outlet (5), a micron-level capture channel (6), a micron-level gas flow channel I (8), a micron-level expansion structure I (23), a micron-level single microsphere capture structure (24), a micron-level contraction structure I (25), a micron-level single cell capture structure (26), a micron-level expansion structure II (27), and a micron-level contraction structure II (28); among them, inlet I (9) and inlet II (10) are both connected to the inlet of the micron-level capture channel (6), and inlet I ( 9) Used to introduce cell suspension, inlet II (10) is used to introduce microsphere suspension; micron-level gas flow channel I (8) and inlet III (11) are connected, and inlet III (11) introduces air; micron-level expansion structure I (23), micron-level contraction structure I (25), micron-level expansion structure II (27) and micron-level contraction structure II (28) are set in micron-level capture channel (6); micron-level single microsphere capture structure (24) and micron-level single cell capture structure (26) are respectively set in the micron-level capture channel (6) on the left and right sides of micron-level contraction structure I (25); the arrangement order is micron-level expansion structure I (23) Micron-level contraction structure I (25) Micron-level expansion structure II (27) and micron-level contraction structure II (28); The control layer includes inlet IV (16), micron-level gas flow channel II (13) and micron-level protrusion structure (14); Inlet IV (16) and micron-level gas flow channel II (13) are connected, and air is introduced through inlet IV (16); The width of micron-level gas flow channel I (8) is wider than the width of micron-level contraction structure II (28); The upper flow layer and the lower control layer are both PDMS thin film structures.

2. The microfluidic control chip for single-cell extracellular vesicle separation and analysis according to claim 1, characterized in that, The micron-scale single microsphere capture structure (24) and the micron-scale single cell capture structure (26) are designed according to the size of the microspheres and cells, and both are composed of two mirror-arranged PDMS micropillars. The length of the micron-scale single microsphere capture structure (24) is 50~100 μm, the width is 20~55 μm, the radius of the built-in groove is 20~30 μm, and the width of the built-in slit is 20~30 μm. The length of the micron-scale single cell capture structure (26) is 15~36 μm, the width is 10~20 μm, the radius of the built-in groove is 5~10 μm, and the width of the built-in slit is 4~6 μm. The distance between the micron-scale single cell capture structure (26) and the micron-scale single microsphere capture structure (24) is 150~200 μm.

3. The microfluidic control chip for single-cell extracellular vesicle separation and analysis according to claim 1, characterized in that, The microfluidic control chip is a specially designed six-row parallel array structure, with each row including 9 units, for a total of 54 units; each unit consists of a micron-level capture channel (6) with a length of 0.25~0.50 cm and a width of 100~200 μm when there are no contraction or expansion structures in the channel, a flow layer micron-level gas flow channel I (8) with a length of 0.35~0.75 cm and a width of 100~200 μm, a micron-level single microsphere capture structure (24), a micron-level single cell capture structure (26), a micron-level expansion structure I (23), a micron-level expansion structure II (27), a micron-level contraction structure I (25), a micron-level contraction structure II (28), and two micron-level protrusion structures (14) of the control layer with a length of 450~900 μm and a width of 150~300 μm, and a control layer micron-level gas flow channel II (13) with a length of 0.25~0.50 cm and a width of 350~400 μm.

4. A microfluidic control chip for single-cell extracellular vesicle separation and analysis according to claim 1, characterized in that, The length of the micron-scale expanded structure I (23) is 150~400 μm and the width is 220~280 μm; the length of the micron-scale expanded structure II (27) is 180~650 μm and the width is 500~600 μm; the length of the micron-scale contracted structure I (25) is 100~250 μm and the width is 150~200 μm. The length of the micron-scale contraction structure II (28) is 550~750 μm and the width is 50~250 μm; the width at the connection between the micron-scale contraction structure II (28) and the micron-scale gas flow channel I (8) is 25~75 μm; the distance between the micron-scale expansion structure I (23) and the micron-scale contraction structure I (25) is 350~400 μm; the distance between the micron-scale contraction structure I (25) and the micron-scale expansion structure II (27) is 100~120 μm.

5. A microfluidic control chip for single-cell extracellular vesicle separation and analysis according to claim 1, characterized in that, The heights of the micron-level trapping channel (6), the micron-level single-cell trapping structure (26), the micron-level single microsphere trapping structure (24), and the first micron-level gas flow channel (8) in the flow layer are all 30~70 μm; the heights of the second micron-level gas flow channel (13) and the micron-level protrusion structure (14) in the control layer are all 30~70 μm; the area of ​​the microfluidic control chip is 1.27 cm². 2 ~10.50 cm 2 .

6. A microfluidic chip imaging system for single-cell extracellular vesicle separation and analysis, characterized in that, The device comprises a microfluidic control chip for single-cell extracellular vesicle separation and analysis as described in any one of claims 1-4, and microinjection pump a, microinjection pump b, pressure controller a, pressure controller b, capillary tube a, capillary tube b, capillary tube c, PFA tube a, PFA tube b, and a fluorescence microscope (17); the microinjection pump a is connected to the inlet I (9) of the microfluidic control chip through capillary tube a, and capillary tube a serves as the inlet channel for cell suspension and matrix gel; the microinjection pump b is connected to the inlet II (10) of the microfluidic control chip through capillary tube b, and capillary tube b serves as the inlet channel for microsphere solution; Pressure controller a is connected to the inlet Ⅲ (11) of microfluidic control chip through PFA tube a, and PFA tube a serves as the air inlet channel; Pressure controller b is connected to the inlet IV (16) of the microfluidic control chip through PFA tube b, with PFA tube b serving as the air inlet channel.

7. A microfluidic chip imaging system for single-cell extracellular vesicle separation and analysis according to claim 6, characterized in that, The capillary tube c serves as a waste liquid channel, with one end connected to the waste liquid outlet (5) of the microfluidic control chip and the other end used for waste liquid discharge; the fluorescence microscope (17) is set directly above the micron-level capture channel (6) of the microfluidic control chip; the outer diameter of capillary tube a, capillary tube b, and capillary tube c is 1.6 mm and the inner diameter is 160 μm; the outer diameter of PFA tube a and PFA tube b is 1.6 mm and the inner diameter is 800 μm.

8. A method for analyzing single-cell extracellular vesicles with zero cross-contamination, characterized in that, The microfluidic chip imaging system for single-cell extracellular vesicle separation and analysis as described in claim 6 or 7 is used to achieve this, specifically including the following steps: Step 1, Preparation of matrix gel: Prepare matrix gel for cell adhesion growth. The matrix gel is supplied by microinjection pump a and connected to the microfluidic control chip inlet I (9) through capillary tube a. Step 2, cell sample preparation: Prepare the cells to be tested into a cell suspension and filter it through a cell filter sieve; Step 3, Preparation of microsphere samples: The aptamer for capturing exovesicles is attached to the surface of the microspheres and a microsphere suspension is prepared. Step 4, Sample preparation: Cell suspension is supplied by microinjection pump a and connected to microfluidic control chip inlet I (9) through capillary tube a; microsphere suspension is supplied by microinjection pump b and connected to microfluidic control chip inlet II (10) through capillary tube b; air is supplied by pressure controller b and connected to microfluidic control chip inlet IV (16) through PFA tube b. Step 5, Capture of single cells and single microspheres: Air is introduced into the micron-level gas flow channel 2 (13) of the control layer through pressure controller b and PFA tube b, causing the PDMS film to bulge; the cell suspension is introduced into the micron-level capture channel (6) of the microfluidic control chip through micro-injection pump a and capillary tube a, and the microsphere suspension is introduced into the micron-level capture channel (6) of the microfluidic control chip through micro-injection pump b and capillary tube b. The cells and microspheres will be captured by the micron-level single cell capture structure (26) and the micron-level single microsphere capture structure (24) in the micron-level capture channel (6); Step 6, droplet generation: Air is supplied by pressure controller a and connected to the microfluidic control chip inlet III (11) through PFA tube a; air is introduced into the micron-level gas flow channel one (8) of the flow layer through pressure controller a and PFA tube a; since the width of the micron-level gas flow channel one (8) is wider than the width of the micron-level contraction structure II (28), the gas will preferentially flow to the micron-level gas flow channel one (8), thereby forming droplets; Step 7, Single-cell culture and capture of external vesicles: The droplet microfluidic control chip containing a single cell and a single microsphere is placed in an incubator for culture, and the external vesicles secreted by the single cell are captured by an aptamer modified on the surface of the microsphere. Step 8, Labeling of single cells and single-cell extracellular vesicles: The prepared fluorescent nanoprobe solution is introduced into the microfluidic control chip to label single cells and single-cell secretory extracellular vesicles; Step 9, Fluorescence imaging analysis of single cells and single extracellular vesicles: Place the microfluidic control chip containing fluorescent probe-labeled single cells and single microspheres on the stage of a fluorescence microscope (17) for observation, and obtain the expression information of cancer-related biomarker proteins on the surface of single cells and single extracellular vesicles.

9. The method for analyzing single-cell extracellular vesicles with zero cross-contamination according to claim 8, characterized in that, In step 4, the injection flow rate of the cell suspension into the microfluidic control chip is 10~100 μL / min, and the injection flow rate of the microsphere solution into the microfluidic control chip is 20~120 μL / min; the pressure of air entering the control layer is 800~1500 mbar.

10. The method for analyzing single-cell extracellular vesicles with zero cross-contamination according to claim 8, characterized in that, In step 6, the pressure in the micron-level gas flow channel through which air enters the flow layer is 10~150 mbar.

Images