Integrated microfluidic system and method for single cell capture, stimulation and analysis

Abstract

The invention relates to the technical field of micro-fluidic chips and cell culture and analysis, in particular to an integrated micro-fluidic system and method for single cell capture, stimulation and analysis, and the integrated micro-fluidic system for single cell capture, stimulation and analysis is formed by integrating two micro-fluidic chips of a front part and a rear part, and the front half part is connected with the rear half part through a capillary hose c. Sorting capture, mechanical stimulation and culture analysis of single cells are achieved through the integrated pretreatment module and the capture culture module, accelerated mixing of cell suspension liquid introduced into the pretreatment module is achieved under the action of curve secondary flow through the first micro-fluidic chip located at the pretreatment module, and the single cells are rapidly captured and cultured through the first micro-fluidic chip located at the position of the first micro-fluidic chip. Cells in the cell suspension are sequenced to a certain extent, the next step is facilitated, and sample cells with required sizes are captured and cultured through a second micro-fluidic chip which consists of a plurality of cell capturing units and is positioned on the rear half part for subsequent observation and analysis.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of microfluidic chip, cell culture and analysis technology, and particularly relates to an integrated microfluidic system and method for single cell capture, stimulation and analysis. BACKGROUND

[0002] In recent years, many cell-related researches have elucidated the complexity of cells, however, population studies sometimes lack the heterogeneity of individual cells, which can lead to inaccurate results and affect the final judgment. By studying the heterogeneity of single cell-related protein expression, further early monitoring, metastasis and personalized prevention of tumors can be carried out. However, single cell research still faces great challenges, such as lack of target analytes and complex cell volume and composition. There are many methods for single cell capture at present, affinity-based methods have been reported for the separation and detection of single circulating tumor cells (CTC), which can effectively identify and capture CTC in blood by using double aptamer modification, but the complexity of real samples hinders its stability and binding affinity; flow-based single cell analysis and cell suspension sampling have also been gradually applied to single cell analysis, but the expensive equipment and experimental devices are not easy to obtain, and the randomness of the sampling method may produce a high probability of cell cluster events, which is not conducive to the high resolution and high throughput of single cell analysis. Therefore, it is valuable to establish a method and device based on stable identification of single cell and accurate separation of single cell.

[0003] Microfluidic chip is a kind of micro device, which integrates micron-sized channels and structures, can accurately manipulate micro fluid, and has wide application in the fields of chemistry, biomedicine, etc. Compared with traditional methods with large volume and long time-consuming, microfluidic technology is gradually applied to cell experiments as a new method for single cell separation and analysis due to its low consumption, integration, high throughput, accurate manipulation and good biocompatibility.

[0004] At present, the single cell separation and analysis method based on microfluidic technology is mainly divided into active and passive methods, although microfluidic separation has significant advantages, but there are still some problems to be optimized.

[0005] Active microfluidic separation methods mainly control the cell trajectory by applying external fields (electric field, acoustic wave, magnetic field, etc.) to achieve precise manipulation and sorting of cells, with ultra-high sorting precision and adjustability, and non-contact manipulation, which to some extent ensures the activity of cells. However, because the active method relies on hardware, it needs an external high-frequency power supply, a precision piezoelectric transducer or a superconducting magnet, resulting in high equipment cost and certain complexity, and is significantly affected by environmental temperature, medium conductivity, etc. At the same time, there is a contradiction between flux and precision, which cannot meet the clinical level cell preparation demand. Long-term exposure to high-intensity electric field may cause cell membrane electroporation or abnormal gene expression, which is not conducive to the survival of subsequent cell culture. The external equipment has standardization challenges, and the parameters of equipment from different manufacturers are not unified, and the cross-platform data comparability is poor.

[0006] Passive microfluidic separation methods mainly rely on microchannel structure design and fluid dynamics to achieve cell sorting, without the need for external energy field intervention, and have unique technical characteristics and application potential. The core advantage of passive sorting is high throughput and low cost, label-free and non-invasive, biocompatibility and low stress, easy integration and scale. Common passive separation methods include deterministic lateral displacement (DLD), inertial focusing, micro-pore / micro-filter membrane screening and hydrodynamic focusing (HDF). Passive microfluidic separation methods achieve sorting based on microstructure, although no external field energy is needed, but there are still significant defects, such as: Single function: only sorting can be achieved, and cannot be integrated with capture, stimulation, culture and analysis; Complex structure: the microstructure design needs to be repeatedly optimized to adapt to different cell sizes; Limited flux: easy to block (such as micro-pore / micro-filter membrane screening) or unable to distinguish cells with similar sizes (such as DLD method).

[0007] And although the above methods can achieve the corresponding purpose, they do not maximize the advantages of microfluidic methods, which greatly limits the further development of microfluidics. In addition to cell sorting, microfluidics also has the functions of capture, extraction, labeling and culture. In summary, the single-cell separation and analysis methods based on microfluidic technology commonly used in existing technologies often have the following problems: Low integration: sample dilution, single-cell capture, stimulation culture and analysis need to be operated step by step, which easily introduces pollution and is low in efficiency; Poor flexibility: it is difficult to quickly adjust the capture parameters according to the required analysis cell size to obtain more accurate measurement results; Insufficient signal amplification: lack of controllable stimulation means for single cells, making it difficult to amplify small cancer signals.

[0008] Therefore, it is necessary to develop a high-throughput, high-integration and high-sensitivity multifunctional single-cell analysis platform to realize one-stop operation from sample processing to signal detection to overcome the problems existing in the prior art in early diagnosis of cancer. SUMMARY

[0009] The application provides an integrated microfluidic system and method for single-cell capture, stimulation and analysis to solve the problems of low integration, poor flexibility and insufficient signal amplification of traditional separation and analysis methods in the prior art.

[0010] The application provides an integrated microfluidic system for single-cell capture, stimulation and analysis, comprising: The pretreatment module comprises a first microfluidic chip having an inlet I, an inlet II and an outlet I, and a micron-scale curved secondary flow structure is further arranged inside the first microfluidic chip. The capture culture module comprises a second microfluidic chip having an inlet III, an outlet II and an outlet III, and a plurality of cell capture units are integrated in the second microfluidic chip, each of which is provided with a plurality of cell capture structures. The microsyringe pump is connected to the pretreatment module through a capillary hose a and a capillary hose b.

[0011] The pretreatment module and the capture culture module are connected through a capillary hose c.

[0012] Preferably, the curved secondary flow structure is composed of a plurality of semicircular curves, and the included angle between the inner wall of the curve and the horizontal direction is 20°.

[0013] Preferably, the plurality of cell capture units are connected in sequence, each of which has a cell capture inlet and three cell capture outlets, and the cell capture structure is arranged between the cell capture inlet and the cell capture outlet.

[0014] Preferably, the cell capture structure arranged in any one of the cell capture units is two, each of which comprises a cell capture small chamber, a cell capture large chamber and a narrow gap groove. One end of the cell capture small chamber is connected to the cell capture inlet, and the other end is connected to the cell capture large chamber through the narrow gap groove.

[0015] Preferably, the cell capture outlet comprises: The first cell capture outlet has one end connected to the cell capture large chamber and the other end connected to the outlet II or the outlet III. The second cell capture outlet.

[0016] Preferably, the cell capture inlet of any one of the cell capture units except the first end and the last end is connected with the second cell capture outlet of the adjacent last cell capture unit; Preferably, the cell capture inlet of the cell capture unit at the first end is connected with the outlet I through the capillary tube c.

[0017] Preferably, the output end of the micro-injection pump is connected with the inlet I through the capillary tube a and connected with the inlet II through the capillary tube b.

[0018] Preferably, the system further comprises: a fluorescence microscope arranged above the capture culture module; a capillary tube d and a capillary tube e connected with the outlet II and the outlet III respectively.

[0019] The application also provides an analysis method of the integrated microfluidic system for single cell capture, stimulation and analysis based on any one of the above, comprising: Step S1, preparing a cell suspension, trypsinizing adherent cells and resuspending with a phosphate buffer; Step S2, diluting and mixing, injecting the cell suspension and the buffer into the pre-treatment module through the inlet I and the inlet II at the same flow rate, and realizing dilution and mixing of the sample cells by using the bend secondary flow structure; Step S3, single cell capture, passing the sample solution after dilution and mixing into the capture culture module through the capillary tube c at a first preset flow rate, and capturing single cells of a required size by using the cell capture structure; Step S4, culture or labeling, culturing the captured single cells or labeling the surface markers of the single cells by using an aptamer / antibody solution containing a fluorescent group; Step S5, imaging analysis, observing the cells after culture or labeling by using a fluorescence microscope, and detecting and analyzing the expression of the surface markers of the single cells.

[0020] Preferably, in the step S4, the process of culturing the captured single cells further comprises: Step S41, applying mechanical stimulation, increasing the flow rate to a second preset flow rate, and applying mechanical stimulation to the captured single cells to promote the generation of deformation and the secretion of exosomes; Step S42, culture medium preparation, collecting the sample cells after applying mechanical stimulation, placing the collected sample cells in a cell culture bottle for culture until the sample cells adhere to the bottle wall and are stable, extracting exosomes from the sample cells by using an ultrahigh-speed centrifuge, and adding the exosomes into a DMEM culture medium without serum and mixing for use; Step S43, in response to step S3, the medium prepared in step S42 is introduced into the capillary tube c and co-cultured with the single cells captured by the cell capture structure.

[0021] The beneficial effects of the present application are as follows: The integrated microfluidic system for single cell capture, stimulation and analysis of the present application realizes the sorting capture, mechanical stimulation and culture analysis of single cells through the integrated pretreatment module and the capture culture module. The first microfluidic chip at the pretreatment module is designed according to the "pirate ship" swing path, so that the cell suspension flowing into the pretreatment module can realize accelerated mixing and sorting of the cells in the cell suspension into a relatively neat cell bundle under the action of the secondary flow in the bend, which is convenient for the next step. The second microfluidic chip in the latter part composed of multiple cell capture units captures and cultures the sample cells of the required size for subsequent observation and analysis. The injection flow rate of the microsyringe pump can also be increased to apply mechanical stimulation to the captured single cells, promote the secretion of exosomes and the expression of related proteins, and amplify some key microsignals, so that cancer can be observed with a higher probability, facilitating early monitoring and prevention of cancer. Further, compared with the traditional active sorting microfluidic chip, no external conditions need to be added, and the differences in power supply or piezoelectric devices do not need to be considered, so that the single cell capture can be performed more simply and quickly. Especially, compared with the traditional passive sorting microfluidic chip, no repeated optimization of the structure is needed, and the simulation structure availability can be used, and the single cell capture and culture can be realized by a relatively simple structure, which saves the cost to a certain extent. BRIEF DESCRIPTION OF DRAWINGS

[0022] In order to more clearly illustrate the specific embodiments of the present application or the technical solutions in the prior art, the drawings needed in the description of the specific embodiments or the prior art will be briefly introduced. Obviously, the drawings in the following description are some embodiments of the present application, and other drawings can also be obtained by those skilled in the art without creative labor.

[0023] Figure 1 The overall structure schematic diagram of the integrated microfluidic system for single cell capture, stimulation and analysis provided by the embodiments of the present application is shown in the figure. Figure 2 The overall structure schematic diagram of the pretreatment module in the embodiments of the present application is shown in the figure. Figure 3 The angle position schematic diagram of the bend secondary flow structure in the embodiments of the present application is shown in the figure. Figure 4Figure 1 is a schematic diagram of the overall structure of a cell capture and culture module according to an embodiment of the present application; Figure 5 Figure 2 is a schematic diagram of the overall structure of a cell capture unit according to an embodiment of the present application; Figure 6 Figure 3 is a schematic diagram of the structure of a cell capture structure in a cell capture unit according to an embodiment of the present application; Figure 7 Figure 4 is a flowchart of an analysis method based on an integrated microfluidic system for single cell capture, stimulation and analysis according to an embodiment of the present application; Figure 8 Figure 5 is a flowchart of a process for culturing a captured single cell according to an embodiment of the present application.

[0024] Reference signs: 1, microsyringe pump; 2, capillary tube b; 3, capillary tube a; 4, pretreatment module; 5, capillary tube c; 6, cell capture and culture module; 7, fluorescence microscope; 8, capillary tube d; 9, capillary tube e; 10, first microfluidic chip; 11, inlet I; 12, inlet II; 13, outlet I; 14, curved secondary flow structure; 15, curved angle schematic; 16, second microfluidic chip; 17, inlet III; 18, outlet II; 19, outlet III; 20, cell capture unit; 21, cell capture inlet; 22, first cell capture outlet a; 23, second cell capture outlet; 24, first cell capture outlet b; 25, cell capture structure; 26, cell capture large chamber; 27, narrow gap groove; 28, cell capture small chamber. DETAILED DESCRIPTION

[0025] The technical solutions of the present application will be described clearly and completely below with reference to the embodiments. Obviously, the described embodiments are only some of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by a person of ordinary skill in the art without creative effort are within the scope of protection of the present application.

[0026] The integrated microfluidic system and method for single cell capture, stimulation and analysis according to an embodiment of the present application will be described below with reference to Figures 1-8

[0027] Reference will be made to Figure 1 ​As shown, the integrated microfluidic system for single-cell capture, stimulation and analysis provided in the embodiment of the present application includes a pre-processing module 4, which is integrated with two microfluidic chips in the front and rear parts. The front half is connected to the rear half by a capillary hose c5. Among them, the first microfluidic chip 10 located in the pre-processing module 4 in the front half is designed according to the "Pirate Ship" swing path. The angle of inclination of the curved part is 20° with the horizontal direction. This part of the chip has two sample inlets and one sample outlet. The two sample inlets are inlet I 11 and inlet II 12 respectively arranged in parallel at the head end of the curved secondary flow structure 14. The input ends of the two sample inlets are both connected to the micro-injection pump 1, and the sample outlet is outlet I 13 arranged at the end of the curved secondary flow structure 14. The sample solution (cell suspension digested with trypsin) containing the cells to be captured and detected is injected into the pre-treatment module 4 from inlet I 11 and inlet II 12 via the microinjection pump 1. The sample cells flow forward with the fluid. At this time, as the transverse path in the curved secondary flow structure 14 gradually lengthens, the sample cells are basically mixed by the time they reach outlet I. The second microfluidic chip 16 located in the rear half has a sample inlet, namely inlet III17, and two sample outlets, namely outlet II18 and outlet III19. The sample cells mixed through the curved secondary flow structure 14 flow out from outlet I13, pass through the capillary hose c5, and flow into inlet III17. From inlet III17, they enter the interior of the second microfluidic chip 16 which is integrated with multiple cell capture units 20, and the sample cells of the required size are captured and cultured through the cell capture structure 25 in the cell capture unit 20 for subsequent observation and analysis.

[0028] The integrated pre-processing module 4 and capture and culture module 6 are used to achieve single cell sorting, capture, mechanical stimulation, and culture analysis. The first microfluidic chip 10 located in the pre-processing module 4, designed based on the "Pirate Ship" swing path, allows the cell suspension entering the pre-processing module 4 to accelerate mixing by utilizing the flow transverse to the downstream fluid streamline under the action of the secondary flow in the bend. At the same time, in this process, the cell suspension is also sorted to a certain extent, forming relatively neat cell bundles, which facilitates the next step. The second microfluidic chip 16 located in the rear half, composed of multiple cell capture units 20, captures and cultures sample cells of the desired size for subsequent observation and analysis. Furthermore, compared with traditional active sorting microfluidic chips, this method does not require the addition of external conditions or consideration of differences in power supplies or piezoelectric devices, making it simpler and faster to capture single cells. In particular, compared with traditional passive sorting microfluidic chips, there is no need to repeatedly optimize the structure and simulate the structural availability. Instead, a relatively simpler structure is used to achieve the capture and culture of single cells, which saves costs to a certain extent. Furthermore, the chip not only achieves single-cell capture, but is also not limited to this function. On the basis of capture and sorting, it can also be cultured and observed in situ, as well as analyze cell behavior. After the observation inside the chip is completed, the activity is not affected, and the cell can be continued to be cultured and analyzed later. By increasing the injection flow rate, mechanical stimulation can be applied to captured single cells to promote the secretion of exosomes and the expression of related proteins, amplifying some key tiny signals, thereby increasing the probability of observing cancerous changes and facilitating early monitoring and prevention of cancer. In particular, not only can high-throughput experiments be achieved, but the capture culture module 6 in the second half can also process multiple tiny culture chambers at the same time, saving time and resources. At the same time, the cell positioning and observation can be carried out through the set fluorescence microscope 7, and the cell growth and interaction process can be monitored in real time, which helps to gain a deeper understanding of cell behavior.

[0029] Please continue reading Figure 2 and Figure 3 ,like Figure 2 and Figure 3 As shown, Figure 2 This is a schematic diagram of the overall structure of the pre-processing module in the embodiment of this application. Figure 3 This is a schematic diagram of the angle position of the secondary flow structure of the bend in the embodiment of the present application; In some specific embodiments, the curved secondary flow structure 14 is composed of a plurality of semicircular curves, and the angle between the inner wall of the curve and the horizontal direction is 20°.

[0030] Specifically, the curved secondary flow structure 14 is a micron-scale path channel similar to a "pirate ship" swinging path composed of at least 8 micron-scale semicircular curves. For example, the curved secondary flow structure 14 shown in this embodiment is a path channel composed of 8 curves for accelerating sample dilution and cell bundle sorting. The channel size in the shown embodiment is 39.60 mm × 27.00 mm, the channel width is 400 μm, and the channel height is 50 μm.

[0031] Please continue reading Figure 4 ,like Figure 4 As shown, it is a schematic diagram of the overall structure of the capture and cultivation module in the embodiment of the present application; In some specific embodiments, a plurality of cell capture units 20 are connected in sequence, each cell capture unit 20 has a cell capture inlet 21 and three cell capture outlets, and the cell capture structure 25 is disposed between the cell capture inlet 21 and the cell capture outlets.

[0032] For example, in this embodiment, the cell capture culture chip (CELL capture culture chip, CELL CC ) is composed of 50 cell capture units 20 connected end to end, and each individual cell capture unit 20 has a cell capture inlet 21, three cell capture outlets and two cell capture structures 25 for capturing single cells of the required size. When integrated together, there are a total of 100 cell capture structures 25 that can be used to capture cells. CC The chip size is approximately 81.72 mm × 29.56 mm, and the channel height is 20 μm.

[0033] Please continue reading Figure 5 and Figure 6 ,like Figure 5 and Figure 6 As shown, Figure 5 This is a schematic diagram of the overall structure of the cell capture unit in the embodiment of this application. Figure 6 Schematic diagram of the structure of the cell capture structure in the cell capture unit of an embodiment of the present application; In some specific embodiments, there are two cell capture structures 25 provided in any one cell capture unit, and each cell capture structure 25 includes a cell capture chamber 28, a cell capture chamber 26, and a narrow gap groove 27; One end of the cell capture chamber 28 is connected to the cell capture inlet 21 , and the other end is connected to the cell capture chamber 26 via a narrow gap groove 27 .

[0034] The inter-chamber radius of the large cell capture chamber 26 is more than ten times the inter-chamber radius of the small cell capture chamber 28. For example, in this embodiment, the radius of the selected cell capture chamber 28 is 60 μm, and the channel gap d at the narrow gap groove 27 is designed to be 4, 8, 12, 16, 18, 20, 22 μm and other sizes that common tumor cells may have for flexible selection. Cells can only enter the large cell capture chamber 26 with a radius of 1000 μm after passing through the narrow gap groove 27. The obvious size contrast between it and the cell capture chamber 28 also promotes the occurrence of mechanical stimulation to a certain extent, providing a positive effect for cells to pass through the narrow gap groove 27.

[0035] In some embodiments, the cell capture outlet comprises: A first cell capture outlet, one end of which is connected to the cell capture chamber 26 and the other end of which is connected to the outlet II 18 or the outlet III 19; like Figure 5As shown, each cell capture unit is provided with two first cell capture outlets, which are divided into first capture cell outlet a22 and first capture cell outlet b24, and the two first cell capture outlets are located on both sides of the cell capture inlet 21 in a herringbone shape. The second cell capture outlet 23 is located between the two cell capture structures 25.

[0036] The cell suspension flowing out of the pre-treatment module 4 flows into the capture culture module 6 after passing through a straight channel formed by the capillary tube c5, and flows into the corresponding cell capture unit 20 at the cell capture inlet 21. When passing through the bifurcation, the sample cells of the corresponding size are intercepted and captured at the narrow gap groove 27 and stay in the cell capture chamber 28 to be stimulated or cultured. After the cell capture chamber 28 at the current position captures the sample cells of the corresponding size, the sample cells that are not captured flow into the next cell capture unit 20 through the second cell capture outlet 23 located in the middle position. When all the cell capture units 20 complete the capture of the sample cells, the micro-injection pump 1 can increase the flow rate of the cell suspension flowing into the capture culture module 6 to apply mechanical stimulation to the captured single cells, until they break through the interception of the narrow gap groove 27 of the corresponding size under the push of the high flow rate of the suspension and enter the cell capture large chamber 26, and then flow to the corresponding outlet II 18 or outlet III 19 for discharge and collection.

[0037] In some embodiments, the cell capture inlet 21 of any cell capture unit 20 except the one at the first end and the one at the end is connected to the second cell capture outlet 23 of the adjacent previous cell capture unit 20; In some embodiments, the cell capture inlet 21 of the cell capture unit 20 at the first end is connected to the outlet I 13 through the capillary tube c5, that is, one end of the capillary tube c5 is connected to the outlet I 13, and the other end is connected to the cell capture inlet 21 at the most first end. The second cell capture outlet 23 of the cell capture unit 20 at the end can be connected to an external recovery container through a corresponding tube.

[0038] In some embodiments, the output end of the micro-injection pump 1 is connected to the inlet I 11 through the capillary tube a3 for introducing the treated cell suspension, and is connected to the inlet II 12 through the capillary tube b2 for introducing the buffer.

[0039] In some embodiments, the system further comprises: A fluorescence microscope 7 is arranged above the capture culture module 6, and is used for fluorescence imaging analysis of the captured single cells in the latter half of the microfluidic chip. The fluorescence microscope 7 is arranged to make the system of the application more perfect. According to actual use requirements, other types of fluorescence microscopes 7, confocal microscopes or other types of detection methods, such as flow cytometry, can be selected. Capillary tubes d8 and e9 are connected with outlet II 18 and outlet III 19 respectively, and are used for collecting the cell samples after mechanical stimulation. The capillary tubes a3, b2, c5, d8 and e9 are all tygon tubes, with an outer diameter of 1.6 mm and an inner diameter of 160 μm.

[0040] The working process of the integrated microfluidic system for single cell capture, stimulation and analysis provided in the application is as follows: The microsyringe pump 1 is connected with the inlet I 11 through the capillary tube a3, and is used for introducing the treated cell suspension. The capillary tube b2 is connected with the inlet II 12, and is used for introducing the buffer. After the cell suspension and the buffer enter the micron-level mixing channel at the same first preset flow rate, the sample solution containing the sample cells is mixed under the action of the secondary flow in the bend, and then flows through four semicircles on the left and right respectively. The mixed sample solution flows out of the front treatment module 4 from the outlet I 13, and the front treatment of the sample cells is completed. The cell suspension flowing out of the front treatment module 4 flows into the capture culture module 6 through a straight channel formed by the capillary tube c5, and then flows into the corresponding cell capture unit 20 through the cell capture inlet 21. When flowing through the branch intersection, the sample cells of the corresponding size are intercepted and captured at the narrow gap groove 27, and then stay in the cell capture chamber 28 to be stimulated or cultured. After the cell capture chamber 28 at the current position captures the sample cells of the corresponding size, the sample cells that are not captured flow into the next cell capture unit 20 through the second cell capture outlet 23 located in the middle position, because the current position is blocked by the corresponding single cell. When all the cell capture units 20 complete the capture of the sample cells, the captured sample cells can be co-cultured by adding a culture medium containing different types of exosomes into the cell capture chamber 28. After the solution of the fluorescence group labeled aptamer / antibody is connected, the detection of the surface biomarkers of the single cells can be realized through the fluorescence microscope 7. When all the cell capture units 20 complete the capture of the sample cells, the flow rate of the cell suspension into the capture culture module 6 can be increased by the micro-injection pump 1 to apply mechanical stimulation to the captured single cells until they break through the narrow gap groove 27 of the corresponding size under the push of the large flow rate of the suspension and enter the cell capture large chamber 26, and then flow to the corresponding outlet II 18 or outlet III 19 for discharge, wherein the capillary tube d8 and the capillary tube e9 are connected to the outlet II 18 and the outlet III 19 respectively to collect the discharged sample cells subjected to mechanical stimulation for subsequent culture observation and corresponding analysis. Please continue to refer to Figure 7 As shown in Figure 7 The flow chart of the analysis method based on the integrated microfluidic system for single cell capture, stimulation and analysis provided by the embodiments of the present application is shown in Specifically, the present application also provides an analysis method based on any of the above integrated microfluidic systems for single cell capture, stimulation and analysis, comprising: Step S1, preparing a cell suspension, trypsinizing adherent cells and resuspending with a phosphate buffer; Step S2, diluting and mixing, injecting the cell suspension and the buffer into the pretreatment module 4 through the inlet I 11 and the inlet II 12 at the same flow rate, and using the bend secondary flow structure 14 to realize the dilution and mixing of the sample cells; wherein the sample cells are single cells of a corresponding size contained in the cell suspension and used for subsequent capture analysis; Step S3, single cell capture, injecting the diluted and mixed sample solution into the capture culture module 6 through the capillary tube c5 at a first preset flow rate, and capturing single cells of a required size by the cell capture structure 25; the sample solution is a cell solution containing sample cells to be captured; Step S4, culture or labeling, culturing the captured single cells or labeling the single cell surface markers by using an aptamer / antibody solution containing a fluorescent group; Step S5, imaging analysis, observing the cells subjected to culture or labeling by the fluorescence microscope 7, and detecting and analyzing the expression of the single cell surface markers.

[0041] The above analysis and detection method is still applicable to the processing and subsequent experiments of the patient's whole blood sample.

[0042] And according to the size of the cells, the selected CELL CC The gap width of the narrow gap groove 27 in the above can be different. Before that, the selected CELL CCWe optimized the flow of cells of different sizes and used seven different tumor cells (A375, Lovo, HepG2, A549, HCCLM3, Hela, and MDA-MB-231 cells) to verify different narrow gaps (d=8, 12μm). We concluded that when the cell size is 13-17μm, a chip with d=8μm is suitable and can be captured at a flow rate of 50μL / min; when the cell size is 18-20μm, a chip with d=12μm is suitable and can be captured at a flow rate of 150μL / min. That is, when the cell size is 13-17μm, a chip with d=8μm can be used in step 3 at a flow rate of 50μL / min; when the cell size is 18-20μm, a chip with d=12μm can be used in step 3 at a flow rate of 150μL / min. The cell size can be measured using a cell counting instrument. In step 1, the cell suspension is prepared by culturing cells in a cell culture flask in a corresponding complete medium until the cells adhere to the wall in the logarithmic growth phase, removing the medium and washing twice, then digesting the cells with trypsin, centrifuging and washing, and finally resuspending the centrifuged product in a phosphate buffer solution to prepare a cell suspension, wherein the phosphate particle concentration is 0.01 mol / LPO4 3- , pH=7.4; In step 9, the specific detection method is as follows: the fluorescence on the surface of the single cell is captured and photographed in dark field mode, the fluorescence intensity is extracted using ImageJ software, and different biomarkers on the surface of the single cell are analyzed based on the fluorescence intensity value to distinguish whether it undergoes carcinogenic transformation or other cellular behaviors.

[0043] Please continue reading Figure 8 ,like Figure 8 As shown, it is a flow chart of culturing captured single cells in an embodiment of the present application; In some specific embodiments, in step S4, the process of culturing the captured single cells further includes: Step S41, applying mechanical stimulation, increasing the flow rate to a second preset flow rate, and applying mechanical stimulation to the captured single cells to promote their deformation and secretion of exosomes; The second preset flow rate is greater than the first preset flow rate, and the selection of the first preset flow rate and the second preset flow rate should be adapted according to the size of the single cell to be captured; Step S42, culture medium preparation, collect the sample cells after mechanical stimulation, collect the sample cells after mechanical stimulation discharged from the outlet II 18 and the outlet III 19 through the capillary soft tube d8 and the capillary soft tube e9, place the collected sample cells in the cell culture bottle for culture until adherent and stable, then extract the exosomes in the ultra-high speed centrifuge, and add to the DMEM culture medium without serum, mix and use; Step S43, connected with step S3, introduce the culture medium prepared in step S42 through the capillary soft tube c5, and co-culture with the single cells captured by the cell capture structure 25 for three days.

[0044] Embodiment 1 An analysis method of an integrated microfluidic system for single cell capture, stimulation and analysis, which adopts the integrated microfluidic system for single cell capture, stimulation and analysis and the corresponding fluorescence microscope 7, comprising the following steps: Step 1, preparation of microsphere suspension: (1) According to the given microsphere original solution concentration, the concentration of the solution required by the experiment is 10 5 particles / mL; (2) Add Tween as diluent, shake well, filter and reserve.

[0045] Step 2, microfluidic chip pretreatment, lubricate the inside of the chip to prevent sedimentation and be more conducive to subsequent experiments; The microfluidic chip pretreatment step is: filtered Tween solution is provided by the microsyringe pump 1, and is introduced into the microfluidic device through the capillary soft tube a3 and the capillary soft tube b2 at a flow rate of 50 μL / min, until the solution flows out from the last outlet II 18 and the outlet III 19 of the device; Step 3, single microsphere capture: introduce the microsphere solution prepared in step 1 and the buffer (the buffer here is Tween) by the microsyringe pump 1, introduce the microsphere solution into the inlet I 11 through the capillary soft tube a3, and introduce the buffer into the inlet II 12 through the capillary soft tube b2, adopt 14.5 μm polystyrene fluorescent microspheres, and the microfluidic chip has a gap width d=8 μm, which is captured at the narrow gap groove 27 position under the condition of 50 μL / min sample flow rate; Step 4, detection of single microsphere capture: place the microfluidic chip on the stage of the fluorescence microscope 7, since the polystyrene fluorescent microspheres are introduced, the fluorescence signal thereof can be collected by the fluorescence microscope 7, and finally the capture condition of the 14.5 μm polystyrene microspheres is obtained; Repeat steps 1-4 to detect the 19.3 μm polystyrene fluorescent microspheres again.

[0046] It should be noted that the polystyrene fluorescent microspheres in this example cannot pass through the narrow gap within the flow rate range allowed by the microsyringe pump 1 to operate, but are all in a captured state; the gap width at the narrow gap groove 27 in the second microfluidic chip 16 is changed to d = 12 μm, and the critical point of the sample flow rate is 150 μL / min. Under the condition of a flow rate lower than 150 μL / min, the 19.3 μm fluorescent microspheres are all in a captured state, and under the condition of a flow rate higher than 150 μL / min, they are in a passing state, i.e., the mechanical extrusion state described.

[0047] Example 2 An analysis method of an integrated microfluidic system for single cell capture, stimulation and analysis, which adopts the integrated microfluidic system for single cell capture, stimulation and analysis and combines the corresponding fluorescence microscope 7, comprises the following steps: Step 1, preparation of a cell sample: cells are prepared into a cell suspension through trypsin digestion; The preparation method of the cell suspension is as follows: (1) In a cell culture bottle, cells are cultured to the logarithmic growth phase using complete culture medium (seven kinds of cells: A375, Lovo, HepG2, A549, HCCLM3, Hela and MDA-MB-231 cells are cultured in this step); (2) The cells are digested from the cell culture bottle by trypsin, and the digestion is stopped by adding complete culture medium, and the solution is collected; (3) The digested cells are collected and centrifuged, and washed twice with a phosphate buffer solution, wherein the phosphate ion particle concentration is 0.01 mol / L and pH = 7.4; (4) The cells in the cell suspension are counted using a cell counter, and the centrifugation product is resuspended with serum-free cell culture medium to prepare a cell suspension with a density of 10 5 cells / mL; Step 2, microfluidic chip pretreatment, lubricating the inside of the chip to prevent sedimentation and facilitate subsequent experiments; The microfluidic chip pretreatment step is: filtered culture medium solution without serum is provided by the microsyringe pump 1, and is introduced into the microfluidic device at a flow rate of 50 μL / min through the capillary soft tube a3 and the capillary soft tube b2, until the solution flows out from the last outlet II 18 and the outlet III 19 of the device; Step 3, single cell capture: the cell suspension solution prepared in step 1 and the buffer solution are introduced by the microsyringe pump 1, the cell suspension solution is introduced into the inlet I 11 by the capillary soft tube a3, and the buffer solution is introduced into the inlet II 12 by the capillary soft tube b2, and then the single cell capture is carried out by introducing the microfluidic chip through the capillary soft tube c5; The gap width of the microfluidic chip is d = 8 μm, and the cells are captured at the position of the narrow gap groove 27 at an injection flow rate of 50 μL / min; Step 4, single cell labeling: the microfluidic channel is washed with a phosphate buffer solution to remove unnecessary substances and avoid affecting observation, and then a fluorescent group modified aptamer solution / antibody solution is introduced into the microfluidic chip for incubation; Step 5, detection of single cell capture: the microfluidic chip is placed on the stage of the fluorescence microscope 7, and the captured single cells are modified with a fluorescent group, so that the fluorescence signal thereof can be collected by the fluorescence microscope 7, and finally the capture conditions of seven different single cells are obtained; Repeat steps 1-5 to detect different single cell suspensions.

[0048] It should be noted that, by verifying seven different tumor cells, the evaluation results of the polystyrene fluorescent microspheres are as follows: when the cell size introduced into the microfluidic device is 13-17 μm, the chip with d = 8 μm is suitable, and the cells can be captured at a flow rate of 50 μL / min; when the cell size introduced into the microfluidic device is 18-20 μm, the chip with d = 12 μm is suitable, and the cells can be captured at a flow rate of 150 μL / min.

[0049] Example 3 An analysis method of an integrated microfluidic system for single cell capture, stimulation and analysis, which combines the integrated microfluidic system for single cell capture, stimulation and analysis with a corresponding fluorescence microscope 7, includes the following steps: Step 1, preparing a cell sample: preparing cells into a cell suspension by trypsin digestion; The preparation method of the cell suspension is as follows: (1) In a cell culture bottle, the cells are cultured to the logarithmic growth phase using complete culture medium; (2) The cells are digested from the cell culture bottle by trypsin, and the digestion is stopped by adding complete culture medium, and the solution is collected; (3) The digested cells are collected and centrifuged, and washed twice with a phosphate buffer solution, wherein the phosphate ion concentration is 0.01 mol / L and pH = 7.4; (4) The cells in the cell suspension are counted using a cell counter, and the centrifuged product is resuspended with serum-free cell culture medium to prepare a cell suspension with a density of 10 5 cells / mL; Step 2, microfluidic chip pretreatment, lubricating the inside of the chip to prevent sedimentation and facilitate subsequent experiments; The microfluidic chip pre-treatment step is: providing the filtered culture medium solution without serum by the micro-injection pump 1, and the solution is introduced into the microfluidic device through the capillary tube a3 and the capillary tube b2 at a flow rate of 50 μL / min, until the solution flows out from the last outlet II 18 and the outlet III 19; Step 3, sample solution dilution and mixing: in the micron-scale channel, the solutions at the inlet I 11 and the inlet II 12 are accelerated and mixed under the action of the secondary flow in the bend, by using the flow transverse to the downstream fluid flow line; meanwhile, the cell suspension is sorted to a certain extent in the process, to become a relatively neat cell bundle, facilitating the capture and analysis of single cells in the next step.

[0050] Step 4, single cell capture: introducing the cell suspension solution prepared in step 1 and the buffer solution by the micro-injection pump 1, the cell suspension solution is introduced into the inlet I 11 by the capillary tube a3, the buffer solution is introduced into the inlet II 12 by the capillary tube b2, and then the single cell capture is performed by introducing the solution into the microfluidic chip through the capillary tube c5; The gap width d of the microfluidic chip is 8 μm, and the single cell is captured at the narrow gap groove 27 position at a sample flow rate of 50 μL / min; Step 5, mechanical stimulation on single cell: after the pre-treatment of the microfluidic chip in step 2, the cell suspension solution prepared in step 1 and the buffer solution are introduced by the micro-injection pump 1, the cell suspension solution is introduced into the inlet I 11 by the capillary tube a3, the buffer solution is introduced into the inlet II 12 by the capillary tube b2, and then the sample flow rate is adjusted to be greater than 50 μL / min to make the single cell deform and pass through the narrow gap to be mechanically extruded; the gap width d of the microfluidic chip is 8 μm; Step 6, extraction of single cell exosome: the cell after mechanical extrusion is placed in a culture bottle for culture, and after stable adhesion, the culture medium is replaced with serum-free culture medium for culture for more than 24 h, the culture medium is extracted, and the exosome is collected by high-speed centrifugation; the collected exosome is diluted to an appropriate concentration with a phosphate buffer solution, and is added into the serum-free culture medium for standby; Step 7, single cell co-culture: introducing the culture medium prepared in step 6 into the single cell captured in step 4, so that the exosome co-cultures with the single cell, and the carcinogenic transformation ability of different kinds of cancer cells is observed; Step 8, single cell labeling: washing the microfluidic channel with a phosphate buffer solution to wash away unnecessary substances and avoid affecting observation, and then introducing the aptamer solution / antibody solution modified with a fluorescent group into the microfluidic chip for incubation; Step 9, detection of single cell capture: the microfluidic chip is placed on the stage of the fluorescence microscope 7, and the captured single cells are modified by the fluorescent group, so that the fluorescence signal of the single cells can be collected by the fluorescence microscope 7, and finally the capture cell behavior state of different single cells is obtained; Step 10, subsequent culture of single cells: the inlet and outlet of the whole microfluidic device are replaced, and the original outlet II 18 and outlet III 19 are converted into inlet IV and inlet V, and inlet I 11 and inlet II 12 are converted into outlet IV and outlet V; the phosphate buffer solution is introduced from the inlet IV and the inlet V, and the solution is collected from the outlet IV and the outlet V, and is placed in a cell culture bottle for continuous culture, and the cells still maintain high activity.

[0051] Specifically, the integrated microfluidic system for single cell capture, stimulation and analysis provided by the embodiments of the present application can achieve the same technical effects by performing the above-mentioned analysis method, which will not be described here.

[0052] In the description of the present application, it should be understood that the orientations or positional relationships indicated by the terms "center", "longitudinal", "transverse", "length", "width", "thickness", "upper", "lower", "front", "rear", "left", "right", "vertical", "horizontal", "top", "bottom", "inner", "outer", "clockwise", "counterclockwise", "axial", "radial", "circumferential" and the like are based on the orientations or positional relationships shown in the drawings, and are only for the convenience of describing the present application and simplifying the description, and therefore cannot be understood as indicating or implying that the devices or elements indicated must have a particular orientation, be constructed and operated in a particular orientation, and therefore cannot be understood as limiting the present application.

[0053] In addition, the terms "first" and "second" are only for descriptive purposes, and cannot be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features. Therefore, the features defined as "first" and "second" can explicitly or implicitly include at least one of the features. In the description of the present application, the meaning of "a plurality of" is at least two, such as two, three, etc., unless otherwise explicitly specified and limited.

[0054] In the present application, unless otherwise explicitly specified and limited, the terms "mounting", "connection", "connection", "fixing" and the like should be understood in a broad sense, for example, can be fixedly connected, or can be detachably connected, or can be integrated; can be mechanically connected, or can be electrically connected or can communicate with each other; can be directly connected, or can be indirectly connected through an intermediate medium; can be the internal communication of two elements or the interaction relationship between two elements, unless otherwise explicitly limited. For those skilled in the art, the specific meaning of the above terms in the present application can be understood according to the specific circumstances.

[0055] In this application, the terms "one embodiment", "some embodiments", "an example", "a specific example", or "some examples" mean that a particular feature, structure, material, or characteristic is included in at least one embodiment or example of the present application. The illustrative appearances of the above-mentioned terms in various places in the specification are not necessarily intended to refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics can be combined in any suitable manner in one or more embodiments or examples. Moreover, the terms "first", "second", "third", etc. are used herein to describe various embodiments or examples and are not necessarily intended to denote a particular order, position, or priority of the elements being described. In addition, the terms "comprises", "comprising", "includes", "including", "has", "having", "contains", "containing", or any other similar word(s), are intended to be inclusive or open ended and not restrictive or exhaustive. In other words, the terms "comprises", "comprising", "includes", "including", "has", "having", "contains", "containing", or any other similar word(s), are intended to cover a special embodiment or example, but not exclude other embodiments or examples. In addition, the terms "an" and "one" are intended to be inclusive or open ended and not restrictive or exhaustive. In other words, the terms "an" and "one" are intended to cover both the singular aspect and the plural aspect, unless the context clearly indicates otherwise.

[0056] Although the embodiments of the present application have been shown and described above, it is understood that the above-described embodiments are exemplary, and are not to be interpreted as limiting the present application, and that the ordinary skilled person in the art can make changes, modifications, replacements, and variations to the above-described embodiments within the scope of the present application.

Claims

1. An integrated microfluidic system for single-cell capture, stimulation, and analysis, characterized in that: include: The pre-processing module comprises a first microfluidic chip having an inlet I, an inlet II and an outlet I, wherein the first microfluidic chip is further provided with a micron-scale curved secondary flow structure; A capture and culture module comprises a second microfluidic chip having an inlet III, an outlet II, and an outlet III, wherein a plurality of cell capture units are integrated in the second microfluidic chip, and each of the cell capture units is provided with a plurality of cell capture structures; A microinjection pump connected to the pre-treatment module via capillary hoses a and b; The pre-treatment module and the capture and cultivation module are connected via a capillary hose c; The curved secondary flow structure is composed of a plurality of semicircular curved channels, and the angle between the inner wall of the curved channel and the horizontal direction is 20°; The plurality of cell capture units are connected in sequence, and each of the cell capture units has a cell capture inlet and three cell capture outlets; The cell capture structure is disposed between the cell capture inlet and the cell capture outlet; There are two cell capture structures provided in any one of the cell capture units, and each of the cell capture structures includes a small cell capture chamber, a large cell capture chamber, and a narrow gap groove.

2. The integrated microfluidic system for single cell capture, stimulation and analysis according to claim 1, characterized in that: One end of the cell capture chamber is connected to the cell capture inlet, and the other end is connected to the cell capture large chamber through the narrow gap groove.

3. The integrated microfluidic system for single cell capture, stimulation and analysis according to claim 2, characterized in that: The cell capture outlet comprises: a first cell capture outlet, one end of which is connected to the cell capture chamber, and the other end of which is connected to the outlet II or the outlet III; Second cell capture outlet.

4. The integrated microfluidic system for single cell capture, stimulation and analysis according to claim 3, characterized in that: The cell capture inlet of any cell capture unit except the first end is connected to the second cell capture outlet of the adjacent previous cell capture unit; Wherein, the cell capture inlet of the cell capture unit at the head end is connected to the outlet I through the capillary hose c.

5. The integrated microfluidic system for single cell capture, stimulation and analysis according to claim 1, characterized in that: The output end of the microinjection pump is connected to the inlet I through the capillary hose a, and is connected to the inlet II through the capillary hose b.

6. The integrated microfluidic system for single cell capture, stimulation and analysis according to claim 1, characterized in that: The system further comprises: a fluorescence microscope, disposed above the capture and culture module; Capillary hose d and capillary hose e are connected to the outlet II and the outlet III respectively.

7. An analysis method based on the integrated microfluidic system for single cell capture, stimulation and analysis according to any one of claims 1 to 6, characterized in that: include: Step S1, preparing a cell suspension by trypsinizing adherent cells and resuspending them in phosphate buffered saline; Step S2: dilution and mixing: inject the cell suspension and buffer solution into the pretreatment module through inlet I and inlet II at the same flow rate, and use the curved secondary flow structure to achieve dilution and mixing of the sample cells; Step S3, single cell capture, passing the diluted and mixed sample solution into the capture and culture module through the capillary tube c at a first preset flow rate, and capturing single cells of a desired size through the cell capture structure; Step S4, culturing or labeling, culturing the captured single cells or labeling the single cell surface markers with an aptamer / antibody solution containing a fluorescent group label; Step S5, imaging analysis, observing the cultured or labeled cells through a fluorescence microscope, and detecting and analyzing the expression of single cell surface markers.

8. The analysis method of the integrated microfluidic system for single cell capture, stimulation and analysis according to claim 7, characterized in that: In step S4, the process of culturing the captured single cells further includes: Step S41, applying mechanical stimulation, increasing the flow rate to a second preset flow rate, and applying mechanical stimulation to the captured single cells to promote their deformation and secretion of exosomes; Step S42, culture medium preparation, collecting sample cells after mechanical stimulation, placing the collected sample cells in a cell culture flask and culturing them until they adhere to the wall and stabilize, then placing them in an ultra-high-speed centrifuge to extract exosomes, and adding them to serum-free DMEM culture medium, mixing them and setting aside for use; Step S43 , following step S3 , introduces the culture medium prepared in step S42 through the capillary tube c and co-cultures it with the single cells captured by the cell capturing structure.

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