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

Abstract

The application relates to the field of microfluidic chips, cell culture and analysis, in particular to an integrated microfluidic system and method for single cell capture, stimulation and analysis, wherein the integrated microfluidic system for single cell capture, stimulation and analysis is composed of two microfluidic chips in front and back parts, and the front half part and the rear half part are connected through a capillary hose c. The application realizes sorting capture, mechanical stimulation and culture analysis of single cells through an integrated pretreatment module and a capture culture module. The first microfluidic chip located at the pretreatment module makes the cell suspension flowing into the pretreatment module realize accelerated mixing under the action of the secondary flow of the bend, and the cells in the cell suspension are sorted to facilitate the next step. The second microfluidic chip located at the rear half part and composed of multiple cell capture units captures and cultures sample cells with a required size 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:

[0007] Single function: only sorting can be achieved, and capture, stimulation, culture and analysis cannot be integrated;

[0008] Complex structure: the microstructure design needs to be optimized repeatedly to adapt to different cell sizes;

[0009] 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).

[0010] 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. Based on the above, the single-cell separation and analysis methods commonly used in existing technologies based on microfluidic technology often have the following problems:

[0011] Low integration: sample dilution, single-cell capture, stimulation culture and analysis need to be operated step by step, which is easy to introduce pollution and low in efficiency;

[0012] Poor flexibility: it is difficult to quickly adjust the capture parameters according to the required analysis cell size to obtain more accurate measurement results;

[0013] Insufficient signal amplification: lack of controllable stimulation means for single cells, making it difficult to amplify small cancer signals.

[0014] 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

[0015] 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.

[0016] The application provides an integrated microfluidic system for single-cell capture, stimulation and analysis, comprising:

[0017] 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.

[0018] 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.

[0019] The microsyringe pump is connected to the pretreatment module through a capillary hose a and a capillary hose b.

[0020] The pretreatment module and the capture culture module are connected through a capillary hose c.

[0021] 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°.

[0022] 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.

[0023] 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.

[0024] 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.

[0025] Preferably, the cell capture outlet comprises:

[0026] a first cell capture outlet connected to the cell capture chamber at one end and connected to the outlet II or the outlet III at the other end;

[0027] a second cell capture outlet.

[0028] Preferably, the cell capture inlet of any one of the cell capture units except the first end and the last end is connected to the second cell capture outlet of the adjacent last cell capture unit.

[0029] Preferably, the cell capture inlet of the cell capture unit at the first end is connected to the outlet I through the capillary tube c.

[0030] Preferably, the output end of the micro-injection pump is connected to the inlet I through the capillary tube a and connected to the inlet II through the capillary tube b.

[0031] Preferably, the system further comprises:

[0032] a fluorescence microscope arranged above the capture culture module.

[0033] a capillary tube d and a capillary tube e connected to the outlet II and the outlet III respectively.

[0034] The application also provides an analysis method for the integrated microfluidic system for single cell capture, stimulation and analysis based on any one of the above, comprising:

[0035] Step S1, preparing a cell suspension, trypsinizing adherent cells and resuspending with a phosphate buffer;

[0036] Step S2, diluting and mixing, injecting the cell suspension and the buffer into the pretreatment 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 curved channel secondary flow structure;

[0037] 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 the cell capture structure;

[0038] 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;

[0039] 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.

[0040] Preferably, in the step S4, the process of culturing the captured single cells further comprises:

[0041] Step S41, applying mechanical stimulation, increasing the flow rate to a second preset flow rate, applying mechanical stimulation to the captured single cell to promote the generation of its deformation and the secretion of exosomes;

[0042] Step S42, culture medium preparation, collecting the sample cells after mechanical stimulation, placing the collected sample cells in a cell culture bottle for culture until they adhere and stabilize, then placing them in an ultracentrifuge to extract exosomes, and adding them to a serum-free DMEM culture medium, mixing well for use;

[0043] Step S43, as in step S3, introducing the culture medium prepared in step S42 through the capillary tube c to co-culture with the single cells captured by the cell capture structure.

[0044] The beneficial effects of the present application are as follows:

[0045] 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 capture culture module. The first microfluidic chip located at the pretreatment module is designed according to the "pirate ship" swing path, so that the cell suspension introduced into the pretreatment module can realize accelerated mixing and sorting of cells in the cell suspension into a relatively neat cell bundle by using the flow transverse to the downstream fluid flow line under the action of the secondary flow in the bend, facilitating the next step. The second microfluidic chip located in the latter part composed of multiple cell capture units captures and cultures sample cells of the required size for subsequent observation and analysis. The mechanical stimulation of the captured single cell can also be applied by increasing the injection flow rate of the microsyringe pump to promote the secretion of its exosomes and the expression of related proteins, amplify some key microsignals, and thus more likely to observe cancer, facilitating early monitoring and prevention of cancer.

[0046] Further, compared with traditional active sorting microfluidic chips, no external conditions need to be added, and differences in power supply or piezoelectric devices do not need to be considered, and single cell capture can be performed more simply and quickly;

[0047] Especially, compared with traditional passive sorting microfluidic chips, there is no need to repeatedly optimize the structure, and the availability of the simulation structure can be used, and the relatively simpler structure is used to realize the capture and culture of single cells, which saves costs to a certain extent. BRIEF DESCRIPTION OF DRAWINGS

[0048] In order to more clearly illustrate the technical solutions in the specific embodiments or prior art of the present application, the drawings needed in the specific embodiments or prior art description will be briefly introduced below. 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.

[0049] Figure 1 The overall structure schematic diagram of the integrated microfluidic system for single cell capture, stimulation and analysis provided in the embodiments of the present application is shown in the figure.

[0050] Figure 2 The overall structure schematic diagram of the pretreatment module in the embodiments of the present application is shown in the figure.

[0051] 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.

[0052] Figure 4 The overall structure schematic diagram of the capture culture module in the embodiments of the present application is shown in the figure.

[0053] Figure 5 The overall structure schematic diagram of the cell capture unit in the embodiments of the present application is shown in the figure.

[0054] Figure 6 The structure schematic diagram of the cell capture structure in the cell capture unit of the embodiments of the present application is shown in the figure.

[0055] Figure 7 The flow chart of the analysis method based on the integrated microfluidic system for single cell capture, stimulation and analysis provided in the embodiments of the present application is shown in the figure.

[0056] Figure 8 The flow chart of the culture of the captured single cell in the embodiments of the present application is shown in the figure.

[0057] Reference signs:

[0058] 1, microsyringe pump; 2, capillary tube b; 3, capillary tube a; 4, pretreatment module; 5, capillary tube c; 6, capture 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, bend secondary flow structure; 15, bend angle schematic diagram; 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

[0059] The technical solutions of the present application will be described clearly and completely below in conjunction with embodiments. Obviously, the described embodiments are only part of, rather than all of, the embodiments of the present application. Based on the embodiments in the present application, all other embodiments obtained by a person of ordinary skill in the art without creative effort belong to the scope of protection of the present application.

[0060] The integrated microfluidic system and method for single cell capture, stimulation and analysis provided in the embodiments of the present application will be described below in conjunction with Figures 1-8

[0061] Referring to FIG. 1, the integrated microfluidic system for single cell capture, stimulation and analysis provided in the embodiments of the present application includes a pretreatment module 4 composed of two microfluidic chips integrated in front and back two parts, and the front half part and the rear half part are connected through a capillary tube c5. The first microfluidic chip 10 at the pretreatment module 4 in the front half part is designed according to a "pirate ship" swing path, and the included angle between the inclined angle of the curved part and the horizontal direction is 20°. The chip has two sample inlets and one sample outlet. The two sample inlets are inlet I 11 and inlet II 12 which are arranged in parallel at the head end of the curved secondary flow structure 14, and the input ends of the two sample inlets are connected with a microsyringe pump 1. The sample outlet is outlet I 13 arranged at the tail end of the curved secondary flow structure 14. Figure 1 The sample solution (trypsin-digested cell suspension) containing the required capture and detection cells is injected into the pretreatment module 4 through inlet I 11 and inlet II 12 by the microsyringe 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 when they flow to the outlet I.

[0062] The second microfluidic chip 16 in the rear half part has one sample inlet, i.e. inlet III 17, and two sample outlets, i.e. outlet II 18 and outlet III 19. The sample cells mixed in the curved secondary flow structure 14 flow out from outlet I 13, pass through the capillary tube c5, and flow into inlet III 17. The sample cells enter the inside of the second microfluidic chip 16 in which a plurality of cell capture units 20 are integrated, and are captured and cultured by the cell capture structure 25 in the cell capture unit 20 to wait for subsequent observation and analysis.

[0063]

[0064] ​​The sorting and capturing, mechanical stimulation and culture analysis of single cells are realized by the integrated pre-processing module 4 and the capturing culture module 6. The first microfluidic chip 10 at the pre-processing module 4 is designed according to the swinging path of the "pirate ship", so that the cell suspension flowing into the pre-processing module 4 can realize accelerated mixing by using the flow transverse to the downstream fluid flow line under the action of the bend secondary flow. At the same time, the cell suspension is sorted to a certain extent to become a relatively neat cell bundle, which is convenient for the next step. The second microfluidic chip 16 at the latter part composed of a plurality of cell capturing units 20 captures and cultures the sample cells of the required size for subsequent observation and analysis.

[0065] Further, compared with the traditional active sorting microfluidic chip, no external conditions need to be added, and the differences of power supply or piezoelectric device do not need to be considered, and the single cell can be captured more simply and quickly.

[0066] Especially, compared with the traditional passive sorting microfluidic chip, no repeated optimization of structure is needed, and the simulation structure availability can be used, and the single cell can be captured and cultured by a relatively simple structure, which saves the cost to a certain extent.

[0067] Further, not only the capture of single cells is realized, but also it is not limited to this function. On the basis of capturing and sorting, in-situ culture and observation can be carried out, and the analysis of cell behavior can be carried out. After the observation in the chip is completed, the activity is still not affected, and the subsequent analysis can be carried out.

[0068] The mechanical stimulation can be applied to the captured single cells by increasing the injection flow rate, promoting the secretion of exosomes and the expression of related proteins, amplifying some key micro signals, and thus more probability of observing cancer is obtained, so that the early monitoring and prevention of cancer can be facilitated.

[0069] Especially, not only high-throughput experiments can be realized, but also the capturing and culture module 6 at the latter part can process multiple micro culture chambers at the same time, saving time and resources. At the same time, the cell positioning and observation can be carried out by the fluorescence microscope 7, the growth and interaction process of the cells can be monitored in real time, and the behavior of the cells can be understood more deeply.

[0070] Please continue to refer to Figure 2 and Figure 3 as shown in Figure 2 and Figure 3 , wherein Figure 2 is the overall structure diagram of the pre-processing module in the embodiment of the application, Figure 3 is the angle position diagram of the bend secondary flow structure;

[0071] In some specific embodiments, the curved secondary flow structure 14 consists of multiple semi-circular curves, with the angle between the inner wall of the curve and the horizontal direction being 20°.

[0072] Specifically, the curved secondary flow structure 14 is a micron-level path channel composed of at least 8 micron-level semi-circular bends, resembling a "pirate ship" swinging path. For example, the curved secondary flow structure 14 shown in this embodiment is a path channel composed of 8 bends used to accelerate sample dilution and cell bundle sorting. In the embodiment shown, the channel size is 39.60 mm × 27.00 mm, the channel width is 400 μm, and the channel height is 50 μm.

[0073] Please continue reading. Figure 4 ,like Figure 4 As shown, it is a schematic diagram of the overall structure of the capture and culture module in an embodiment of this application;

[0074] In some specific embodiments, multiple cell capture units 20 are connected in sequence, each cell capture unit 20 having a cell capture inlet 21 and three cell capture outlets, and a cell capture structure 25 is disposed between the cell capture inlet 21 and the cell capture outlets.

[0075] For example, in this embodiment, the cell capture culture chip (CELL) is composed of the second microfluidic chip 16 in the latter half. CC It consists of 50 cell capture units 20 connected end to end, and each individual cell capture unit 20 has one cell capture inlet 21, three cell capture outlets, and two cell capture structures 25 for capturing single cells of the required size. Integrating them, there are a total of 100 cell capture structures 25 for capturing cells. CC The chip size is approximately 81.72 mm × 29.56 mm, and the channel height is 20 μm.

[0076] Please continue reading. Figure 5 and Figure 6 ,like Figure 5 and Figure 6 As shown, where, Figure 5 This is a schematic diagram of the overall structure of the cell capture unit in an embodiment of this application. Figure 6 This is a schematic diagram of the cell capture structure in the cell capture unit of this application embodiment;

[0077] In some specific embodiments, there are two cell capture structures 25 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.

[0078] 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 through a narrow gap groove 27.

[0079] The intercompartmental radius of the large cell capture chamber 26 is more than ten times that of the intercompartmental radius of the small cell capture chamber 28. For example, in this embodiment, the radius of the selected small cell capture chamber 28 is 60 μm. The channel gap d at the narrow gap groove 27 is designed with multiple sizes that common tumor cells may exist, such as 4, 8, 12, 16, 18, 20, and 22 μm, 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 the large cell capture chamber 26 and the small 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.

[0080] In some specific embodiments, the cell capture outlet includes:

[0081] The first cell capture outlet has one end connected to the cell capture chamber 26 and the other end connected to outlet II 18 or outlet III 19.

[0082] like Figure 5 As shown, each cell capture unit is provided with two first cell capture outlets, namely first cell capture outlet a22 and first cell capture outlet b24. The two first cell capture outlets are located in a V-shape on both sides of the cell capture inlet 21.

[0083] The second cell capture outlet 23 is located between the two cell capture structures 25.

[0084] The cell suspension flowing from the pretreatment module 4 flows into the capture culture module 6 after passing through a straight channel formed by a capillary tube c5, and then flows into a corresponding cell capture unit 20 through the cell capture inlet 21. When flowing through the fork inlet, sample cells of the corresponding size are intercepted and captured at the narrow gap groove 27 and remain in the cell capture chamber 28 to await stimulation or culture. After capturing the sample cells of the corresponding size, the cell capture chamber 28 at the current position is blocked by a corresponding single cell, and the uncaptured sample cells are transported through the channel located at the... The second cell capture outlet 23 in the middle position flows into the next cell capture unit 20. After all cell capture units 20 have completed the capture of sample cells, the flow rate of the cell suspension introduced 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 deform under the impetus of the high-flow suspension and break through the interception of the corresponding size narrow gap groove 27 and enter the cell capture chamber 26. Then, they flow through the first cell capture outlet to the corresponding outlet II 18 or outlet III 19 for discharge and collection.

[0085] In some embodiments, the cell capture inlet 21 of any one cell capture unit 20 except the first and the last is connected with the second cell capture outlet 23 of the adjacent last cell capture unit 20;

[0086] In some embodiments, the cell capture inlet 21 of the cell capture unit 20 at the first end is connected with the outlet I 13 through a capillary tube c5, i.e. one end of the capillary tube c5 is connected with the outlet I 13 and the other end is connected with the cell capture inlet 21 at the first end;

[0087] The second cell capture outlet 23 of the cell capture unit 20 at the last end can be connected with an external recovery container through a corresponding tube.

[0088] In some embodiments, the output end of the micro-injection pump 1 is connected with the inlet I 11 through a capillary tube a3 for introducing the treated cell suspension and is connected with the inlet II 12 through a capillary tube b2 for introducing the buffer.

[0089] In some embodiments, the system further comprises:

[0090] A fluorescence microscope 7 is arranged above the capture culture module 6 for performing fluorescence imaging analysis on the captured single cells in the latter half of the microfluidic chip. The fluorescence microscope 7 is arranged to make the system function of the present 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.

[0091] A capillary tube d8 and a capillary tube e9 are connected with the outlet II 18 and the outlet III 19 respectively 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.

[0092] The working process of the integrated microfluidic system for single cell capture, stimulation and analysis provided in the present application is as follows:

[0093] The micro-injection pump 1 is connected with the inlet I 11 through a capillary tube a3 for introducing the treated cell suspension and is connected with the inlet II 12 through a capillary tube b2 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 curve, flows through the four semicircles respectively on the left and right sides, and is mixed. The mixed sample solution flows out of the outlet I 13 from the pre-treatment module 4, completing the pre-treatment of the sample cells.

[0094] The cell suspension flowing out of the pre-processing module 4 flows into the capture culture module 6 after passing through a straight channel formed by the capillary tube c5, and flows into a corresponding cell capture unit 20 at the cell capture inlet 21. When passing through the bifurcation, sample cells of a corresponding size are intercepted and captured at the narrow gap groove 27 and stay in the cell capture chamber 28 for stimulation or culture. After the cell capture chamber 28 at the current position is captured with sample cells of a corresponding size, the sample cells that are not captured flow into the next cell capture unit 20 through the second cell capture outlet 23 at the middle position. After all the cell capture units 20 complete the capture of sample cells, the captured sample cells can be co-cultured by adding a culture medium containing different types of exosomes in the cell capture chamber 28, and then the detection of the surface biomarkers of single cells can be realized by fluorescence microscope 7 after connecting a solution of fluorescent group-labeled aptamer / antibody.

[0095] When all the cell capture units 20 complete the capture of sample cells, the flow rate of the cell suspension flowing into the capture culture module 6 can be increased by the micro-injection pump 1 to apply mechanical stimulation to the captured single cells. When the single cells are deformed and break through the interception of the narrow gap groove 27 of a corresponding size under the push of the high flow rate of the suspension, they enter the cell capture large chamber 26 and flow to the corresponding outlet II 18 or outlet III 19 for discharge through the first cell capture outlet. 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.

[0096] Please continue to refer to Figure 7 As Figure 7 shown, it is a flow chart of an analysis method based on the integrated microfluidic system for single cell capture, stimulation and analysis provided by the embodiments of the present application;

[0097] Specifically, the present application also provides an analysis method based on any one of the above integrated microfluidic system for single cell capture, stimulation and analysis, comprising:

[0098] Step S1, preparing a cell suspension, trypsinizing adherent cells and resuspending with a phosphate buffer;

[0099] Step S2, diluting and mixing. The cell suspension and the buffer are injected into the pre-processing module 4 through the inlet I 11 and the inlet II 12 at the same flow rate, and the dilution and mixing of the sample cells are realized by using the bend secondary flow structure 14. The sample cells are single cells of a corresponding size contained in the cell suspension and can be used for subsequent capture analysis;

[0100] Step S3, single cell capture, the sample solution after mixing and dilution is introduced into the capture culture module 6 through the capillary tube c5 at a first preset flow rate, and the single cells of the required size are captured by the cell capture structure 25; the sample solution is a cell solution containing the sample cells to be captured;

[0101] Step S4, culture or labeling, the captured single cells are cultured or the surface markers of the single cells are labeled by the solution containing the fluorescent group labeled aptamer / antibody;

[0102] Step S5, imaging analysis, the cells after culture or labeling are observed by the fluorescence microscope 7, and the expression of the surface markers of the single cells is detected and analyzed.

[0103] The above analysis and detection method is still applicable to the processing and subsequent experiments of the whole blood sample of the patient.

[0104] And according to the size of the cells, the selected CELL CC The gap width of the narrow gap groove 27 can be different, and before that, the CELL CC is optimized for cells of different sizes to flow through, and 7 different tumor cells (A375, Lovo, HepG2, A549, HCCLM3, Hela and MDA-MB-231 cells) are used to verify different narrow gaps (d=8, 12 μm), and finally it is concluded that when the cell size is 13-17 μm, the chip with d=8 μm can be used to capture at a flow rate of 50 μL / min; when the cell size is 18-20 μm, the chip with d=12 μm can be used to capture at a flow rate of 150 μL / min, that is, when the cell size is 13-17 μm, the chip with d=8 μm can be selected in step 3, and the flow rate is 50 μL / min; when the cell size is 18-20 μm, the chip with d=12 μm is selected in step 3, and the flow rate is 150 μL / min, and the size of the cells can be measured by a cell counting instrument;

[0105] In step 1, the preparation method of the cell suspension is as follows: in a cell culture bottle, the cells incubated in the corresponding complete culture medium are adhered to the logarithmic growth phase, the culture medium is removed and washed twice, then the cells are digested with trypsin, washed after centrifugation, and finally the centrifuged product is resuspended with a phosphate buffer solution to prepare a cell suspension, wherein the phosphate particle concentration is 0.01 mol / L PO4 3- , pH=7.4;

[0106] In step 9, the specific detection method is as follows: the fluorescence on the surface of single cells is captured and photographed in dark field mode, the fluorescence intensity is extracted through ImageJ software, and the different biomarkers on the surface of single cells are analyzed according to the fluorescence intensity value to distinguish whether they are carcinogenically transformed or other cell behaviors.

[0107] Please continue to refer to Figure 8 As Figure 8 shown, it is a flow chart of culturing the captured single cells in the embodiments of the present application;

[0108] In some specific embodiments, the process of culturing the captured single cells in step S4 further includes:

[0109] Step S41, applying mechanical stimulation, increasing the flow rate to a second preset flow rate, applying mechanical stimulation to the captured single cells to promote the generation of deformation and the secretion of exosomes;

[0110] Wherein, 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 to the size of the single cells to be captured;

[0111] Step S42, culture medium preparation, collecting the sample cells after mechanical stimulation, collecting 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, placing the collected sample cells in a cell culture bottle for culture until they adhere to the wall and are stable, then placing them in an ultrahigh-speed centrifuge to extract exosomes, and adding them to a DMEM culture medium without serum and mixing for use;

[0112] Step S43, connecting step S3, introducing the culture medium prepared in step S42 through the capillary soft tube c5, and co-culturing with the single cells captured by the cell capture structure 25 for three days.

[0113] Embodiment 1

[0114] An analysis method of an integrated microfluidic system for single cell capture, stimulation and analysis, which adopts an integrated microfluidic system for single cell capture, stimulation and analysis combined with a corresponding fluorescence microscope 7, including the following steps:

[0115] Step 1, preparation of microsphere suspension:

[0116] (1) According to the given concentration of the microsphere original solution, the concentration of the solution required for the experiment is 10 5 particles / mL;

[0117] (2) Add Tween as a diluent, shake well, filter and reserve.

[0118] Step 2, microfluidic chip pretreatment, lubricate the inside of the chip, prevent sedimentation, and make it more conducive to subsequent experiments;

[0119] The microfluidic chip pretreatment step is as follows: the 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;

[0120] Step 3, single microsphere capture: the microsphere solution prepared in step 1 and the buffer (here, the buffer is Tween) are introduced by the microsyringe pump 1, the microsphere solution is introduced into the inlet I 11 by the capillary soft tube a3, and the buffer is introduced into the inlet II 12 by the capillary soft tube b2, the 14.5 μm polystyrene fluorescent microspheres are captured at the narrow gap groove 27 position of the microfluidic chip at a sample flow rate of 50 μL / min;

[0121] Step 4, detection of single microsphere capture: the microfluidic chip is placed 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;

[0122] Repeat steps 1-4 to detect the 19.3 μm polystyrene fluorescent microspheres again.

[0123] It should be noted that in this example, the polystyrene fluorescent microspheres cannot pass through the narrow gap within the sample flow rate range allowed by the microsyringe pump 1, but are all in a captured state; the gap width at the narrow gap groove 27 of the second microfluidic chip 16 is changed to d=12 μm, and the critical 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.

[0124] Example 2

[0125] An analysis method of an integrated microfluidic system for single cell capture, stimulation and analysis, which adopts an integrated microfluidic system for single cell capture, stimulation and analysis and a corresponding fluorescence microscope 7, and includes the following steps:

[0126] Step 1, preparation of a cell sample: cells are prepared into a cell suspension by trypsin digestion;

[0127] The preparation method of the cell suspension is as follows:

[0128] (1) In the cell culture bottle, the cells are cultured to logarithmic growth phase using complete medium (this step is to culture seven kinds of cells: A375, Lovo, HepG2, A549, HCCLM3, Hela and MDA-MB-231 cells);

[0129] (2) The cells are digested from the cell culture bottle by trypsin, and the digestion is stopped by adding complete medium, and the solution is collected;

[0130] (3) The cells after digestion are collected and centrifuged, and washed twice with phosphate buffer solution, wherein the phosphate ion particle concentration is 0.01 mol / L, and pH=7.4;

[0131] (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;

[0132] Step 2, microfluidic chip pretreatment, lubricate the inside of the chip, prevent sedimentation, and be more conducive to subsequent experiments;

[0133] The microfluidic chip pretreatment step is: filtered medium solution without serum is provided by microsyringe pump 1, and is introduced into the microfluidic device through capillary soft tube a3 and capillary soft tube b2 at a flow rate of 50 μL / min, until the solution flows out from outlet II 18 and outlet III 19 of the device;

[0134] Step 3, single cell capture: introduce the cell suspension solution prepared in step 1 and the buffer solution by microsyringe pump 1, the cell suspension solution is introduced into inlet I 11 by capillary soft tube a3, and the buffer solution is introduced into inlet II 12 by capillary soft tube b2, and then introduced into the microfluidic chip through capillary soft tube c5 for single cell capture;

[0135] The gap width d of the microfluidic chip is 8 μm, and the captured single cell is at the position of the narrow gap groove 27 under the sampling flow rate of 50 μL / min;

[0136] Step 4, single cell labeling: wash the microfluidic channel with phosphate buffer solution to wash away unnecessary substances and avoid affecting observation, and then introduce the fluorescent group modified aptamer solution / antibody solution into the microfluidic chip for incubation;

[0137] Step 5, detection of single cell capture: place the microfluidic chip on the stage of fluorescence microscope 7, since the captured single cell is modified by the fluorescent group, the fluorescence signal thereof can be collected by fluorescence microscope 7, and finally the capture conditions of seven different single cells are obtained;

[0138] Repeat steps 1-5 to detect different single cell suspensions again.

[0139] It should be noted that the evaluation results of the auxiliary polystyrene fluorescent microspheres are verified by seven different tumor cells: when the cell size passing into the microfluidic device is 13-17 μm, the chip suitable for d=8 μm can be captured at a flow rate of 50 μL / min; when the cell size passing into the microfluidic device is 18-20 μm, the chip suitable for d=12 μm can be captured at a flow rate of 150 μL / min.

[0140] Example 3

[0141] An analysis method of an integrated microfluidic system for single cell capture, stimulation and analysis, which adopts an integrated microfluidic system for single cell capture, stimulation and analysis and a corresponding fluorescence microscope 7, comprises the following steps:

[0142] Step 1, preparing a cell sample: preparing cells into a cell suspension through trypsin digestion;

[0143] The preparation method of the cell suspension is as follows:

[0144] (1) In a cell culture bottle, the cells are cultured to the logarithmic growth phase using complete culture medium;

[0145] (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;

[0146] (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;

[0147] (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;

[0148] Step 2, microfluidic chip pretreatment, lubricating the inside of the chip to prevent sedimentation and facilitate subsequent experiments;

[0149] The microfluidic chip pretreatment step is: the filtered culture medium solution without serum is provided by the microsyringe pump 1, and is passed 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 Ⅱ18 and the outlet Ⅲ19 of the device;

[0150] Step 3, sample solution dilution mixing: in the micron channel, the solutions of inlet I 111 and inlet II 112 are mixed by the secondary flow of the bend, using the flow transverse to the downstream fluid flow line, achieving accelerated mixing; at the same time, the cell suspension is also sorted to a certain extent, becoming a relatively neat cell bundle, facilitating the next step of single cell capture analysis.

[0151] Step 4, single cell capture: introduce the cell suspension solution prepared in step 1 and the buffer solution by micro-injection pump 1, the cell suspension solution is introduced into inlet I 111 by capillary tube a3, the buffer solution is introduced into inlet II 112 by capillary tube b2, and then the single cell capture is carried out by introducing the capillary tube c5 into the microfluidic chip;

[0152] The gap width d of the microfluidic chip is 8 μm, and the captured single cell is at the position of the narrow gap groove 27 under the sampling flow rate of 50 μL / min;

[0153] Step 5, mechanical stimulation on single cell: after the pretreatment of the microfluidic chip in step 2, introduce the cell suspension solution prepared in step 1 and the buffer solution by micro-injection pump 1, the cell suspension solution is introduced into inlet I 111 by capillary tube a3, the buffer solution is introduced into inlet II 112 by capillary tube b2, and then the microfluidic chip is introduced by capillary tube c5, the sampling flow rate is adjusted to be greater than 50 μL / min, so that the single cell is deformed and squeezed by the narrow gap; the gap width d of the microfluidic chip is 8 μm;

[0154] 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 hours, the culture medium is extracted, and the exosome is collected by high-speed centrifugation; the collected exosome is diluted to an appropriate concentration with phosphate buffer solution and added to serum-free culture medium for standby;

[0155] Step 7, single cell co-culture: introduce 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 cancer cells is observed;

[0156] Step 8, single cell labeling: wash the microfluidic channel with phosphate buffer solution to wash away unnecessary substances and avoid affecting observation, and then introduce the aptamer solution / antibody solution modified with fluorescent groups into the microfluidic chip for incubation;

[0157] Step 9, detection of single cell capture: place the microfluidic chip on the stage of fluorescence microscope 7, since the captured single cell is modified with fluorescent groups, the fluorescence signal of the single cell can be collected by fluorescence microscope 7, and finally the capture cell behavior state of different single cells is obtained;

[0158] Step 10, single cell follow-up culture: replace the inlet and outlet of the whole microfluidic device, the original outlet II 18 and outlet III 19 are transformed into inlet IV and inlet V, inlet I 11 and inlet II 12 are transformed into outlet IV and outlet V; the phosphate buffer solution is introduced from the inlet IV and the inlet V, 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.

[0159] 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.

[0160] In the description of the present application, it should be understood that 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 indicate the orientation or positional relationship based on the orientation or positional relationship shown in the drawings, and are only for the convenience of describing the present application and simplifying the description, and do not indicate or imply that the device or element referred to must have a particular orientation, be constructed and operated in a particular orientation, and therefore cannot be understood as a limitation on the present application.

[0161] In addition, the terms "first", "second" are only for descriptive purpose, and cannot be understood as indicating or implying relative importance or implicitly indicating the number of the technical features indicated. Therefore, the features defined with "first", "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.

[0162] 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 fixed connection, or detachable connection, or integral; can be mechanical connection, or electrical connection or communication with each other; can be directly connected, or 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.

[0163] 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.

[0164] 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 preprocessing module includes a first microfluidic chip with inlet I, inlet II and outlet I, and the first microfluidic chip also has a micron-level curved secondary flow structure inside; The capture culture module includes a second microfluidic chip with inlet III, outlet II and outlet III. The second microfluidic chip integrates multiple cell capture units, and each cell capture unit is provided with multiple cell capture structures. A micro-injection pump is connected to the pretreatment module via capillary tubing a and capillary tubing b. The pretreatment module and the capture culture module are connected by a capillary tube c. The curved secondary flow structure is composed of multiple semi-circular curves, and the angle between the inner wall of the curve and the horizontal direction is 20°. Multiple cell capture units are connected in sequence, and each cell capture unit has one cell capture inlet and three cell capture outlets; The cell capture structure is disposed between the cell capture inlet and the cell capture outlet; The cell capture structure in any one of the cell capture units is two, and each cell capture structure includes a cell capture chamber, a cell capture chamber and a narrow gap groove; One end of the cell capture chamber is connected to the cell capture inlet, and the other end is connected to the cell capture chamber through the narrow gap groove. The cell capture outlet includes: The first cell capture outlet has one end connected to the cell capture chamber and the other end connected to outlet II or outlet III; Second cell capture exit; The cell capture inlet of any of the cell capture units, except at the head end, is connected to the second cell capture outlet of the adjacent previous cell capture unit; The cell capture inlet of the cell capture unit at the head end is connected to the outlet I via the capillary tube c.

2. The integrated microfluidic system for single-cell capture, stimulation, and analysis according to claim 1, characterized in that, The output end of the micro-injection pump is connected to inlet I via capillary tube a, and to inlet II via capillary tube b.

3. The integrated microfluidic system for single-cell capture, stimulation, and analysis according to claim 1, characterized in that, The system also includes: A fluorescence microscope is positioned above the capture and culture module; Capillary hoses d and e are connected to outlet II and outlet III, respectively.

4. An analytical method based on the integrated microfluidic system for single-cell capture, stimulation, and analysis according to any one of claims 1-3, characterized in that, include: Step S1: Prepare a cell suspension by digesting adherent cells with trypsin and resuspending them with phosphate buffer. Step S2, dilution and mixing: The cell suspension and buffer solution are injected into the pretreatment module through inlet I and inlet II at the same flow rate. The sample cells are diluted and mixed by using the curved secondary flow structure. Step S3, Single cell capture: The diluted and mixed sample solution is introduced into the capture culture module through capillary tube c at a first preset flow rate, and single cells of the required size are captured through the cell capture structure. Step S4, culture or labeling: The captured single cells are cultured or labeled with aptamer / antibody solutions containing fluorescently labeled markers on the surface of the single cells; Step S5, Imaging Analysis: The cultured or labeled cells are observed using a fluorescence microscope, and the expression of single-cell surface markers is detected and analyzed.

5. The analytical method for an integrated microfluidic system for single-cell capture, stimulation, and analysis according to claim 4, characterized in that, In step S4, the process of culturing the captured single cells further includes: Step S41: Apply mechanical stimulation to increase the flow rate to a second preset flow rate, thereby applying mechanical stimulation to the captured single cells to promote their deformation and exosome secretion. Step S42, culture medium preparation: collect sample cells after mechanical stimulation, place the collected sample cells in a cell culture flask and culture until they adhere to the wall and stabilize. Then, place them in an ultra-high speed centrifuge to extract exosomes and add them to DMEM culture medium without serum. Mix well and set aside for use. Step S43, following step S3, introduces the culture medium prepared in step S42 through capillary tube c, and co-cultures it with the single cells captured by the cell capture structure.

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