Cell detection method based on micro-fluidic chip

Abstract

The invention discloses a cell detection method based on a microfluidic chip, relates to the technical field of microfluidics, and aims to improve the capture efficiency, reduce the total detection time and improve the binding efficiency of cells and antibodies by combining specific flow velocity sample introduction and specific temperature incubation based on the microfluidic chip with a specific structure. By optimizing the pretreatment process and the antibody coating process at the same time, the operation efficiency can be greatly improved, the overall process time is greatly shortened compared with that of a traditional method, and the method is suitable for time-sensitive immune cells. By adopting the micro-fluidic chip and the detection method, the immune cells can be effectively captured, and the cell viability is good, so that the micro-fluidic chip and the detection method have an important application prospect.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of microfluidic technology, in particular to a cell detection method based on a microfluidic chip. BACKGROUND

[0002] Immune cells play a key role in human immune response, and their capture and detection are of great significance for disease diagnosis, immune research and drug development. Traditional cell capture and detection methods have problems such as low efficiency, complex operation and large cell damage. In recent years, microfluidic technology has attracted widespread attention in the biomedical field due to its high throughput, high sensitivity and low sample consumption. However, due to the limitations of traditional chip structure and the shortcomings of detection process, there are still deficiencies in cell capture efficiency and detection efficiency, and the overall process is difficult to shorten, with a total detection time of 6-24 hours, which is difficult to apply to the detection of time-sensitive immune cells. SUMMARY

[0003] The main purpose of the present application is to provide a cell detection method based on a microfluidic chip, which improves the chip structure and detection method steps to improve the cell capture rate and detection efficiency, and reduces cell damage, especially for the detection of immune cells.

[0004] To achieve the above purpose, the present application provides a cell detection method based on a microfluidic chip, which comprises a capture region, and the capture region array is provided with microcolumns, wherein the number of microcolumns in each 400 mm2 of the capture region area is 15000-18000, and the microcolumn spacing is 45-55 μm. The cell detection method based on the microfluidic chip comprises the following steps: S1: injecting a solvent into the microfluidic chip for pretreatment and cleaning; S2: diluting the antibody to obtain an antibody diluent, and injecting the antibody diluent into the microfluidic chip to make the antibody diluent fully cover the surface of the chip, and completing the coating of the antibody; S3: incubating the microfluidic chip with completed antibody coating; S4: processing the cells to be detected which can specifically bind to the antibody into a cell suspension, centrifuging and resuspending the cell suspension after staining and labeling, filtering and injecting into the microfluidic chip, the injection flow rate is 80-120 μl / min, and the cells to be detected are fixed after incubation at 36.5-37.5 ℃ for 0.8-1.2 h; S5: detecting the cells to be detected fixed after staining in step S4.

[0005] The term microcolumn spacing in the microfluidic chip refers to the distance between adjacent microcolumn structures.

[0006] The solvents used for pretreatment of the microfluidic chip in step S1, including solvents used for cleaning, activation, surface modification, etc. in the pretreatment, are exemplarily double-distilled water / laboratory-grade ultrapure water, alcoholic solvents, phosphate buffered saline (PBS), polylysine, etc.

[0007] The antibody mentioned in step S2 can specifically bind to the cells to be detected. As an example, the antibody is one of CD3 antibody and CD4 antibody, and the cells to be detected are, for example, T cells that can specifically bind to the CD3 antibody, MOLT-4 (human acute T lymphoblastic leukemia cells), CCRF-CEM (human acute lymphoblastic leukemia T cells), etc. The cells to be detected are preferably immune cells, and further preferably, when the antibody is a CD3 antibody, the cells to be detected are Jurkat cells. The CD3 antibody is an important transmembrane glycoprotein on the surface of T cells, forms a complex with the T cell receptor (TCR), and is involved in T cell activation signaling. The CD3 antibody can specifically recognize the CD3 antigen on the surface of the cells to be detected (Jurkat cell surface), and the antigen binding site is located in the variable region (VH / VL) of the antibody.

[0008] The traditional microfluidic chip, such as the capture area of 20 mm x 20 mm in the paper "Capture and detection of intracellular estrogen receptor in circulating tumor cells of breast cancer" by Wang Lihua, contains 32200 microcolumn structures with a microcolumn spacing of 30 um, which follows the design logic of "high-density microcolumns to improve capture area", but the dense microcolumns cause a sharp increase in fluid resistance, which may cause uneven cell flow rate, and some cells flow through the capture area without contacting the antibody, resulting in a low capture rate of cells to be detected. Based on the above reasons, the traditional microfluidic chip usually uses a low flow rate of 15 uL / min for sample injection, and the long detection time limits its application in immune cell detection scenarios (immune cells have a short physiological life span, are easily affected by the environment, and usually only survive for a few hours to a few days in vitro). The present application redesigns the microfluidic chip by using a specific ratio of capture area to microcolumn structure and microcolumn spacing to form a sparse and orderly distribution, which not only improves the uniformity of the flow rate in the chip and reduces collision loss, but also delays the effective contact time between the cells to be detected and the antibody, solving the technical defects of traditional microfluidic chips caused by the narrow microcolumn spacing, i.e. a sharp increase in fluid resistance, causing uneven cell flow rate, and some cells flowing away without fully contacting the antibody; at the same time, dense microcolumns increase the probability of cell and column collision, leading to an increase in cell damage rate and the formation of blockage due to cell clumps; and because the microcolumn spacing is too wide, the contact probability between the cells and the antibody on the microcolumn surface is reduced, resulting in a sharp decrease in capture efficiency; at the same time, the wide channel causes insufficient fluid shear force, and the cells are prone to local accumulation due to gravity, which increases the risk of non-specific adhesion. The microfluidic column chip structure of the present application, combined with a specific sample injection flow rate and incubation temperature in the sample injection process, solves the contradiction between "narrow and blocked" and "wide and leaked" in traditional microfluidic chips, improves the capture rate and specific binding rate, reduces cell damage, and realizes efficient sampling, thereby reducing the total time from sample processing to detection completion, improving detection efficiency, and being applicable to immune cell detection scenarios that are sensitive to detection time.

[0009] As a preferred, the cells to be detected are immune cells; the antibody is one of CD3 antibody and CD4 antibody; in step S1, alcohol solvent is injected into the microfluidic chip for pretreatment, and then double distilled water or experimental grade ultrapure water is used for cleaning. The alcohol solvent is injected to dissolve and remove oil stains on the surface of the microfluidic chip, and then double distilled water or experimental grade ultrapure water is used to flush the residual alcohol solvent, creating a good channel environment for the passage and maintenance of immune cells.

[0010] Further preferably, in step S1, anhydrous ethanol is injected into the microfluidic chip for pretreatment, the injection flow rate is 150-200 uL / min, and then double distilled water or experimental grade ultrapure water is used for cleaning; in step S2, the antibody is CD3 antibody; and the cells to be detected are T cells that can specifically bind to CD3 antibody.

[0011] Anhydrous ethanol is one of the common polar organic solvents in the pretreatment of PDMS surface, which can effectively improve the antibody coating rate of PDMS microfluidic chip by surface modification and solution spreading, and also can enhance the hydrophilicity in a short time. As a pretreatment reagent of PDMS surface, anhydrous ethanol can dissolve residual uncrosslinked oligomers, expose more siloxane skeleton to increase active sites, and form temporary hydrogen bonds with the PDMS surface to temporarily enhance the surface polarity, thereby promoting the combination of antibodies containing amino, carboxyl and other polar groups through hydrophobic interaction, electrostatic attraction or hydrogen bonds. At the same time, the antibody solution tends to flow in the form of "droplets" in the PDMS microchannel due to hydrophobicity, resulting in uneven coating. Anhydrous ethanol is miscible with water, which can reduce the surface tension of the solution and make the solution more easily spread in the microchannel to form a uniform liquid film, ensuring the full contact of the antibody with the surface and reducing the deviation of the capture signal of T cells (such as Jurkat cells) caused by local insufficient CD3 antibody coating.

[0012] In the pretreatment step of the traditional microfluidic chip, the flow rate of the solution injected into the chip is usually 100 μL / min based on the design of a relatively dense microcolumn array. However, the combination of the microfluidic chip structure of the present application can improve the washing efficiency, significantly reduce the pretreatment time, and improve the impurity removal rate and antibody coating efficiency. Through the synergistic effect of fluid shear force and surface energy, the best balance between washing efficiency and chip damage is achieved.

[0013] Further preferably, in step S2, the dilution ratio of the antibody is 1:80-500, and the flow rate of the injection into the microfluidic chip is 150-200 μl / min; in step S3, the microfluidic chip is incubated at 30-37℃ for 2.5-3.5 h. The use of temperature-induced antibody conformation optimization combined with flow rate regulation achieves directional arrangement of antibody molecules, thereby improving the specific binding rate.

[0014] Further preferably, in step S1, double distilled water is used to wash 3 times at a flow rate of 100 μl / min; in step S2, the dilution ratio of the antibody is 1:100, and the flow rate of the injection into the microfluidic chip is 200 μl / min; in step S3, the microfluidic chip is incubated at 37℃ for 3 h, and then washed 2 times with double distilled water at a flow rate of 50-100 μl / min; in step S4, after sampling, the microfluidic chip is incubated at 37℃ for 1 h, and then washed 1 time with double distilled water at a flow rate of 100 μl / min. By using high flow rate to inject the antibody into the specific loose microcolumn structure, combined with flow rate regulation to achieve directional arrangement of antibody molecules, the specific binding rate is improved. The specific pretreatment process improves the washing efficiency, improves the impurity removal rate and antibody coating efficiency, and the specific sampling process regulates the Reynolds number to make the cells pass through the capture area in a single column, which comprehensively improves the cell and antibody binding efficiency and maximizes the cell viability.

[0015] Further preferably, in step S4, the process of processing the cell suspension is: centrifuging the cells to be detected, removing the supernatant and collecting the cells, and resuspending the centrifuged cells with a staining solution to form a cell suspension; The process of centrifuging and resuspending the cell suspension after staining and labeling is: (1) staining at room temperature to achieve double labeling of Hoechst and Dil; (2) resuspending the labeled cell suspension in (1) after centrifugation with culture solution, repeating twice, and filtering with a flow filter before sampling; The process of fixing the cells to be detected is: fixing the cells to be detected with a 4% paraformaldehyde solution.

[0016] The present scheme adopts double labeling of Dil (cell membrane) and Hoechst (nucleus), optimizes the resuspension process, and compared with the traditional single labeling method, the fluorescence signal intensity is improved, the accuracy of cell structure recognition is greatly improved, and the basis for subsequent three-dimensional positioning is provided. Moreover, through multi-dimensional information complementation, the core pain points of "fragment misjudgment, signal attenuation, and spatial positioning loss" in the traditional method are solved.

[0017] As preferred, in step S5, the ability of the stained and fixed cells to be detected to bind with the antibody and the cell state are detected by using a flow cytometer, and the cell capture condition is observed by using a fluorescence microscope or a full-automatic cell imager.

[0018] The present scheme optimizes the detection timing and instrument cooperation, and compared with the traditional single microscope detection, the data dimension is increased, and the detection accuracy is improved. The integrated scheme is not a simple device superposition, but through optimization of the concentration of the fixing solution and the detection timing, the synergistic effect of cell morphology preservation and signal acquisition is achieved.

[0019] Preferably, the size of the microfluidic chip is 30 mm x 60 mm, wherein the capture area is 20 mm x 20 mm, and contains 17689 microcolumns, the spacing of the microcolumns is 50 um, the height is 40 um, and the diameter is 100 um.

[0020] Compared with the prior art, the core beneficial effects of the present application are: The microfluidic column chip structure of the present application, in cooperation with the specific sampling flow rate and incubation temperature in the sampling process, solves the contradiction of "narrow blockage and wide leakage" in the traditional microfluidic chip, improves the capture rate and specific binding rate, realizes efficient sampling, and thus reduces the total time length of the detection method from sample processing to detection completion, improves the detection efficiency. By simultaneously optimizing the pretreatment process and the antibody coating process, the operation efficiency can be greatly improved, and the overall process time is shortened compared with the traditional method, and the shortest time from sample processing to detection completion is only 4.3h, and the number of washing times is reduced, and the cell loss rate is reduced.

[0021] The application can realize efficient capture of immune cells and provide a reliable platform for subsequent cell analysis by optimizing the microfluidic chip structure design and detection method steps. Experiments show that the microfluidic chip and detection method in the application can effectively capture immune cells with good cell viability, and have important application prospects. BRIEF DESCRIPTION OF DRAWINGS

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

[0023] Figure 1 An embodiment structure diagram of the microfluidic chip in Embodiment 1 of the present application; Figure 2 A control diagram for detecting the binding ability of the CD3 antibody with a fluorescent label to Jurkat cells by a flow cytometer in the present application and a control of Embodiment 2; Figure 3 A diagram for capturing cells in the microfluidic chip in Embodiment 2 by a full-automatic cell imager (10 times shooting): a. Fluorescent shooting for capturing cell nuclei, blue fluorescence is jurkat cell nuclei labeled by Hoechst33342 dye. b. Fluorescent shooting for capturing cell membranes, red fluorescence is jurkat cell membranes labeled by Dil dye, c. Bright field shooting for capturing cells, d. Synthesis diagram of fluorescent and bright field capturing cells; Figure 4 A diagram for capturing cells in the microfluidic chip in Embodiment 2 by a full-automatic cell imager (20 times shooting): a. Fluorescent shooting for capturing cell nuclei, blue fluorescence is jurkat cell nuclei labeled by Hoechst33342 dye, b. Fluorescent shooting for capturing cell membranes, red fluorescence is jurkat cell membranes labeled by Dil dye, c. Bright field shooting for capturing cells, d. Synthesis diagram of fluorescent and bright field capturing cells; Figure 5 A diagram for capturing cells observed by a fluorescent microscope in Comparative Example 1-1 in the present application; Figure 6 A diagram for capturing cells observed by a fluorescent microscope in Comparative Example 1-2 in the present application; Figure 7 A diagram for capturing cells observed by a fluorescent microscope in Comparative Example 2 in the present application.

[0024] The objectives, functional features and advantages of the present application will be further described with reference to the embodiments and the accompanying drawings. DETAILED DESCRIPTION

[0025] The technical solutions in the embodiments of the present application will be clearly and completely described below. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all the other embodiments obtained by a person of ordinary skill in the art without creative work shall fall within the protection scope of the present application. In addition, the technical solutions of various embodiments can be combined with each other, but it shall be considered that the combination of technical solutions does not exist and is not within the protection scope of the present application when the combination of technical solutions appears to be contradictory or unachievable.

[0026] The present application provides a cell detection method based on a microfluidic chip. The microfluidic chip comprises a capture area, and the capture area is arranged with microcolumns. The number of microcolumns in each 400 mm 2 of the capture area is 15000-18000, and the spacing between the microcolumns is 45-55 μm. The cell detection method based on the microfluidic chip comprises the following steps: S1: injecting a solvent / solution into the microfluidic chip for pretreatment and cleaning; S2: diluting an antibody to obtain an antibody diluent, and injecting the antibody diluent into the microfluidic chip so that the antibody diluent fully covers the surface of the chip, thereby completing antibody coating; S3: incubating the microfluidic chip after the antibody coating is completed; S4: processing the cells to be detected which can specifically bind to the antibody into a cell suspension, staining and labeling the cell suspension, centrifuging and resuspending, filtering, and then injecting the sample into the microfluidic chip at a flow rate of 80-120 μl / min, and then incubating at 36.5-37.5°C for 0.8-1.2 h, and then fixing the cells to be detected; S5: detecting the cells to be detected after staining and fixing in step S4.

[0027] The cells to be detected are immune cells; the antibody is one of CD3 antibodies and CD4 antibodies; and the solvent is one of anhydrous ethanol, double-distilled water / experimental grade ultrapure water, an alcohol solvent, phosphate buffered saline, and polylysine. In a preferred embodiment, in step S1, the microfluidic chip is injected with anhydrous ethanol for pretreatment at a flow rate of 150-200 μl / min, and then cleaned with double-distilled water or experimental grade ultrapure water; in step S2, the antibody is a CD3 antibody; and the cells to be detected are T cells which can specifically bind to the CD3 antibody. In a preferred embodiment, the T cells are Jurkat cells.

[0028] In step S2, the dilution ratio of the antibody is 1:80-500, and the flow rate of injecting the microfluidic chip is 150-200 μl / min; in step S3, the microfluidic chip is incubated at 30-37℃ for 2.5-3.5 h.

[0029] In the preferred embodiment, in step S1, the double distilled water is used for washing 3 times at a flow rate of 100 μl / min; in step S2, the dilution ratio of the antibody is 1:100, and the flow rate of injecting the microfluidic chip is 200 μl / min; in step S3, the microfluidic chip is incubated at 37℃ for 3 h, and then washed 2 times with double distilled water at a flow rate of 50-100 μl / min; in step S4, after the sample is injected, the microfluidic chip is incubated at 37℃ for 1 h, and then washed 1 time with double distilled water at a flow rate of 100 μl / min.

[0030] In step S4, the process of processing the cell suspension is as follows: the cells to be detected are digested and centrifuged, the supernatant is removed and the cells are collected, and the centrifuged cells are resuspended with a staining solution to form a cell suspension. The process of centrifuging and resuspending the cell suspension after staining and labeling is as follows: (1) staining at room temperature to realize double labeling of Hoechst and Dil; (2) centrifuging the labeled cell suspension in (1), resuspending with culture solution, repeating twice, and filtering with a flow filter before sample injection; The process of fixing the cells to be detected is as follows: 4% paraformaldehyde solution is used to fix the cells to be detected.

[0031] In step S5, the flow cytometer is used to detect the binding ability of the stained and fixed cells to be detected to the antibody and the state of the cells, and the fluorescence microscope or the full-automatic cell imaging instrument is used to observe the cell capture.

[0032] In the preferred embodiment, the size of the microfluidic chip is 30 mm × 60 mm, the capture area is 20 mm × 20 mm, the microfluidic chip contains 17689 microcolumns, the spacing of the microcolumns is 50 μm, the height of the microcolumns is 40 μm, and the diameter of the microcolumns is 100 μm.

[0033] The technical solutions of the present application are further described in detail in combination with specific embodiments or comparative examples. In the following embodiments and comparative examples, the raw materials, reagents and the like not specifically stated are obtained by market purchase, and the same batch of raw materials or reagents is used in the embodiments and comparative examples. The preparation methods not specifically stated are prepared by using conventional and well-known preparation methods.

[0034] The necessary raw materials or instruments involved in the specific embodiments or comparative examples are described as follows: Detection items: (1) Capture rate: the cell capture is observed by a fluorescence microscope or a full-automatic cell imaging instrument; (2) Specific binding rate and cell morphology: detected by flow cytometry, for example, Figure 2 shows the fluorescence intensity distribution of Jurkat cells without binding with fluorescently labeled antibody, and the fluorescence intensity distribution of Jurkat cells with binding with fluorescently labeled antibody. The horizontal axis of the figure is the fluorescence intensity, and the vertical axis is the cell number. The maximum excitation / emission wavelength is 650 nm / 660 nm. The blank control group reflects the fluorescence signal distribution of Jurkat cells without binding with fluorescently labeled antibody, which is used as a benchmark for the experiment to determine the level of non-specific fluorescence signal. Compared with the control group without adding antibody, the binding of CD3 fluorescent antibody with Jurkat in the experimental group is observed, which detects the binding ability of the antibody with Jurkat and reflects the state of Jurkat cells. Figure 2

[0035] Example 1 A microfluidic chip-based cell detection method is provided, and the size of the microfluidic chip is 30 mm × 60 mm, wherein the capture area is 20 mm × 20 mm and contains 17689 microcolumns. The spacing of the microcolumns is 50 μm, the height is 40 μm, and the diameter is 100 μm, as shown in Figure 1. Figure 1

[0036] The specific steps are as follows: S1: Inject anhydrous ethanol into the microfluidic chip for pretreatment; the flow rate is set to 150 μl / min, and the injection is continued for 10 min, followed by washing twice with double distilled water at a flow rate of 100 μl / min.

[0037] S2: Dilute the CD3 antibody to 1:80 to obtain an antibody diluent, and inject the microfluidic chip at a flow rate of 150 μl / min to ensure that the antibody diluent fully covers the surface of the chip, and complete the coating of the antibody; S3: Incubate the microfluidic chip at 37℃ for 2.5 hours, and then wash twice with double distilled water at a flow rate of 100 μl / min.

[0038] S4: Digest and centrifuge the Jurkat cells (600 g, 4℃, 5 min), remove the supernatant and collect the cells, resuspend the centrifuged cells with the staining solution to form a cell suspension, and stain at room temperature for 8 min to achieve double labeling of Hoechst and Dil, resuspend the labeled cell suspension with culture solution, repeat twice, filter with a flow filter, and then inject the sample into the microfluidic chip at a flow rate of 80 μl / min. After incubation at 36.5℃ for 1 hour, wash once with double distilled water at a flow rate of 100 μl / min, and fix the cells to be detected with 4% paraformaldehyde solution for 20 min; S5: Detect the stained and fixed cells to be detected in step S4.

[0039] ​​The flow cytometer is used to detect the ability of the stained and fixed cells to be detected to combine with the antibody and the cell state, and then the full-automatic cell imager is used to observe the cell capturing.

[0040] Embodiment 2 The cell detection method based on the microfluidic chip is provided, and the microfluidic chip is referred to the embodiment 1.

[0041] The specific steps are as follows: S1: The microfluidic chip is injected with anhydrous ethanol for pretreatment; the flow rate is set to 180 μl / min, and the process lasts for 10 minutes, and then the microfluidic chip is washed twice with double distilled water at a flow rate of 100 μl / min.

[0042] S2: The CD3 antibody is diluted to 1:200 to obtain an antibody diluent, and the microfluidic chip is injected with the antibody diluent at a flow rate of 180 μl / min, so that the antibody diluent fully covers the surface of the chip, and the antibody coating is completed; S3: The microfluidic chip is incubated at 30℃ for 3.5 hours, and then the microfluidic chip is washed twice with double distilled water at a flow rate of 50 μl / min.

[0043] S4: The Jurkat cells are centrifuged (600 g, 4℃, 5 minutes), the supernatant is removed, and the cells are collected, the centrifuged cells are resuspended with a staining solution to form a cell suspension, and the cell suspension is stained at room temperature for 8 minutes to realize double labeling of Hoechst and Dil, the labeled cell suspension is centrifuged, resuspended with a culture solution, and repeated twice, filtered by a flow filter, and then injected into the microfluidic chip at a flow rate of 100 μl / min, and then incubated at 37℃ for 1 hour, and then washed once with double distilled water at a flow rate of 100 μl / min, and then the cells to be detected are fixed with a 4% paraformaldehyde solution for 20 minutes. S5: The cells to be detected after being stained and fixed in the step S4 are detected.

[0044] The flow cytometer is used to detect the ability of the stained and fixed cells to be detected to combine with the antibody and the cell state, and then the full-automatic cell imager is used to observe the cell capturing.

[0045] Embodiment 3 The cell detection method based on the microfluidic chip is provided, and the microfluidic chip is referred to the embodiment 1.

[0046] The specific steps are as follows: S1: The microfluidic chip is injected with anhydrous ethanol for pretreatment; the flow rate is set to 200 μl / min, and the process lasts for 3 minutes, and then the microfluidic chip is washed once with double distilled water at a flow rate of 100 μl / min.

[0047] S2: Dilute the CD3 antibody to 1:500 to obtain an antibody diluent, inject the microfluidic chip at a flow rate of 200 μl / min, so that the antibody diluent fully covers the chip surface, and complete the coating of the antibody; S3: Incubate the microfluidic chip at 35°C for 3 hours, and then wash twice with double distilled water at a flow rate of 80 μl / min.

[0048] S4: Digest and centrifuge the Jurkat cells (600 g, 4°C, 5 minutes), remove the supernatant and collect the cells, resuspend the centrifuged cells with a staining solution to form a cell suspension, and stain at room temperature for 8 minutes to achieve double labeling of Hoechst and Dil, centrifuge the labeled cell suspension, resuspend with culture solution, repeat twice, filter with a flow cytometer, and then inject into the microfluidic chip at an injection flow rate of 120 μl / min, incubate at 37.5°C for 1 hour after injection, and then wash once with double distilled water at a flow rate of 100 μl / min, and fix the cells to be tested with a 4% paraformaldehyde solution for 20 minutes.

[0049] S5: Detect the stained cells to be tested in step S4.

[0050] Detect the ability of the stained and fixed cells to be tested to bind to the antibody and the state of the cells by using a flow cytometer, and observe the cell capture by using a full-automatic cell imager.

[0051] Comparative Examples 1-1 to 1-2 The microfluidic chip and detection steps refer to Example 2, except that the injection process parameters in step S4 are different.

[0052] In Comparative Example 1-1, the injection flow rate is 15 μL / min; In Comparative Example 1-2, the incubation temperature after injection is 25°C.

[0053] Comparative Example 2 In Comparative Example 2, the microfluidic chip and detection steps refer to Example 2, except that no incubation is performed after injection.

[0054] Comparative Example 3 In Comparative Example 3, a conventional microfluidic chip is used, and the existing detection method steps are used.

[0055] The conventional microfluidic chip has a chip size of 30 mm x 60 mm, and a capture area of 20 mm x 20 mm; Microcolumn array: contains 32200 microcolumns, material is PDMS; microcolumn parameters: diameter 100 μm, height 40 μm, spacing between microcolumns in each row 30 μm; fluid channel: width 50 μm, depth 40 μm; the existing detection method steps are: the chip is flushed with 100 μL / min of anhydrous ethanol for 3 times, 5 min, washed with double distilled water at the same flow rate for 3 times, 5 min pretreatment; after dilution at 1:50, 50 μL / min is injected, 25℃ incubation for 3h, PBS washing for 3 times; after cell digestion, resuspend with culture solution, centrifuge at 1000r / min for 5min to adjust the concentration to 5×10 4 6 / mL, sample 1ml; sample at 10 μL / min, 25℃ incubation for 30min, PBS washing; after 4% paraformaldehyde fixation for 30min, PBS washing for 3 times, 5min each time; single fluorescent dye staining for 10min, detected by microscope.

[0056] After detecting the fixed cells to be detected in Examples 1-3 and Comparative Examples 1-3, the overall detection time of Examples 1-3 is between 4.3-5 hours, and the overall detection time of Comparative Example 3 is 7 hours. The detection time of the present scheme is obviously shortened compared with the traditional chip + traditional detection method, while ensuring high capture efficiency and specificity, the detection throughput is greatly improved. As can be seen from the images of Comparative Example 2 and Comparative Examples 1-1, 1-2 and 2, by optimizing the antibody coating concentration and incubation conditions, the antibody is distributed in a more suitable state on the chip surface, which can fully expose the specific binding sites, accurately recognize and bind to the antigens on the surface of target cells, and effectively reduce the problems of antibody aggregation and non-specific site occupation. Avoid the interference of "false capture" on the results, so that the proportion of target cells in the finally captured cells is higher, and the detection result can more truly reflect the situation of target cells, thereby improving the capture rate and specific binding rate, and providing accurate sample guarantee for subsequent qualitative and quantitative analysis. Moreover, by comparing Examples 1-3 and Comparative Example 3, it is found that the use of the detection sequence in the present scheme and the paraformaldehyde solution in step S5 helps to improve the cell preservation rate; the present scheme uses Hoechst and Dil double labeling, the fluorescence signal intensity is improved, and the cell structure recognition accuracy is greatly improved compared with Comparative Example 3; by optimizing the detection time sequence and cooperating with 4% paraformaldehyde solution, the cell morphology preservation and signal acquisition are realized.

[0057] Comparative Example 2 and Comparative Examples 1-1-1-2, when the sample flow rate decreases, such as Comparative Example 1-1, the capture efficiency decreases, the cell viability is damaged, and the detection time is prolonged, and the image thereof is shown in Figure 5 When the incubation temperature after sample injection decreases, such as Comparative Example 1-2, the capture rate decreases, and the image thereof is shown in Figure 6As shown, high sample flow rate and high incubation temperature are required to achieve the balance between capture rate, binding rate, cell state maintenance and efficiency.

[0058] As shown in the comparison between Example 2 and Comparative Example 2, the cell capture rate decreased after sample injection without incubation, and the image is shown in FIG. 6. Figure 7

[0059] The above description is only preferred embodiments of the present application, and does not limit the patent scope of the present application. Any equivalent structure transformation based on the content of the present application, or direct / indirect application in other related technical fields, are included in the patent protection scope of the present application.​

Claims

1. A microfluidic chip-based cell detection method, characterized in that, The microfluidic chip comprises a capture region, and the capture region is arranged with micro-pillars, wherein the number of micro-pillars per 400 mm2 of the capture region area is 15000-18000, and the micro-pillar spacing is 45-55 μm; The detection method based on the microfluidic chip comprises the following steps: S1: The microfluidic chip is injected with a solvent for pretreatment and cleaning; S2: The antibody is diluted to obtain an antibody diluent, and the antibody diluent is injected into the microfluidic chip to fully cover the surface of the chip, and the antibody coating is completed; S3: The microfluidic chip with completed antibody coating is incubated; S4: The cells to be detected capable of specifically binding to the antibody are processed into a cell suspension, the cell suspension is dyed and labeled, then centrifuged and resuspended, filtered, and then injected into the microfluidic chip for sampling, the sampling flow rate is 80-120 μl / min, and after sampling, the cells to be detected are fixed at 36.5-37.5°C for 0.8-1.2 h; S5: The cells to be detected dyed and fixed in step S4 are detected.

2. The microfluidic chip-based cell detection method according to claim 1, wherein, The cells to be detected are immune cells; the antibody is one of CD3 antibody and CD4 antibody; in step S1, an alcohol solvent is injected into the microfluidic chip for pretreatment, and then double distilled water or experimental grade ultrapure water is used for cleaning.

3. The cell detection method based on the microfluidic chip according to claim 2, wherein in step S1, the microfluidic chip is pretreated by injecting anhydrous ethanol at a flow rate of 150-200 μl / min, and then cleaned with double distilled water or experimental grade ultrapure water; in step S2, the antibody is CD3 antibody, and the cells to be detected are T cells capable of specifically binding to CD3 antibody.

4. The microfluidic chip-based cell detection method according to claim 3, wherein, In step S2, the dilution ratio of the antibody is 1:80-500, and the flow rate of injection into the microfluidic chip is 150-200 μl / min; In step S3, the microfluidic chip is incubated at 30-37°C for 2.5-3.5 h.

5. The microfluidic chip-based cell detection method according to claim 4, wherein, In step S1, the microfluidic chip is cleaned with double distilled water at a flow rate of 100 μl / min for 3 times; In step S2, the dilution ratio of the antibody is 1:100, and the flow rate of injection into the microfluidic chip is 200 μl / min; In step S3, the microfluidic chip is incubated at 37°C for 3 h, and then cleaned with double distilled water at a flow rate of 50-100 μl / min for 2 times; In step S4, after sampling, the cells to be detected are incubated at 37°C for 1 h, and then cleaned with double distilled water at a flow rate of 100 μl / min for 1 time.

6. The microfluidic chip-based cell detection method according to claim 1 or 2 or 3 or 4 or 5, wherein, In step S4, the process of processing into a cell suspension is: digesting and centrifuging the cells to be detected, removing the supernatant and collecting the cells, resuspending the centrifuged cells with a staining solution to form a cell suspension; The process of centrifuging and resuspending the cell suspension after dyeing and labeling is: (1) dyeing at room temperature to realize Hoechst and Dil double labeling; (2) centrifuging the labeled cell suspension in (1), resuspending with culture solution, repeating twice, and filtering with a flow filter before sampling; The process of fixing the cells to be detected is: fixing the cells to be detected with 4% paraformaldehyde solution.

7. The microfluidic chip-based cell detection method according to claim 6, wherein, The cell to be detected is a Jurkat cell.

8. The microfluidic chip-based cell detection method according to claim 6, wherein, In step S5, the flow cytometer is used to detect the binding ability of the fixed and dyed cell to be detected with the antibody and the cell state, and then the fluorescence microscope or the full-automatic cell imaging instrument is used to observe the cell capture.

9. The microfluidic chip-based cell detection method according to claim 1, wherein, The microfluidic chip has a size of 30 mm*60 mm, wherein the capture area is 20 mm*20 mm, and contains 17689 microcolumns with a spacing of 50 microns, a height of 40 microns and a diameter of 100 microns.

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