A method of cell culture, detection and screening

Abstract

This disclosure relates to the fields of life sciences and medicine. Specifically, it relates to a method for cell culture, detection, and screening. The method for capturing cell secretions includes: culturing single cells under suitable conditions; and subjecting the single-cell culture system to a controllable vortexing treatment with a first affinity reagent, said affinity reagent specifically binding to the secretions of the single cells.

Description

A method for cell culture, detection and screening Technical Field

[0001] The present application relates to the fields of life science and medicine. Specifically, the present application relates to a method for cell culture, detection and screening. Background Art

[0002] Currently, single-cell research is of great significance in the fields of life sciences and medicine, especially in drug development, clinical diagnosis, and precision treatment. However, due to the heterogeneity of cell populations, specific cells need to be screened, characterized, and recovered, and secretions from single living cells (such as proteins, antibodies, cytokines, etc.) need to be detected with high sensitivity, high efficiency, rapidity, and low cost. Traditional protein analysis methods have many limitations, including high sample abundance requirements, limited detection types, high costs, and effects on cell activity and proliferation.

[0003] Currently, several technologies are available for single-cell protein analysis, such as flow cytometry, antibody microarrays, and encoded microbead-capture protein detection. However, these methods have various limitations, including low cell viability, complex operations, lack of flexibility, and cell damage. Furthermore, they cannot simultaneously meet the requirements of high sensitivity, low cost, and high efficiency and speed.

[0004] Therefore, developing a method that can efficiently capture secretions from single living cells without affecting cell activity and proliferation ability, and that has the characteristics of high sensitivity, low cost, and flexibility is of great significance for single-cell research.

[0005] Summary of the Invention

[0006] This application is made based on the inventor's discovery of the following problems and facts:

[0007] Currently, traditional methods for analyzing secreted proteins (such as enzyme-linked immunosorbent assay, western blotting, and mass spectrometry) typically target large numbers of mixed cell samples to obtain average values ​​for detection. However, these methods have several drawbacks, such as high protein sample abundance requirements, limited detectable species, high costs, and the fact that some endpoint tests are damaging to cells.

[0008] In order to detect proteins more accurately, a variety of new technologies have emerged in recent years, including flow cytometry, antibody microarray-based detection methods, and encoded microbead-capture protein detection methods.

[0009] Although flow cytometry enables precise analysis of protein expression in single cells, the high shear forces and charged sheath fluids often reduce cell viability after analysis, hindering subsequent culture. Furthermore, its detection capabilities are limited by the number of fluorescent channels and can only detect cell membrane and plasma proteins, not secreted proteins.

[0010] On the other hand, antibody microarrays use a substrate to capture the target and then detect it through fluorescence signals, which can characterize a variety of cytokines or proteins. However, their preparation is complex, and the antibody types are pre-set, which cannot be flexibly adjusted during experiments.

[0011] The present application aims to solve at least one of the above problems. To this end, one object of the present invention is to provide a means for cell culture, detection and screening at high efficiency and low cost.

[0012] Specifically, this application provides the following technical solutions:

[0013] In the first aspect of the present application, the present application proposes a method for capturing cell secretions. According to an embodiment of the present application, the method includes: culturing a single cell under suitable conditions; subjecting a first affinity reagent to a controllable rotational flotation treatment in a single cell culture treatment system, wherein the affinity reagent can specifically bind to the secretions of the single cell. According to the above method of an embodiment of the present application, the first affinity reagent is suspended in the single cell culture treatment system through a controllable rotational flotation treatment, and the secretions of the single cell are aggregated at the same time, and the first affinity reagent can bind to the secretions in a concentrated and more concentrated manner, thereby achieving faster, more efficient and more accurate capture of cell secretions, and also achieving the capture of low-concentration secretions. Moreover, the above method according to an embodiment of the present invention will not cause mechanical damage to the cells to be tested, is conducive to maintaining the activity of the cells to be tested, and continuously captures the secretions, and the captured cells can continue to be cultured.

[0014] The "suitable conditions" described herein refer to conditions suitable for the expression of the cell secretions described herein. Those skilled in the art will readily appreciate that conditions suitable for the expression of cell secretions include, but are not limited to, a suitable cell culture environment, a suitable cell culture time, and the like.

[0015] The "spinning and flotation treatment" described in this application refers to the controllable rotation of the single cell culture treatment system, and the controllable substances include but are not limited to cells, secretions or affinity reagents.

[0016] The "first affinity reagent" described in the present application is selected from reagents / groups that can capture / bind to the secretion to be detected, such as antibodies, aptamers, etc.

[0017] According to an embodiment of the present application, the method for capturing cell secretions may further include at least one of the following technical features:

[0018] According to an embodiment of the present application, the single cell is obtained by separation using a microfluidic chip method, a microwell array combined with a centrifugal gravity pore method, or an acoustic tweezers resonator method. In one example of the present application, the above method can be used to achieve an array distribution of single cells or to distribute single cells in each microwell, such as a PDMS array microwell plate substrate, a PS microwell plate, a cell culture plate, etc. In other examples of the present application, microwell plates with a diameter of 20 to 1000 μm can be applied.

[0019] In some preferred examples of this application, the single cells are isolated using an acoustic tweezers resonator. The inventors used an acoustic tweezers resonator device to separate single cells. Because the acoustic tweezers resonator uses the principle of sound waves to separate cells, it can avoid external forces directly acting on the cells, thereby affecting cell activity.

[0020] According to an embodiment of the present application, the acoustic tweezers resonator separation parameters are selected from a power of 50-500mW and a frequency of 1-50GHz. In some examples of the present application, the power may be 50mW, 100mW, 150mW, 200mW, 250mW, 300mW, 400mW, 450mW, or 500mW depending on the volume and type of the cells; and the frequency may be 1GHz, 5GHz, 10GHz, 15GHz, 20GHz, 25GHz, 30GHz, 35GHz, 40GHz, 45GHz, or 50GHz. Under the above-mentioned acoustic tweezers resonator separation parameters according to an embodiment of the present invention, the efficiency of cell separation and cell integrity can be significantly improved, and the single cell separation effect can be significantly improved.

[0021] In some preferred examples of the present application, the acoustic tweezers resonator separation parameters are selected from power of 50-300 mW and frequency of 1.5 GHz-2.5 GHz.

[0022] According to an embodiment of the present application, the first affinity reagent is provided in the form of microbeads, and the first affinity reagent is coated on the outer surface of the microbeads. In one example of the present application, cell secretions are captured using microbeads coated with the first affinity reagent.

[0023] According to an embodiment of the present application, the first affinity reagent is selected from antibodies.

[0024] According to an embodiment of the present application, the first affinity reagent is selected from aptamers.

[0025] According to embodiments of the present application, the microbeads are selected from at least one of polystyrene beads, Luminex™ beads, magnetic beads, and microrods. In the present application, the microbeads include, but are not limited to, the aforementioned examples. In some examples of the present application, microbeads with a diameter of 100 nm to 10 μm can be coated with an affinity reagent.

[0026] According to an embodiment of the present application, the first affinity reagent is provided in the form of cells, and the first affinity reagent is expressed on the outer surface of the cell membrane. In one example of the present application, the first affinity reagent expressed by the cells is used to capture cell secretions.

[0027] According to an embodiment of the present application, the first affinity reagent is selected from an antibody or a polypeptide. In an example of the present application, the antibody or polypeptide is expressed by the cell on the outer surface of the cell membrane.

[0028] According to an embodiment of the present application, the rotation and floating treatment is achieved by an acoustic tweezers resonator. The inventor uses an acoustic tweezers resonator device to controllably oscillate or rotate the single-cell culture treatment system, so that the first affinity reagent rotates and floats in the culture system. At the same time, the single-cell secretions are also aggregated during the rotation of the culture system. At the same time, the secretions rise from the bottom of the culture system to the top during the rotation and aggregation process, so that the first affinity reagent can be captured. Since the acoustic tweezers resonator is based on the principle of sound waves to achieve rotation, floating and aggregation, it can avoid the problem that some existing means cannot completely collect single-cell secretions, thereby promoting the capture efficiency of the first affinity reagent for secretions.

[0029] According to an embodiment of the present application, the rotational flotation treatment is carried out under conditions of a power of 250 to 350 mW and a frequency of 1.5 to 2.5 GHz. It can be understood that the power of the rotational flotation treatment is different depending on the target substance to be enriched. When the acoustic tweezers resonator is used to realize the rotational flotation of the first affinity reagent, the above-mentioned power according to the embodiment of the present invention can ensure the rotational effect and avoid damage to cells and antibodies, the above-mentioned frequency according to the embodiment of the present invention can enhance the rotational effect, and the above-mentioned embodiment of the present invention provides a more stable rotational effect. In a preferred example of the present application, the rotational flotation treatment is carried out under conditions of a power of 300 mW and a frequency of 2 GHz. After a large number of experiments, the inventors found that under the above-mentioned rotational flotation treatment parameters according to the embodiment of the present invention, the best rotational flotation effect of the first affinity reagent and the single-cell secretion, as well as the best aggregation effect of the single-cell secretion, were achieved.

[0030] In one example of the present application, the principle and device for capturing single-cell secretions are shown in FIG1 . The vortex generated by the acoustic fluidic tweezers controls the microbeads coated with the first affinity reagent and moves them into a single microwell. The capture of single-cell secretions is achieved by adjusting the parameters of the acoustic tweezers resonator.

[0031] According to an embodiment of the present application, the microbeads coated with the first affinity reagent are pre-enriched by an acoustic tweezers resonator. In one example of the present application, the microbeads coated with antibodies may be preferentially enriched by an acoustic tweezers resonator.

[0032] According to an embodiment of the present application, the method further includes injecting magnetic beads coated with the first affinity reagent into the single-cell culture system through the inlet of the acoustic tweezers resonator microfluidic chip. Since each single cell is distributed in different microwells or microchannels, the single-cell secretions can be captured by injecting the magnetic beads coated with the first affinity reagent into a single microwell or microchannel through the inlet of the acoustic tweezers resonator microfluidic chip.

[0033] According to the embodiments of the present application, the single cell comprises at least one selected from T cells, B cells, hybridoma cells, CHO cells, and tumor cells. According to the embodiments of the present application, the single cell is only illustrative, and in some other embodiments, other types of cells may also be selected for secretion capture.

[0034] According to embodiments of the present application, the single cell secretions include at least one selected from antibodies, cytokines, growth factors, exosomes, nucleic acids, or other small molecules. In some examples of the present application, other single cell secretions may also be captured, such as hormones, enzymes, extracellular vesicles, and metabolites.

[0035] According to an embodiment of the present application, the acoustic tweezers resonator microfluidic chip includes: a micropore array chip, a microfluidic channel layer and an acoustic tweezers resonator array chip stacked in sequence along the thickness direction, the surface of the micropore array chip close to the microfluidic channel layer is recessed to form a plurality of micropores, and the plurality of micropores form a micropore array for accommodating cells and / or cell secretions; the microfluidic channel layer includes a fluid channel, a plurality of in situ culture through-holes penetrating the microfluidic channel layer along the thickness direction and connected to the fluid channel, and an inlet and an outlet formed on the surface of the fluid channel layer, the fluid channel is formed from the inlet along the thickness direction. The direction of the in situ culture through-holes intersecting with the direction extends to the outflow outlet; the multiple in situ culture through-holes form an in situ culture through-hole array; the acoustic tweezers resonator array chip includes multiple acoustic wave resonators, and the multiple acoustic wave resonators form an acoustic wave resonator array; the in situ culture through-holes are arranged in a one-to-one correspondence with the micropores and one end along the thickness direction is connected to the micropores, and the other end corresponds to the acoustic wave resonator and is used to receive the acoustic waves emitted by the acoustic wave resonator, the dimension of the in situ culture through-holes along the direction perpendicular to the thickness direction is larger than that of the micropores, and the projection of the acoustic wave resonator along the thickness direction covers at least part of the in situ culture through-holes and at least part of the micropores.

[0036] In some examples of the present application, the single cell system is added to the microwell for single cell culture.

[0037] According to an embodiment of the present application, the row spacing and column spacing of the micropore array are independently 100 μm to 1 cm; the depth of the micropores is 10 to 100 μm; and the pore diameter of the micropores is 10 to 75 μm.

[0038] According to an embodiment of the present application, the micropore array chip includes: a substrate; a dielectrophoresis electrode layer, which is arranged on the substrate and contains dielectrophoresis electrodes; and the micropore array, which is arranged on the dielectrophoresis electrode layer.

[0039] According to an embodiment of the present application, the dielectrophoretic electrode layer includes a plurality of dielectrophoretic electrodes, which form a dielectrophoretic electrode array; the dielectrophoretic electrodes in the dielectrophoretic electrode array correspond one-to-one to the micropores in the micropore array; and each of the dielectrophoretic electrodes is provided with an independent switch.

[0040] According to an embodiment of the present application, the acoustic wave resonator array and the in situ culture through-hole array are embedded and sealed, and the acoustic wave resonator is located in the in situ culture through-hole.

[0041] According to an embodiment of the present application, each acoustic wave resonator in the acoustic wave resonator array is provided with an independent switch; the row spacing and column spacing of the acoustic wave resonator array are independently 100 μm to 1 cm; each of the acoustic wave resonators can form an acoustic wave generating area facing the in situ culture through hole, and the area of ​​the acoustic wave generating area is not less than 300 μm 2 .

[0042] According to an embodiment of the present application, the row spacing and column spacing of the in situ culture through-hole array are independently 100 μm to 1 cm; the height of the in situ culture through-hole is 10 to 5000 μm; the in situ culture through-hole is cylindrical with a diameter of 100 to 500 μm; the volume of the in situ culture through-hole is 8×10 4 ~10×10 8 μm 3 .

[0043] According to an embodiment of the present application, the micropores, the in situ culture through-holes and the acoustic wave resonators correspond one to one, and the micropore array, the in situ culture through-hole array and the acoustic wave resonator array have consistent periodic distribution.

[0044] According to an embodiment of the present application, the fluid channel extends in a direction perpendicular to the thickness and is formed by being recessed from one side of the microfluidic channel layer close to the micropore array; the width of the fluid channel is 10 to 250 μm, and the recessed depth of the fluid channel is 10 to 250 μm; the inlet and outlet are respectively arranged on both sides of the microfluidic channel layer in a direction perpendicular to the thickness.

[0045] In the second aspect of the present application, the present application proposes a method for detecting cell secretions. According to an embodiment of the present application, the method includes: culturing a single cell under suitable conditions; subjecting a first affinity reagent and a second affinity reagent to a controllable spin float treatment in a single cell culture treatment system, wherein the first affinity reagent and the second affinity reagent can specifically bind to the secretions of the single cell. According to an embodiment of the present application, by using this method, flexible detection of single cell secretions can be achieved, the sensitivity of secretion detection can be improved, and the efficiency of single cell screening can be improved. This method can perform multiple rounds of detection of multiple cell secretions while maintaining cell activity, thereby realizing single-cell secretome research. At the same time, after the cells are detected, they can still be recovered, cultured and amplified.

[0046] According to an embodiment of the present application, the above method for detecting cell secretions may further include at least one of the following technical features:

[0047] According to an embodiment of the present application, the first affinity reagent is coupled to microbeads or expressed on the outer surface of the cell membrane, and the second affinity reagent is coupled to a marker; the marker is used to detect the content of secretions from the single cell to be tested. According to an embodiment of the present application, the first affinity reagent and the second affinity reagent are used to achieve flexible detection of single cell secretions, improve the sensitivity of secretion detection, and enhance the efficiency of single cell screening.

[0048] According to an embodiment of the present application, the second affinity reagent is selected from antibodies.

[0049] According to an embodiment of the present application, the fluorescent label is selected from at least one of a fluorescent dye, an enzyme labeling molecule, and streptavidin coupled to a fluorescent group.

[0050] According to an embodiment of the present application, the fluorescent labeling treatment is performed in the following manner: the captured product is mixed with a second affinity reagent that is bound to a fluorescent dye, and the second affinity reagent has the activity of binding to the secretions of the single cell.

[0051] In one example of this application, the detection principle, as shown in Figure 2, involves microbeads coated with a first affinity reagent capturing single-cell secretions. A fluorescently labeled second affinity reagent then binds to the single-cell secretions to form an antibody complex. This complex is then excited by the fluorescent marker, enabling detection of the single-cell secretions. Without the antibody complex, the fluorescent marker cannot be detected; only with the antibody complex can fluorescence be detected.

[0052] According to an embodiment of the present application, the method further includes: performing amplification and culture treatment on single cells with high fluorescence signals to obtain single cell clones.

[0053] In its third aspect, the present application proposes a method for screening target cells. According to an embodiment of the present application, the method for detecting cell secretions described in the second aspect of the present application is used to detect and process secretions from multiple single cells to be tested; the target cells are identified based on the fluorescence signal detection results. According to an embodiment of the present application, the above method can screen for target cells capable of secreting target secretions from unknown cells to be tested.

[0054] According to an embodiment of the present application, the above-mentioned target cell screening method may further include at least one of the following technical features:

[0055] According to an embodiment of the present application, the above method further comprises: culturing the target cells to obtain target cell clones. Through this step, a single clone of the target cells is obtained.

[0056] According to an embodiment of the present application, the above method further includes: collecting the target cells to obtain a plurality of target cells.

[0057] In one example of the present application, the target cells are screened and obtained using the above-mentioned target cell screening method, and the target single cells are separated and collected by culturing the target cells in situ or using an acoustic tweezers resonator.

[0058] In one example of the present application, during the in situ culture of target cells, the fluid inlet of the cell culture microporous array chip can be used to supplement the nutrients required by the target cells, such as culture medium, and the target cells can be recovered after the cells proliferate to a certain number.

[0059] In one example of the present application, the method further includes performing sequencing processing on the recovered target cells.

[0060] It should be noted that the features and technical effects described in this article for different aspects can be used as reference for each other and will not be repeated here.

[0061] Additional aspects and advantages of the present invention will be set forth in part in the description which follows and, in part, will be obvious from the description which follows, or may be learned by practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0062] The above and / or additional aspects and advantages of the present application will become apparent and easily understood from the description of the embodiments in conjunction with the following drawings, in which:

[0063] Figure 1 is a schematic diagram of the single-cell secretion capture principle and physical device according to an embodiment of the present application; Figure 1a is a schematic diagram of the principle of an ultra-high frequency resonator generating vortices to capture secretions; Figure 1b is a physical picture of the acoustic tweezers resonator (the core component is a pentagonal chip); Figure 1c is a schematic diagram of the pentagonal chip detecting aggregated single-cell secretions, including 101 single cells to be tested, 102 cell culture container, 103 cell secretions, 104 antibody-coated microbeads, and 105 automatic displacement module (connected to the acoustic tweezers resonator to control particle movement).

[0064] FIG2 is a schematic diagram of the detection principle of detecting cell secretions based on the acoustic fluidic tweezers technology according to one embodiment of the present application.

[0065] FIG3 is a schematic diagram of the structure of the acoustofluidic microfluidic chip according to one embodiment of the present application; the left side is a main structure diagram; the right side is a spatial decomposition diagram.

[0066] FIG4 is a flow chart of a detection method for detecting cell secretions based on an acoustic tweezers resonator according to an embodiment of the present application.

[0067] FIG5 is a microscopic photograph of controllable manipulation of a single cell based on an acoustic tweezers resonator according to one embodiment of the present application.

[0068] FIG6 is a schematic diagram of a process for detecting multiple cytokines secreted by T cells based on an acoustic tweezers resonator according to an embodiment of the present application.

[0069] FIG7 is a proliferation curve diagram of a single cell after being treated with an acoustic tweezers resonator according to an embodiment of the present application.

[0070] FIG8 is a microscopic photograph of a fluorescent particle group aggregated by the acoustic tweezers, showing the function of the acoustic tweezers resonator for aggregating microparticles according to one embodiment of the present application; wherein the dotted box represents the acoustofluidic tweezers chip;

[0071] FIG9 is a microscope photograph of a single cell distribution unit according to one embodiment of the present application;

[0072] FIG10 is a microscopic photograph of the detection results of single-cell microbeads capturing cell secretions in microwells according to one embodiment of the present application;

[0073] FIG11 is a microscopic photograph of the detection results of fluorescent complexes captured by microspheres in a microwell array according to one embodiment of the present application; wherein A is the detection result of fluorescent complexes captured by microspheres in a microwell array; B is the detection result after magnification of the microwell array;

[0074] FIG12 is a microscope photograph of in situ cultured and expanded multifunctional T cells in microwells according to one embodiment of the present application. DETAILED DESCRIPTION

[0075] The following describes embodiments of the present invention in detail, examples of which are shown in the accompanying drawings, wherein the same or similar reference numerals throughout represent the same or similar elements or elements having the same or similar functions. The embodiments described below with reference to the accompanying drawings are exemplary and are intended to be used to explain the present invention, and are not to be construed as limiting the present invention.

[0076] As used herein, unless otherwise indicated, the singular forms "a," "an," and the like include plural referents (more than one); "a set" or "a plurality" refers to two or more.

[0077] In this document, unless otherwise specified, the terms "first", "second", "third", "fourth", etc. are used for descriptive purposes only and are not to be understood as indicating or implying relative importance or implicitly indicating the number of technical features indicated; features specified as "first", "second", etc. may explicitly or implicitly include one or more of the said features.

[0078] In this document, unless otherwise specified, the term "acoustic tweezers resonator" refers to a device that uses sound waves to generate a force field, thereby precisely manipulating tiny objects. Based on the properties of sound waves, it uses the principle of resonance to generate an acoustic field to locate, manipulate, and separate microscale objects (such as cells, microparticles, or microfluids). In this application, the acoustic tweezers resonator is used to achieve single-cell separation and secretion capture.

[0079] In this application, unless otherwise specified, the term "spin-flotation treatment" refers to spinning and floating the affinity reagent and cells or cell secretions to aggregate the cell secretions, and enhancing the interaction between the secretions and the affinity reagent based on in situ vibration rotation (which can also be understood as stirring).

[0080] Methods for capturing cell secretions

[0081] In one aspect of the present application, a method for capturing cell secretions is proposed. The method comprises: using an acoustic tweezers resonator to separate cell clusters (or cell groups) so that single cells are distributed in different microcavities (or micropores). The single cells in the microcavity are placed under suitable conditions for culture treatment to promote the secretion of secretions by the single cells in the microcavity. The acoustic tweezers resonator parameters are then adjusted to perform a rotational floating (rotational floating) treatment on the microbeads coated with the first affinity reagent, and the microbeads are controlled to enter the microcavity. Based on the characteristics of sound waves, the principle of resonance is utilized to aggregate cell secretions, and the microbeads coated with the first affinity reagent are controlled to capture cell secretions. Since the capture method described in the present application is based on sound waves to control the movement and aggregation of cells, microbeads, and cell secretions, the operation is simple and convenient, and no mechanical damage is caused to the cells. More importantly, the detection of low-concentration secretions can be achieved.

[0082] The microbeads described in this application can be any shape suitable for the experimental conditions, such as round, square, flaky, elliptical, porous, cylindrical or irregular; the material of the microbeads can be magnetic beads, silica, polymers, metals, etc.

[0083] In one example of the present application, contact-free controlled reactions between different compounds can also be achieved based on the present method.

[0084] In one example of the present application, in the field of microorganisms, contactless cultivation of microorganisms can be achieved based on the present method.

[0085] For ease of understanding, the overall structure and capture principle of the acoustic tweezers resonator are described in detail below with reference to FIG. 1 to FIG. 3 .

[0086] The overall structure of the acoustic tweezers resonator includes a micropore array chip 100, a microfluidic channel layer 200, and an acoustic wave resonator array chip 300, which are sequentially stacked along the thickness direction.

[0087] Microwell array chip

[0088] The microwell array chip 100 has a plurality of microwells 110 formed in a recessed manner on the side of the microfluidic channel layer 200. The plurality of microwells form a microwell array for accommodating cells and / or cell secretions. For example, cell secretions include, but are not limited to, enzymes, antibodies, cytokines, growth factors, exosomes, and the like.

[0089] The plurality of micropores 110 can be arranged along the X and Y directions to form a micropore array. The array arrangement can be linear, circular, rectangular, polygonal, or spherical, preferably rectangular. For example, the row spacing and column spacing of the micropore array are each independently 100 μm to 1 cm, specifically 100 μm, 300 μm, 500 μm, 1 cm, or a range formed by any two endpoints, preferably 500 μm to 1 cm.

[0090] The micropores 110 in the micropore array are used to accommodate cells and / or cell secretions, and can specifically accommodate a single or a small number (such as 2-4) cells, thereby being used not only for single cell analysis but also for multi-cell interaction analysis. The shape of the micropores can be a circular or polygonal geometric structure, preferably a circular structure. For example, the depth of the micropores 110 is 10 to 100 μm, specifically 10 μm, 20 μm, 50 μm, 80 μm, 100 μm or any two point values ​​as the range value formed by the endpoint value, preferably 10 to 50 μm; the pore size of the micropores 110 is 10 to 75 μm, specifically 10 μm, 25 μm, 50 μm, 70 μm, 75 μm or any two point values ​​as the range value formed by the endpoint value, preferably 10 μm to 25 μm.

[0091] The microwell array can be a conventional microwell array or a microwell array with dielectrophoresis (DEP) function.

[0092] Microfluidic channel layer

[0093] The microfluidic channel layer 200 includes a fluid channel 210, a plurality of in situ culture through-holes 220 that penetrate the microfluidic channel layer 200 along the thickness direction and are connected to the fluid channel 210, and an inlet 230 and an outlet 240 formed on the surface of the fluid channel layer 200. The fluid channel 210 extends from the inlet 230 to the outlet 240 along a direction intersecting the thickness direction; the plurality of in situ culture through-holes 220 form an in situ culture through-hole array.

[0094] In situ culture through-holes 220 are arranged in a one-to-one correspondence with microwells 110, with one end along the thickness direction communicating with microwells 110 and the other end corresponding to acoustic resonator 310 and used to receive bulk acoustic waves emitted by the acoustic resonator. The in situ culture through-holes not only facilitate in situ cell culture but also provide an independent reaction environment, ensuring mutual isolation of samples and preventing crosstalk between acoustic and fluidic signals, thereby improving the precision of cell and / or cell secretion manipulation. In some embodiments, the microwell array and the in situ culture through-hole array have the same periodic distribution.

[0095] The dimension of the in situ culture through-hole 220 perpendicular to the thickness direction is larger than that of the micro-hole 110. As a result, the bulk acoustic wave generates a wider range of sound waves, which helps to better act on the cells and / or cell secretions in the micro-hole; on the other hand, it also helps to provide a suitable environment for cell capture and in situ culture in the in situ culture through-hole.

[0096] Acoustic wave resonator array chip

[0097] The acoustic wave resonator array chip 300 includes multiple acoustic wave resonators 310, which form an acoustic wave resonator array. Specifically, the multiple acoustic wave resonators are laid out in the X and Y directions on a single-crystal silicon wafer substrate to form an acoustic wave resonator array. A radio frequency signal source can provide bulk acoustic wave signals to the acoustic wave resonators. Each acoustic wave resonator can be independently switched, allowing for selective capture of cells and / or cell secretions by controlling one or more target cells.

[0098] In the present invention, the distribution of the acoustic resonators in the acoustic resonator array can be one-to-one corresponding to the micropores, or one micropore can correspond to multiple acoustic resonators, or multiple micropores can correspond to multiple acoustic resonators. It is preferred that the micropores in the micropore array correspond to the acoustic resonators in a one-to-one distribution. In some embodiments, the acoustic resonator 310 can correspond one-to-one to the in situ culture through-hole 220, that is, one acoustic resonator 310 corresponds to one in situ culture through-hole 220; or one acoustic resonator 310 corresponds to multiple in situ culture through-holes 220. It can be flexibly selected according to actual conditions, and a one-to-one correspondence is preferred, thereby achieving precise manipulation. Preferably, the micropores 110, the in situ culture through-hole 220 and the acoustic resonator 310 correspond one-to-one, and the micropore array, the in situ culture through-hole array and the acoustic resonator array have the same periodic distribution. In this way, the samples are isolated from each other, providing an independent reaction / culture environment, avoiding crosstalk between acoustic fluid signals, and facilitating the manipulation of cells and / or cell secretions and cell culture.

[0099] The projection of the acoustic wave resonator 310 along the thickness direction covers at least a portion of the in situ culture through-hole 220 and at least a portion of the microwell 110. Thus, on the one hand, the range of the acoustic wave generated by the bulk acoustic wave is large, which helps to better act on the cells and / or cell secretions in the microwell; on the other hand, it also provides a suitable environment for in situ cell culture in the in situ culture through-hole.

[0100] The acoustic wave resonator 310 can be arranged inside the in situ culture through-hole 220, or outside the in situ culture through-hole 220 and near the opening of the in situ culture through-hole 220, so that the in situ culture through-hole 220 receives the bulk acoustic wave emitted by the acoustic wave resonator, that is, the cell and / or cell secretion capture potential well is located inside the in situ culture through-hole 220, so as to achieve manipulation. In some embodiments, the acoustic wave resonator array and the in situ culture through-hole array are formed in a chimeric and sealed manner, and the acoustic wave resonator 310 is located inside the in situ culture through-hole 220. In this way, the bulk acoustic wave emitted by the acoustic wave resonator can better act on the cells and / or cell secretions in the micropores 110. In other embodiments, each acoustic wave resonator 310 in the acoustic wave resonator array is provided with an independent switch. Thus, independent manipulation is facilitated.

[0101] The projection of the acoustic wave resonator 310 along the thickness direction covers at least part of the in situ culture through-hole 220 and at least part of the microwell 110. This helps to receive the bulk acoustic waves emitted by the acoustic wave resonator in the in situ culture through-hole, thereby better acting on the cells in the microwell.

[0102] Methods for detecting cell secretions

[0103] In another aspect, this application proposes a method for detecting cell secretions. This method comprises capturing cell secretions using the aforementioned method for capturing cell secretions, fluorescently labeling the captured product, and then determining the content or type of the cell secretions based on the intensity or type of the fluorescent signal generated by the fluorescently labeled captured product. This method enables multi-round, three-dimensional dynamic detection of cell secretions, as well as multiple flexible detection capabilities.

[0104] In one example of the present application, the fluorescent label is selected from at least one of a fluorescent dye, an enzyme labeling molecule, and streptavidin coupled with a fluorescent group.

[0105] In one example of the present application, the fluorescent labeling treatment is performed in the following manner: the captured product is mixed with a second affinity reagent that is conjugated with a fluorescent dye, and the second affinity reagent has an activity of binding to the secretions of the single cell.

[0106] In one example of the present application, the second affinity agent can be selected from antibodies.

[0107] In one example of the present application, the aforementioned method for detecting cell secretions further includes: amplifying and culturing single cells that meet the target fluorescent signal to obtain single cell clones.

[0108] In another aspect of the present application, a method for detecting cell secretions is provided. The method comprises: culturing a single cell under suitable conditions so that the single cell secretes the cell secretions;

[0109] The first affinity reagent and the second affinity reagent are then subjected to a controllable spin float treatment in a single cell culture processing system, where the first affinity reagent and the second affinity reagent can specifically bind to the secretions of the single cell; wherein the first affinity reagent is coupled to the microbeads, and the second affinity reagent is coupled to the label;

[0110] By detecting the fluorescent signal of the marker, the content of the secretion of the single cell to be tested is determined.

[0111] In one example of the present application, during the capture of cell secretions, microbeads coated with different first affinity reagents can be used to capture different cell secretions, and then second affinity reagents containing different fluorescent labels (such as those that can produce fluorescence of different colors after excitation) are used to bind to different types of cell secretions, and the type of cell secretions is determined based on the difference in fluorescence color.

[0112] In another example of the present application, during the capture of cell secretions, microbeads coated with the same first affinity reagent are used to capture the same cell secretions, and then the same fluorescent-labeled antibody is used to bind to the cell secretions, and the content of the cell secretions is determined based on the fluorescence intensity.

[0113] In another example of the present application, the capture parameters may be adjusted and the changes in fluorescence intensity may be compared to determine the optimal capture parameters.

[0114] In another example of the present application, the optimal protein expression and secretion conditions can be screened by changing the culture conditions and comparing the changes in fluorescence intensity.

[0115] In one example of the present application, the aforementioned method for detecting cell secretions further includes: amplifying and culturing single cells that meet the target fluorescent signal to obtain single cell clones.

[0116] For ease of understanding, the technical solution of this application is described in detail below with reference to FIG4 .

[0117] Step 301: Obtaining a single-cell distribution unit: Using an acoustic tweezers resonator, separate cell clusters and distribute the single cells in different microcavities to form a single-cell distribution array. The single cells in the microcavities are then cultured under conditions suitable for protein expression and secretion to promote protein secretion by the single cells in the microcavities.

[0118] Step 302: The acoustic tweezers resonator manipulates the antibody-coated microbeads to enter the single-cell microcavity: the acoustic tweezers resonator parameters are adjusted to perform a rotational levitation (rotational floating) process on the microbeads coated with the first affinity reagent, and the microbeads are controlled to enter the single-cell microcavity.

[0119] Step 303, the acoustic tweezers resonator aggregates cell secretions and enhances the microbead capture capability: This step is based on the characteristics of acoustic waves and utilizes the principle of resonance to aggregate cell secretions and control the microbeads coated with the first affinity reagent to capture cell secretions.

[0120] Step 304 , adding fluorescently labeled antibodies to bind to the captured secretory proteins: binding to cell secretions by adding fluorescently labeled antibodies.

[0121] Step 305 , detecting corresponding signals: exciting the fluorescent marker, and detecting the fluorescent signal generated based on the fluorescent marker.

[0122] Step 306 , recovering the clones of interest: amplifying and culturing the single cells that meet the target fluorescence signal to obtain single cell clones.

[0123] Target cell screening method

[0124] In another aspect, this application proposes a method for screening target cells. The method comprises: detecting and processing secretions from multiple single cells to be tested using the method for detecting cell secretions described above; and identifying the target cells based on the fluorescence signal detection results. According to embodiments of the application, the above method can screen for target cells capable of secreting target secretions from unknown cells to be tested.

[0125] In one example of the present application, the method further comprises: culturing the target cells to obtain target cell clones. Through this step, a single clone of the target cells is obtained.

[0126] For ease of understanding, the above method is described in detail by taking the screening of multifunctional cytokine-secreting cells (target cells) as an example.

[0127] 1) Adjusting the acoustic tweezers resonator separation parameters (e.g., power of 50-500 mW, frequency of 1-50 GHz) to separate the cell clusters.

[0128] 2) Incubate and culture the cells to be tested separated into individual microwells to activate the cells to be tested.

[0129] 3) After the cells to be tested are activated, the parameters of the acoustic tweezers resonator rotation processing are adjusted (such as power of 250-350 mW and frequency of 1.5-2.5 GHz) to capture the secretions of the cells to be tested, and fluorescence is monitored using a fluorescence microscope.

[0130] 4) After the fluorescence detection, the microbeads are removed so that the detected cells can continue to be cultured and expanded.

[0131] 5) Repeat steps 1) to 4) to perform multiple rounds of capture and detection of the cells to be tested, achieving multiplexed detection of cell secretions and recording fluorescence intensity in real time. Based on the fluorescence intensity detection results, the multifunctional antibody-secreting cells are identified and recovered, cultured, and expanded to obtain a monoclonal multifunctional cytokine-secreting cell.

[0132] Advantages compared to existing technologies

[0133] Current methods for analyzing secretions using a combination of microfluidics and photodielectrophoresis present several challenges: complex optical path design, high costs, and prolonged cell manipulation. Furthermore, this method cannot achieve three-dimensional manipulation of particles, requiring only two-dimensional movement to a common flow channel for cell recovery. In contrast, the cell secretion detection method proposed in this application enables three-dimensional control of cells, microbeads, and cell secretions, and requires significantly less time.

[0134] There is currently a multi-omics detection method for single-cell secretory proteins based on microfluidic design, and its equipment structure includes an upper part with an antibody array (for protein detection) and a rectangular groove at the bottom (for limiting single cells). Although this method can detect multiple secretory factors of single cells, it cannot maintain cell activity, nor can it recover and amplify cells. In addition, the preparation of this method is cumbersome, and the chip array needs to be pre-coated with antibodies and fixed with a panel, which cannot be flexibly adjusted. In contrast, the cell secretion detection method proposed in this application uses sound waves to control the movement and aggregation of cells, microbeads and cell secretions. This method is convenient and flexible to operate, and does not cause mechanical damage to cells.

[0135] Currently, there is a method based on microfluidic design that uses gravity to force single cells into micropores for single cell culture and analysis. However, this method requires mechanical manipulation of the capillary to absorb the cell population, which is difficult to operate and causes significant damage to the cells. In addition, the degree of automation is not high and the efficiency is low. In contrast, the method described in this application that uses sound waves to control the movement and aggregation of cells, microbeads, and cell secretions achieves a high degree of automation, flexible and convenient operation, and high-efficiency cell secretion detection. This method does not cause mechanical damage to the cells and can also be used to recover and culture single cells that produce target secretions.

[0136] The embodiments of the present invention will be described in more detail below, and examples of the embodiments are shown in the accompanying drawings. The embodiments described below with reference to the accompanying drawings are exemplary and are intended to be used to explain the present invention, but are not to be construed as limiting the present invention. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in this area or according to the product specifications. Reagents or instruments used that do not specify the manufacturer are conventional products that can be obtained commercially.

[0137] The reagents used in the following examples are shown in Table 1.

[0138] Table 1

[0139] Example 1: Detection of antibody secretion by a single B cell based on acoustic tweezers resonator

[0140] This example demonstrates the process of detecting secretions of a single B cell based on the principle of acoustic tweezers resonator.

[0141] 1. Obtaining Single B Cell Distribution Arrays

[0142] By capturing and manipulating a single B cell into a microwell using an acoustic tweezers resonator connected to an automatic displacement module (using the combined effect of the DEP force and fluid force generated by the microfluidic chip in the acoustic tweezers resonator to achieve single-cell entry into the well and form a single-cell array), a distributed microwell array of single B cells was obtained;

[0143] The separation parameters are set as follows:

[0144] The resonator frequency is 1-50 GHz, and the power is 50-500 mW. In this embodiment, the optimal parameters are set to 1.5 GHz-2.5 GHz, and the power is 50-300 mW. As shown in FIG9 , the single-cell distribution microwell array is obtained, and one cell to be tested is distributed in each microwell.

[0145] 2. B cell activation

[0146] incubating the B cells in the microwell array under conditions favorable for antibody expression;

[0147] The incubation conditions include: culture medium RPMI 1640 + 10% FBS + IL-2 (50ng / ml) + 50uM β-mercaptoethanol, adding the corresponding immune antigen to activate B cells, and incubating them in a constant temperature of 37°C and 5% carbon dioxide incubator for 12-24 hours to achieve an activated state.

[0148] 3. Rotation and floating treatment

[0149] An acoustic tweezers resonator is used to capture microbeads coupled to the first affinity reagent (biotin-modified anti-IgG secondary antibody coupled to streptavidin PS microbeads, with a diameter of 5 μm) and introduce them individually or multiple times into the microwells. The acoustic tweezers resonator parameters are adjusted to keep the microbeads stable in the vortex to capture antibody proteins secreted by B cells.

[0150] The parameters for the acoustic tweezers resonator rotation and floating processing are set as follows:

[0151] The resonator frequency is 1.5 GHz and the power is 300 mW.

[0152] 4. Second affinity reagent (fluorescently labeled secondary antibody / fluorescently labeled antigen) binds to the secretion

[0153] Add fluorescently labeled antigens or fluorescently labeled secondary antibodies (the fluorescent group can be Alexa Fluo.488, 594, 647 or Cy3, Cy5, etc., diluted with PBS, the ratio is 1:50-1:1000), and bind to the microspheres that capture the cell-secreted antibodies.

[0154] 5. Secreted Protein Characterization

[0155] Wash with DPBS to remove unbound fluorescently labeled secondary antibodies or antigens. Use fluorescence microscopy to image and measure fluorescence intensity to characterize the amount of cell-secreted protein. Turn on the acoustic tweezers resonator to aggregate the antibody proteins secreted by the cells, as shown in Figure 1a. The vortex generated by the acoustic tweezers resonator can aggregate particles in the culture medium.

[0156] The acoustic tweezers resonator parameters are set as follows:

[0157] The resonator frequency is 2 GHz and the power is 200 mW.

[0158] 6. Target cell recovery

[0159] Cells with high fluorescence intensity were screened, their coordinates marked, cultured in situ, and recovered after single colonies formed (Figure 5). High-performing single colonies were recovered and transferred to 96-well plates for further expansion. Cell proliferation was observed over seven days, as shown in Figure 7, demonstrating that cell manipulation using acoustofluidic tweezers did not affect cell viability.

[0160] Example 2: Detection of multiple cytokines secreted by T cells based on acoustic tweezers resonator

[0161] This example exemplifies the process of detecting multiple cytokines secreted by T cells based on the principle of acoustic tweezers resonator ( FIG. 6 ).

[0162] 1. Obtaining a single T cell distribution unit array

[0163] A single T cell is manipulated using acoustic fluidic tweezers (105), and a single cell is placed into a hole of a microwell array to achieve single cell distribution;

[0164] The acoustic tweezers resonator separation parameters are set as follows:

[0165] The resonator frequency is 2.5 GHz and the power is 300 mW.

[0166] 2. Activate T cells

[0167] T cells in the microwell array were placed under conditions that favored cytokine expression (e.g., CD3 / CD28 activated microbeads (Dynabeads TMThe cells were then co-incubated with Human T-Activator CD3 / CD28 (1:1) for activation for 24 hours. Similarly, a mixture of capture microspheres coupled to different antibodies (anti-IFN-γ (2 μm diameter), anti-TNF-α (5 μm diameter), and anti-IL-2 (8 μm diameter)) was introduced into the microwells using acoustofluidic tweezers. The different diameters of the microspheres coupled to the different antibodies allowed for differentiation during the experiment. After 24 hours of co-incubation, T cells secreting cytokines IFN-γ, TNF-α, and IL-2 were captured. As shown in Figure 10, three capture microspheres with different diameters were introduced into the cell microwells.

[0168] 3. Fluorescently labeled antibodies binding to cytokines

[0169] Add the corresponding antibody to bind to the protein captured by the beads (such as Human IL-2Alexa 647-conjμgated Antibody, 1:500 dilution; recombinant PE Anti-TNF alpha antibody, 1:500 dilution; recombinant Alexa 488 fluorescent Anti-Interferon gamma antibody, 1:1000 dilution). Unbound antibody dye was washed away with PBS. If the microbeads captured IL-2 cytokine, the microbeads would form microbeads@IL-2@Human IL-2Alexa The 647-conjμgated Antibody complex is displayed as 647 fluorescence on the 8μm microbeads. If the microbeads capture the TNF-α cytokine, the microbeads will form a microbead@TNF-α@Biotin anti-human TNF-α@PE fluorescent complex, which is displayed as 532 fluorescence on the 5μm microbeads. If the microbeads capture the IFN-γ cytokine, the microbeads will form a microbead@IFN-γ@anti-human IFN-γ-Alex 488 fluorescent complex, which is displayed as 488 fluorescence on the 2μm microbeads.

[0170] 4. Cytokine Characterization

[0171] The acoustic tweezers resonator is turned on to promote the binding of the antibody on the capture microspheres with the secreted cytokine to be detected, thereby achieving the effect of aggregation and signal enhancement (402). As shown in FIG8 , the fluorescence intensity is found to be enhanced and different from the background, indicating that the acoustic fluid tweezers have the effect of aggregating particles.

[0172] The images are taken on a fluorescence microscope (403) and the fluorescence intensities of different microspheres are detected, which correspond to the different cytokine contents secreted by the cells.

[0173] The acoustic tweezers resonator parameters are set as follows:

[0174] The resonator frequency was 3 GHz and the power was 200 mW. The results are shown in Figure 10. The fluorescence of the three microspheres with different diameters in the microwells represents the expression levels of different cytokines. Figure 11 shows the fluorescent complex captured by the detection microspheres in the microwell array. T cell secretion of cytokines was detected in the microwells. The microspheres captured the target cytokines and formed an antibody complex with the fluorescent secondary antibody for detection. The vortex effect of the resonator promoted the binding and emitted a more obvious fluorescence.

[0175] 5. Target cell recovery

[0176] The target cells are cultured in situ. During the culture period, the culture medium can be supplemented using the fluid inlet of the cell culture microwell array chip. After 3-7 days of culture, the cells proliferate to a certain number. Figure 12 shows the multifunctional T cell clones cultured and expanded in situ in the microwells. The positive cells in the microwells located in step 4 are recovered to a 96-well plate and cultured in RPMI 1640 + 10% FBS + 1% Glutamax medium or directly proceed to the next step of sequencing and other subsequent processing.

[0177] Example 3: Detection of Antibody Secretion by Spleen Cells of Immunized Mice Based on Acoustic Tweezers Resonator

[0178] This example exemplifies the process of screening B cells that can secrete anti-CD3 antibodies from the spleen cells of CD3-immunized mice based on the principle of acoustic tweezers resonator.

[0179] 1.1. Enrichment of spleen cells from immunized mice

[0180] Mice were immunized with human CD3 protein as an antigen, and their spleen cells were separated, washed and enriched to obtain a spleen cell suspension of the immunized mice, which was then cultured in RPMI1640+10% FBS medium for later use.

[0181] 2. Obtaining a single mouse spleen cell distribution array

[0182] Mouse spleen cells were collected and resuspended to a concentration of 3*10^5 cells / mL to 2*10^6 cells / mL. They were then introduced into microwells using an acoustic tweezers resonator microfluidic chip (the combined effect of the DEP force and fluid force generated by the microfluidic chip in the acoustic tweezers resonator enables single cell entry into the wells to form a single cell array). This yielded a distributed microwell array of single spleen cells.

[0183] The separation parameters are set as follows:

[0184] The resonator frequency is 1.5 GHz and the power is 400 mW;

[0185] 2.3. Jurkat cell introduction

[0186] This example utilizes the high expression of CD3 protein on the Jurkat cell membrane as a capture carrier for CD3 antibodies (primary affinity reagent). Jurkat cells are enriched and resuspended in culture medium. FITC-conjugated secondary antibodies (goat anti-mouse IgG antibody, (H+L)FITC conjugated, 1:1000, as the second affinity reagent) are added and introduced into the microwells of the microfluidic chip in step 1. The cells are then incubated with mouse spleen cells for 1-2 hours to allow B cells to secrete sufficient antibodies.

[0187] 4. Rotation and floating treatment

[0188] The acoustic tweezers resonator parameters were adjusted to keep Jurkat stable in the vortex, thereby capturing the anti-CD3 antibody protein secreted by B cells. At the same time, the 488 fluorescently modified secondary antibody also bound to the Jurkat cells, displaying the 488 fluorescence of the Jurkat cell membrane.

[0189] The parameters for the acoustic tweezers resonator rotation and floating processing are set as follows:

[0190] The resonator frequency is 1.5GHz and the power is 300mW.

[0191] 5. Target cell recovery

[0192] Cells with high fluorescence intensity were screened out, their coordinates were marked, and they were recovered.

[0193] Furthermore, the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of the technical features being referred to. Thus, a feature defined as "first" or "second" may explicitly or implicitly include at least one such feature. In the description of the present invention, "plurality" means at least two, such as two, three, etc., unless otherwise specifically defined.

[0194] In the description of this specification, the reference terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" mean that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art can combine and combine different embodiments or examples described in this specification and features of different embodiments or examples without contradiction.

[0195] Although the embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and are not to be construed as limitations on the present invention. A person skilled in the art may change, modify, replace and modify the above embodiments within the scope of the present invention.

Claims

1. A method for capturing cell secretions, characterized in that: include: Cultivate the single cells under appropriate conditions; The first affinity reagent is subjected to a controllable spin-float treatment in a single cell culture treatment system, wherein the affinity reagent can specifically bind to the secretion of the single cell.

2. The method according to claim 1, characterized in that The single cell is separated and obtained by a microfluidic chip method, a micropore array combined with a centrifugal gravity pore method, or an acoustic tweezers resonator method.

3. The method according to claim 2, characterized in that The single cell is separated and obtained based on the acoustic tweezers resonator method.

4. The method according to claim 3, characterized in that The acoustic tweezers resonator separation parameters are selected from power of 50-500 mW and frequency of 1-50 GHz; Preferably, the acoustic tweezers resonator separation parameters are selected from power of 50-300 mW and 1.5 GHz-2.5 GHz.

5. The method according to claim 1, wherein The first affinity reagent is provided in the form of microbeads, and the first affinity reagent is coated on the outer surface of the microbeads; Optionally, the first affinity reagent is selected from an antibody or an aptamer.

6. The method according to claim 5, characterized in that The microbeads are selected from at least one of polystyrene beads, Luminex™ beads, magnetic beads and microrods; Optionally, the microbeads have a diameter of 100 nm to 10 μm.

7. The method according to claim 1, characterized in that The first affinity reagent is provided in the form of cells, and the first affinity reagent is expressed on the outer surface of the cell membrane; Optionally, the first affinity reagent is selected from an antibody or a polypeptide; Optionally, the spin-floating process is achieved by an acoustic tweezers resonator.

8. The method according to claim 7, characterized in that The spin-floating treatment is carried out under the conditions of a power of 250 to 350 mW and a frequency of 1.5 to 2.5 GHz; Preferably, the spin-floating treatment is performed at a power of 300 mW and a frequency of 2 GHz.

9. The method according to any one of claims 5 to 8, characterized in that The microbeads coated with the first affinity reagent are preliminarily enriched by passing through an acoustic tweezers resonator.

10. The method according to claim 9, characterized in that The method further includes injecting the magnetic beads coated with the first affinity reagent into the single cell culture system through the inlet of the acoustic tweezers resonator microfluidic chip.

11. The method according to claim 1, wherein The single cell comprises at least one selected from T cells, B cells, hybridoma cells, CHO cells and tumor cells.

12. The method according to claim 1, characterized in that The secretion of the single cell includes at least one selected from antibodies, cytokines, growth factors and exosomes.

13. The method according to claim 7, 9 or 10, characterized in that: The acoustic tweezers resonator microfluidic chip comprises: a micropore array chip, a microfluidic channel layer and an acoustic tweezers resonator array chip which are sequentially stacked along the thickness direction. The surface of the micropore array chip close to the microfluidic channel layer is concave to form a plurality of micropores, and the plurality of micropores form a micropore array for accommodating cells and / or cell secretions; The microfluidic channel layer comprises a fluid channel, a plurality of in situ culture through-holes penetrating the microfluidic channel layer along the thickness direction and communicating with the fluid channel, and an inlet and an outlet formed on a surface of the fluid channel layer, wherein the fluid channel extends from the inlet to the outlet along a direction intersecting the thickness direction; the plurality of in situ culture through-holes form an in situ culture through-hole array; The acoustic tweezers resonator array chip includes a plurality of acoustic wave resonators, and the plurality of acoustic wave resonators form an acoustic wave resonator array; The in situ culture through hole is arranged in a one-to-one correspondence with the microhole, and one end along the thickness direction is connected to the microhole, and the other end corresponds to the acoustic wave resonator and is used to receive the acoustic wave emitted by the acoustic wave resonator. The dimension along the direction perpendicular to the thickness is larger than that of the micropore, and the projection of the acoustic wave resonator along the thickness direction covers at least a portion of the in situ culture through-hole and at least a portion of the micropore; Optionally, the row spacing and column spacing of the microwell array are each independently 100 μm to 1 cm; The depth of the micropores is 10 to 100 μm; The pore size of the micropores is 10 to 75 μm; Optionally, the microwell array chip comprises: substrate; a dielectrophoretic electrode layer, the dielectrophoretic electrode layer being disposed on the substrate and comprising dielectrophoretic electrodes; and The micropore array is provided on the dielectrophoresis electrode layer; Optionally, the dielectrophoretic electrode layer includes a plurality of dielectrophoretic electrodes, and the plurality of dielectrophoretic electrodes form a dielectrophoretic electrode array; The dielectrophoresis electrodes in the dielectrophoresis electrode array correspond one to one with the micropores in the micropore array; Each of the dielectrophoresis electrodes is provided with an independent switch; Optionally, the acoustic wave resonator array and the in situ culture through-hole array are embedded and sealed, and the acoustic wave resonator is located in the in situ culture through-hole; Optionally, each acoustic wave resonator in the acoustic wave resonator array is provided with an independent switch; The row spacing and column spacing of the acoustic wave resonator array are independently 100 μm to 1 cm; Each of the acoustic wave resonators can form an acoustic wave generating area facing the in situ culture through hole, and the area of ​​the acoustic wave generating area is not less than 300 μm 2 ; Optionally, the row spacing and column spacing of the in situ culture through-hole array are each independently 100 μm to 1 cm; The height of the in situ culture through-hole is 10 to 5000 μm; The in situ culture through-hole is cylindrical and has a diameter of 100 to 500 μm; The volume of the in situ culture through-hole is 8×10 4 ~10×10 8 μm 3 ; Optionally, the micropores, the in situ culture through-holes and the acoustic wave resonators correspond to each other one by one, and the micropore array, the in situ culture through-hole array and the acoustic wave resonator array have consistent periodic distribution; Optionally, the fluid channel extends perpendicular to the thickness direction and is formed by being recessed from a side of the microfluidic channel layer close to the microwell array; The width of the fluid channel is 10 to 250 μm, and the depth of the depression of the fluid channel is 10 to 250 μm; The inlet and the outlet are respectively arranged on two sides of the microfluidic channel layer perpendicular to the thickness direction.

14. A method for detecting cell secretions, characterized in that: include: Capturing secretions of a single cell to be tested using the method according to any one of claims 1 to 13; The captured products are fluorescently labeled; as well as Based on the fluorescence signal, the content of the secretion of the single cell to be tested is determined.

15. A method for detecting cell secretions, characterized in that: include: Cultivate the single cells under appropriate conditions; performing a controllable spin-float treatment on a first affinity reagent and a second affinity reagent in a single cell culture processing system, wherein the first affinity reagent and the second affinity reagent can specifically bind to the secretion of the single cell; Optionally, the first affinity reagent is coupled to microbeads or expressed on the outer surface of a cell membrane, and the second affinity reagent is coupled to a label; The content of the secretion of the single cell to be tested is detected using a marker. The method according to claim 15 , wherein target cells are screened based on the content of the cell secretions.

17. The method according to claim 15, characterized in that The marker is a fluorescent marker; 18. The method according to claim 14, characterized in that The fluorescent label is selected from at least one of a fluorescent dye, an enzyme labeling molecule and streptavidin coupled with a fluorescent group.

19. The method according to claim 14, wherein The fluorescent labeling process is carried out in the following manner: mixing the captured product with a second affinity reagent conjugated with a fluorescent dye, wherein the second affinity reagent has an activity of binding to the secretion of the single cell; Optionally, the second affinity reagent is selected from an antibody.

20. The method according to claim 14, wherein Further including: Single cells with high fluorescence signals are expanded and cultured to obtain single cell clones.

21. A method for screening target cells, characterized in that: include: Detecting and processing a plurality of single cell secretions to be detected using the method for detecting cell secretions according to any one of claims 14 to 20; The target cells are determined based on the fluorescence signal detection results.

22. The method according to claim 21, characterized in that Further including: The target cells are cultured to obtain target cell clones.

23. The method according to claim 21, characterized in that Further including: The target cells are collected to obtain multiple target cells.

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