Single-cell antibody chip, preparation method, image analysis method and device

Abstract

The embodiment of the present application relates to the technical field of cell chip, and discloses a single cell antibody chip, which is characterized by comprising: a chip substrate, wherein a biological recognition area and a physical function area are arranged on the chip substrate; a first group of core points is arranged in the biological recognition area, the first group of core points comprises a plurality of biological recognition core points, each biological recognition core point is pre-coated with different markers for capturing and detecting a to-be-detected substance; a second group of core points is arranged in the physical function area, the second group of core points comprises a plurality of physical function core points, the physical function core points comprise first positioning core points and second positioning core points, and the display states of the first positioning core points and the second positioning core points are different. In the embodiment of the present application, the single cell antibody chip can capture and label single cells, and the chip substrate can be directionally identified by using the positioning core points, and the imaging result can be uniformly processed by using the quantitative core point array, so that the expression level of the protein can be better quantitatively detected.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of single cell chip, in particular to a single cell antibody chip, a preparation method, an image analysis method and a device. BACKGROUND

[0002] Single cell technology takes a single cell as a unit to analyze gene expression, RNA transcription, protein distribution, cell activity and physiological and biochemical indicators. The single cell sequencing technology, which is currently developing rapidly, can perform high-throughput sequencing analysis of genetic information such as genome, transcriptome and epigenome at the single cell level, and obtain the gene expression and distribution of each cell.

[0003] Traditional sequencing methods (chip expression profiling analysis and second-generation sequencing) extract DNA or RNA from a large number of sample cells for sequencing, and the data obtained after sequencing is the average value of all cells, which loses the heterogeneity between different cell individuals (the functions of the same type of cells are not completely the same). Single cell sequencing extracts single cells of the same type of tissue for analysis (transcriptome, epigenome, proteome, and spatiotemporal characteristics of gene expression analysis, other functional analysis), which can record the different forms of functions of the same type of cells at different times and different sites, and the interaction with the surrounding environment. Single cell sequencing technology labels single cells with tens of thousands of suspended microbeads with unique and independent Barcode labels, so that each single cell-derived DNA or RNA has a uniform Barcode "identity code". DNA or RNA with the same Barcode is a nucleic acid fragment from the same cell, so that each cell-derived DNA / RNA fragment can be distinguished in subsequent bioinformatics analysis, achieving the purpose of single cell genome and transcriptome identification. With the current technical level, 30,000 cells can be input, and the gene expression profile of up to 20,000 cells can be obtained. The average gene detection amount is as high as 3,000 RNA, and the average is about 1,000 RNA; or nearly 10,000 cell whole genome data. The single sample test data volume reaches 500 GB or even several TB.

[0004] At present, international research in the field of single-cell technology has a very deep and extensive application in single-cell sequencing of DNA and RNA, but the research or application related to the protein level is relatively very scarce. The current technology generally uses a millimeter-sized antibody chip to analyze the total protein of a sample, and cannot distinguish the specific cell from which the protein originates. For example, most protein chips or antibody chips can achieve the labeling of hundreds or even thousands of antibodies on a microcarrier to capture the proteins of a sample, and indicate the content of up to thousands of proteins by the method of immunohistochemical coloration or fluorescence coloration. However, this method is also the content of the total protein of the sample, and cannot distinguish the protein originating from a single cell.

[0005] In the aspect of single-cell protein detection, at present, domestic scientists separate single cells one by one and analyze the protein of single cells by mass spectrometry technology, but this technology has the following problems: first, the throughput is low and cannot complete the test of thousands of cells; second, the sensitivity is limited based on the mass spectrometry technology; third, secreted proteins cannot be detected because secreted proteins are located outside the cell and are removed during single-cell separation. Similarly, the detection of secreted proteins is also a technical problem that cannot be solved by almost all the foregoing methods on single cells.

[0006] Although there are many methods for analyzing proteomes at present, none of them is for analyzing or detecting single cells. In the field of single cells, only one technology can complete the high-throughput analysis of the proteome of a single cell, especially secreted proteins, at the single-cell level. The current microfluidic single-cell capture technology. This technology pre-labels an antibody array of more than 30 kinds on a chip. During the test, cell suspension (more than 30,000 cells are required) is distributed to the "microchamber" of the chip through a microfluidic pipeline. After the cell is captured in the "microchamber", it is incubated and cultured for a period of time, and secreted proteins are captured by the "antibody array" around the cell through background free diffusion or Brownian motion. Because secreted proteins are free in the liquid, the cell capture density cannot be too high to prevent the secreted proteins of a cell from diffusing to the "microchamber" around other cells and causing cross interference. Therefore, the average number of captured cells of the chip is only 800 cells. This greatly limits the detection throughput of the method. At the same time, the pre-coated antibody array on the microfluidic chip has a certain spatial difference with the capture position of the cell, so the data stability between two microchambers is not satisfactory. SUMMARY

[0007] In view of the defects, the embodiment of the present application discloses a single-cell antibody chip, which uses a positioning core point to identify the direction of the chip substrate and uses a quantitative core point array to realize the uniformization processing of the imaging result, thereby better quantitatively detecting the expression level of the protein.

[0008] The first aspect of the embodiment of the present application discloses a single-cell antibody chip, comprising:

[0009] a chip substrate, wherein a biological recognition area and a physical function area are arranged on the chip substrate; a first group of core points is arranged on the biological recognition area, the first group of core points comprises a plurality of biological recognition core points, and each biological recognition core point is pre-coated with a different marker for capturing and detecting a to-be-detected substance;

[0010] a second group of core points is arranged on the physical function area, the second group of core points comprises a plurality of physical recognition core points, the physical recognition core points comprise a first positioning core point and a second positioning core point, and the display states of the first positioning core point and the second positioning core point are different, and the first positioning core point and the second positioning core point are used for identifying the direction of the chip substrate.

[0011] As an optional implementation, in the first aspect of the embodiment of the present application, the physical function area further comprises a quantitative core point array, and a standard fluorescent substance with an equal gradient concentration is sequentially added at each concentration core point of the quantitative core point array; the quantitative core point array is used for normalizing the relative expression level of a protein.

[0012] As an optional implementation, in the first aspect of the embodiment of the present application, the marker is any one of a protein antibody, a nucleic acid probe or a fluorescent point, and the display states are different in shape, size or color.

[0013] As an optional implementation, in the first aspect of the embodiment of the present application, the chip substrate is a microarray with a size of 1*1 um, the thickness of the chip substrate is any value in a range from 1 um to 5 um, the first positioning core point and the second positioning core point are respectively located on two sides of a same edge row of the chip substrate, and the chip is a liquid suspension chip.

[0014] The second aspect of the embodiment of the present application discloses a preparation method of a single-cell chip, comprising:

[0015] A single-cell suspension is prepared according to a laboratory standard operation file, and the concentration of the single-cell suspension is tested by using a cell counter or a hemocytometer to determine the cell concentration data in the single-cell suspension;

[0016] A cell suspension with a preset number of cells is sucked according to the cell density;

[0017] A single-cell droplet is prepared, a single-cell antibody chip pre-coated with an antibody combination is added during the preparation process, and a single-cell droplet parameter is adjusted so that each single-cell droplet carries a single-cell antibody chip; and the number of single-cell antibody chips is greater than the number of cells.

[0018] The single-cell containing liquid suspension is incubated under different conditions for a set time according to cell culture requirements or test requirements to release proteins to the outside of the cells and bind to the corresponding chip;

[0019] An antigen-antibody binding inhibitor and a membrane breaker are added to the single-cell droplet mixture, the cell droplet is broken, the single-cell chip is released into the liquid, and the lysed liquid is transferred to a centrifuge tube for centrifugal precipitation and impurity removal;

[0020] A plurality of antibody premixes are used to label the proteins, and incubation is performed at room temperature for a preset time, and the operations of first antibody washing, second antibody incubation, and second antibody washing are sequentially performed;

[0021] The antibody-labeled chip is subjected to color development imaging.

[0022] As an optional implementation, in the second aspect of the embodiment of the present application, the color development imaging of the antibody-labeled chip comprises:

[0023] The washed chip is resuspended with 1ml PBS, and the resuspended liquid is uniformly added to a 12-well or 6-well cell culture plate. If the liquid is not enough to cover the entire plate, PBS is added to evenly cover the plate.

[0024] After standing for 5 minutes to allow the chip to uniformly precipitate at the bottom of the cell culture plate, a bottom-reading scanner or a microscopic imaging system is used to perform panoramic imaging of the chip under bright field imaging conditions.

[0025] The impurity removal operation comprises:

[0026] The supernatant is removed, and cell debris, reagents from the previous step, proteins after cell lysis, and impurities are removed.

[0027] The chip is resuspended with 1ml PBS, and centrifuged at 100g for 1 minute to remove the supernatant. This step is repeated 3 times to remove impurities.

[0028] The third aspect of the embodiment of the present application discloses a single-cell chip image analysis method, comprising:

[0029] Each initial image information of the antibody chip in the suspension is obtained by a display imaging system, and the initial image information is rearranged to obtain chip image information in a uniform direction; the chip image information comprises a biological recognition area for capturing and detecting the detected substance and a physical functional area for marking the chip image information;

[0030] The chip image information is normalized according to the quantitative chip point array of the physical functional area, so that all chip image signals are modulated into images with the same intensity or color.

[0031] performing fluorescence signal intensity analysis or staining optical density analysis on each identification dot in the biological recognition area of the modulated chip image information to obtain signal intensity data of each identification dot;

[0032] performing bioinformatics analysis on the signal intensity data obtained by the analysis to obtain a dot analysis result.

[0033] As an optional implementation, in the third aspect of the embodiment of the present application, the image rearrangement on the initial image information to obtain chip image information of a unified direction comprises:

[0034] identifying a positioning dot in the physical function area of the initial image information to determine position information and shape information of the positioning dot;

[0035] determining positive-negative state information of the initial image information according to the shape information;

[0036] determining direction state information of the initial image information according to the position information;

[0037] performing image flipping and image rotation on the corresponding initial image information according to a preset image direction, the positive-negative state information and the direction state information to obtain chip image information of a unified direction.

[0038] As an optional implementation, in the third aspect of the embodiment of the present application, the normalization processing on the chip image information according to the quantitative dot array of the physical function area comprises:

[0039] obtaining signal intensity of the quantitative dot array at the physical function area of all chip image information, and constructing quantitative standard information according to the signal intensity;

[0040] performing normalization processing on the brightness of the fluorescence dot or the staining dot of all biological recognition areas according to the quantitative standard information.

[0041] The fourth aspect of the embodiment of the present application discloses an electronic device, comprising: a memory storing executable program code; a processor coupled with the memory; the processor invokes the executable program code stored in the memory, and is used for executing the image analysis method of the single cell chip disclosed in the first aspect of the embodiment of the present application.

[0042] The fifth aspect of the embodiment of the present application discloses a computer readable storage medium storing a computer program, wherein the computer program causes a computer to execute the image analysis method of the single cell chip disclosed in the third aspect of the embodiment of the present application.

[0043] Compared with the prior art, the embodiment of the present application has the following beneficial effects:

[0044] The single-cell antibody chip in the embodiment of the present application can capture and label single cells, and the chip substrate is directionally identified by using a positioning core point, and the imaging result is homogenized by using a quantitative core point array, so that the expression level of the protein is better quantitatively detected. BRIEF DESCRIPTION OF DRAWINGS

[0045] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the drawings needed in the embodiments will be briefly introduced as follows. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can be obtained by those skilled in the art without creative labor on the basis of these drawings.

[0046] Figure 1 is a structural schematic diagram of the single-cell antibody chip disclosed by the embodiment of the present application;

[0047] Figure 2 is another structural schematic diagram of the single-cell antibody chip disclosed by the embodiment of the present application;

[0048] Figure 3 is still another structural schematic diagram of the single-cell antibody chip disclosed by the embodiment of the present application;

[0049] Figure 4 is a flowchart of the preparation method of the single-cell chip disclosed by the embodiment of the present application;

[0050] Figure 5 is a flowchart of the image analysis method of the single-cell chip disclosed by the embodiment of the present application;

[0051] Figure 6 is a flowchart of the image positioning adjustment disclosed by the embodiment of the present application;

[0052] Figure 7 is a schematic diagram of the image presentation effect disclosed by the embodiment of the present application;

[0053] Figure 8 is a flowchart of the image flipping and rotating disclosed by the embodiment of the present application;

[0054] Figure 9 is a schematic diagram of different exposure conditions disclosed by the embodiment of the present application;

[0055] Figure 10 is a single-cell microbubble mode diagram disclosed by the embodiment of the present application;

[0056] Figure 11is a structural schematic view of an image analysis device of a single cell chip provided by an embodiment of the present application.

[0057] Figure 12 is a structural schematic view of an electronic device provided by an embodiment of the present application.

[0058] Label: 1, chip substrate; 2, biological recognition area; 3, physical function area; 31, first positioning dot; 32, quantitative dot array; 33, second positioning dot; DETAILED DESCRIPTION

[0059] The technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments of the present application. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative work fall within the scope of protection of the present application.

[0060] It should be noted that the terms "first", "second", "third", "fourth" and the like in the specification and claims of the present application are used to distinguish different objects, rather than to describe a specific order. The terms "include" and "have" and any variations thereof in the embodiments of the present application are intended to cover non-exclusive inclusion, for example, a process, method, system, product or device including a series of steps or units need not be limited to only those steps or units clearly listed, but can include other steps or units not clearly listed or inherent to the process, method, product or device.

[0061] Embodiment one

[0062] As shown in Figure 1 , Figure 2 and Figure 3 , the present application provides a single cell antibody chip, comprising:

[0063] A chip substrate 1, wherein a biological recognition area 2 and a physical function area 3 are arranged on the chip substrate 1; a first dot group is arranged on the biological recognition area 2, the first dot group comprises a plurality of biological recognition dots, and each biological recognition dot is pre-coated with a different marker for capturing and detecting a to-be-detected substance;

[0064] A second dot group is arranged on the physical function area 3, the second dot group comprises a plurality of physical recognition dots, the second dot group comprises a first positioning dot 31 and a second positioning dot 32, and the display states of the first positioning dot 31 and the second positioning dot 32 are different, and the first positioning dot 31 and the second positioning dot 32 are used for identifying the direction of the chip substrate 1.

[0065] In the embodiment of the present application, the biological recognition area 2 and the physical function area 3 are provided. Specifically, the first n-1 rows are the biological recognition area 2, and each of the core points represents a protein or nucleic acid. The core points are pre-coated with antibodies, probes, fluorescence, and other microstructures or substances. The core points provided in the biological recognition area 2 are mainly used for capturing and detecting proteins, nucleic acids, or other detected substances. When performing specific settings, the nth row is the physical function area 3 of the chip, wherein the first and last core points of the nth row are positioning core points, which are respectively referred to as "positioning core point L" and "positioning core point R". The purpose of the settings is mainly to determine the chip orientation and rotation angle.

[0066] Each of the core points of the biological recognition area 2 can be coated with protein antibodies, nucleic acid probes, and other various chemical substances or special structures. For example, if the detected substance is a protein, the corresponding antibodies can be pre-coated. The number of detectable proteins can reach (n-1)*m according to the design of the chip. Taking a 10*10 chip as an example, there are 9*10, that is, 90 proteins for detection in the biological recognition area 2, excluding the physical function area 3. The presence or absence of the protein can be determined according to the presence or absence of the fluorescence signal or the staining signal of each core point. The amount of the protein can be quantified according to the strength of the fluorescence signal or the staining signal.

[0067] If the detected substance is DNA or RNA, the reverse probes can be pre-coated on the chip to capture the RNA or DNA lysed from a single cell, so as to detect the presence and abundance of the DNA or RNA. If the detected substance is other chemical substances, biochemical substances, or special structures or microspheres, different markers can also be provided on the core points to mark or capture these substances or structures. The presence or absence of the detected substance or the abundance thereof can be determined according to the presence or absence of the signal of the core points.

[0068] More preferably, the physical function area 3 further comprises a quantitative core point array 32, and a standard fluorescent substance with an equal gradient concentration is sequentially added to each concentration core point of the quantitative core point array 32; the quantitative core point array 32 is used for normalizing the relative expression level of the protein.

[0069] The middle core point in the nth row is "quantitative core point array 32", which is used to add standard substances of different concentrations. After the chip is combined with the target protein and labeled by immunolabeling or probe labeling, panoramic imaging will be performed by a panoramic scanner, whether it is bright field (optical density) imaging or dark field (fluorescence) imaging, which will involve image exposure problems. For the same sample, underexposure will cause low signal, and overexposure will cause signal to exceed the upper limit, resulting in reduced data accuracy. Especially for fluorescence imaging, exposure time, filter configuration and machine settings can cause certain deviations in image brightness, resulting in poor repeatability of experimental data between different batches. We provide an external "quantitative standard" for the image through the quantitative core point array 32. By adjusting the brightness and contrast of all images, the brightness of all fluorescent core points or stained core points is normalized, so that errors caused by different machine settings in the imaging step or different hardware can be excluded. A "uniform" standard is provided for the image. For example, the first point of the quantitative core point, the brightness of this point of all chips should be the same when the same group of experiments is imaged again. Because of different shooting conditions between different groups, it is necessary to adjust all the points to the same brightness through post-processing. The brightness of other core points also changes.

[0070] In addition, by comparing the light intensity or optical density of the core points in the biological recognition area 2 with the standard core points, we can also quantify all core points on the chip for the same group of experiments or different groups of experiments. Therefore, the quantitative core point array in the physical function area 3 can not only serve as a "uniform" standard for all chip images, but also as a "standard curve" standard for core point quantification on the chip.

[0071] More preferably, the marker is any one of a protein antibody or a nucleic acid probe or a fluorescent dot, and the different display states are different shapes, different sizes or different colors.

[0072] That is, the display state can be designed in different shapes when designing specifically, such as a rectangular shape and a circular shape, or two circles with different radii, or even color-coded markers.

[0073] More preferably, the size of the chip substrate 1 is a 1*1 um microarray, and the thickness of the chip substrate 1 is any value in the range of 1-5 um; the first positioning core point 31 and the second positioning core point 32 are respectively located on the same edge row of the chip substrate 1; and the chip is a liquid suspension chip.

[0074] In the implementation, the suspended chip is wrapped with the cell in a liquid drop in the liquid image suspended state, but is deposited and laid on a plane in the final detection to facilitate the "scanning" imaging of the two-dimensional chip. However, the chip must involve the "direction problem" when being deposited, and thus the following arrangement modes are generated: Figure 7 The front of the chip and different rotation angles of the front, and the back of the chip and different rotation angles. In the actual test, the "positioning core point" of the two chip functional areas is used as the reference to rotate, flip and image all the chips. When being deposited, the chips are overlapped with each other, and thus the concentration needs to be controlled to remove the overlapped chips after imaging. In the specific design, the images are translated, rotated and flipped, and are uniformly arranged with the "positioning core point L" located at the lower left and the "positioning core point R" located at the lower right, and the images are unified. The normalized images are used to identify the signal and the signal strength of the core point of the "biological identification area 2".

[0075] The "chiral suspended single cell protein chip" designed in the embodiment has the following characteristics: "chirality", "suspension", "high throughput", "multi-parameter" and "uniformity".

[0076] I. Chirality: The method uses a solid-phase chip, which has directionality, and does not need to be directionally identified, but only needs to be read and analyzed after imaging. The liquid-phase suspended chip designed in the application is laid on the bottom of the cell culture plate in different directions and angles when being imaged, and thus the direction of the chip must be identified by the "positioning core point".

[0077] II. Suspension: Unlike the solid-phase chip, the chip uses the "one-dimensional microbead" liquid drop method of the traditional single cell sequencing to wrap the "two-dimensional chip" and the single cell in a liquid drop, so that the cell does not need to be distributed on the solid-phase chip through the microfluidic pipeline. The surface "oil film" between each liquid drop is isolated, and the single cell suspension liquid drop is generated at the same time, and the cell is allowed to exist in the whole system. Thus, the problem of cell density and distance of the solid-phase chip is avoided. Compared with the solid-phase chip, the suspended chip system can capture more cells at one time.

[0078] Compared with the traditional biological chip, the chip designed in the application has the following characteristics:

[0079] 1. Single Cell: Traditional microarrays, whether protein microarrays or nucleic acid microarrays, analyze biological samples, tissues, or blood as a whole. They can only detect the relative "total amount" of protein, nucleic acid, or other substances in a biological sample. They cannot detect the specific cell from which these proteins, nucleic acids, etc., originate. This invention allows a single microarray and a single cell to be encapsulated within the same microreaction chamber, i.e., the aforementioned "droplet," enabling the detection of molecules and substances in a closed, three-dimensional space.

[0080] The diagram of microdroplets in suspension is shown below. Figure 10 As shown, within the same reaction suspension system, there may be tens of thousands of such microdroplets, or microbubbles. Each microbubble contains a single cell and a single-cell chip that reacts with it. The reactions between the chip and intracellular and extracellular substances all take place within a single-cell microdroplet. This allows the gene transcription, protein expression, and even metabolic information of each single cell to be visualized through the reactions on a single chip. This enables the identification and analysis of physiological and biochemical indicators of tens of thousands of cells from the same sample.

[0081] 2. Liquid imaging suspension chip: It has higher capture efficiency and increases the number of cells captured by 100 times.

[0082] Traditional microarrays are mostly solid-phase chips, requiring microfluidic channels or micropores to capture single cells on a two-dimensional plane. Cells need to be spaced apart to prevent cross-interference. This results in a very limited number of cells that can be "laid out" on a single plane, significantly impacting capture efficiency. For example, the cell proteomics chip designed by Isoplexis, with an initial capacity of 30,000 cells, only managed to capture approximately 800 cells.

[0083] The liquid imaging microchip designed in this invention adopts the principle of liquid suspension to uniformly distribute cells and microchips in three-dimensional space, achieving higher capture efficiency. It is expected to capture approximately 30,000 cells and capture 10,000 to 15,000 single cells.

[0084] 3. Chiral chip: Like human hands, it has the function of "left and right hand, front and back" recognition.

[0085] Traditional chips mostly use solid-state chips, which require fixed orientation and angle for identification during reading. Therefore, unless there is an operational error, orientation and orientation issues are generally not involved. However, in a liquid imaging suspension system, the chip is uniformly distributed in a three-dimensional system at different angles and rotational planes. During the chip detection and imaging stage, the chip settles to the bottom of the plate, which also raises the issue of orientation and rotation angle.

[0086] The chip designed in the application can complete the identification of the front and back of the "chirality" and the rotation angle through two "positioning core points", so as to facilitate automatic rotation and flip of the image after imaging.

[0087] 4. Quantifiable chip: standard light intensity, optical density identification correction point

[0088] Ordinary chips only determine relative fluorescence intensity and optical density, and require that all experimental conditions and imaging conditions remain completely consistent, and generally need very fixed machines or special equipment for image imaging or scanning. Such equipment is generally very expensive and is a closed system. Most chip reading and scanning equipment on the market generally costs more than 2 million yuan.

[0089] The chip designed in the application has standard "quantitative core point matrix", and allows different batches of experiments to be imaged and scanned on different devices under different detection conditions. Only image normalization is needed based on the quantitative core point before analysis, so as to complete the correction and normalization of chip images of different devices and different imaging conditions.

[0090] 5. Single and few colors multi-function: only one kind of fluorescence and color marking can complete the functions of direction identification, quantitative management, multiple bioinformatics detection and detection.

[0091] The chip designed in the application has direction identification core points, quantitative core point arrays and n*m multiple biological identification matrices. Therefore, the chip can complete all functions such as positioning, quantification and multiple analysis only by using one color of fluorescence, luminosity, optical density and other optical markers. At the same time, the optical marker of a single color makes imaging more convenient and fast. The requirement for the scanner is relatively simpler, and the microchip is more suitable for application and promotion on various detection devices. At the same time, the chip point has an upgrade space for multiple colors. In addition to multi-color marking, multi-color fluorescence marking is also allowed to achieve more functions. The design of the embodiment of the application is applicable to single-cell omics field based on liquid image suspension chirality, in addition to the above-mentioned protein and antibody chip, metabolic chip and gene DNA / RNA chip. The method for single-cell or multi-cell research based on liquid image suspension chirality has potential application.

[0092] The chip mentioned in the embodiment of the application has direction identification core points, quantitative core point arrays and n*m multiple biological identification matrices. Therefore, the chip can complete all functions such as positioning, quantification and multiple analysis only by using one color of fluorescence, luminosity, optical density and other optical markers. At the same time, the optical marker of a single color makes imaging more convenient and fast. The requirement for the scanner is relatively simpler, and the microchip is more suitable for application and promotion on various detection devices. At the same time, the chip point has an upgrade space for multiple colors. In addition to multi-color marking, multi-color fluorescence marking is also allowed to achieve more functions.

[0093] In this embodiment of the invention, the single-cell antibody chip can capture and label single cells, and uses positioning core points to mark the orientation of the chip substrate and uses a quantitative core point array to achieve uniform processing of imaging results, thereby better quantifying the detection of protein expression levels.

[0094] Example 2

[0095] like Figure 4 As shown, this embodiment of the invention provides a method for preparing a single-cell chip, comprising:

[0096] S101: Prepare a single-cell suspension according to the laboratory standard operating procedure, and determine the cell concentration data in the single-cell suspension by using a cell counter or hemocytometer.

[0097] This step mainly involves the preparation of single-cell suspensions, including the single-cell formation of biological samples such as tissues and blood. Different laboratories may have different procedures for single-cell formation of different tissue samples; please refer to relevant literature or laboratory SOP documents for details. Cell counting is performed using a cell counter or hemocytometer to test cell concentration.

[0098] S102: Draw a cell suspension of a preset number of cells according to the cell density; draw a cell suspension of approximately 30,000 cells according to the cell density.

[0099] S103: Prepare single-cell droplets by adding pre-coated antibody arrays to single-cell antibody chips during the preparation process. Adjust the single-cell droplet parameters to ensure that each single-cell droplet carries a single-cell antibody chip, and the number of single-cell antibody chips is greater than the number of cells. Prepare single-cell droplets using 10xGenomics, Syngenomics, or other brands, and add pre-coated antibody arrays to single-cell antibody chips during the preparation process. The number of single-cell chips should be higher than the number of cells. Adjust the droplet parameters using different brands to ensure that each cell carries one chip. Empty droplets containing a single chip are allowed as blank or negative controls.

[0100] S104: A liquid suspension containing single cells is temporarily incubated under different conditions for a set time according to cell culture requirements or testing needs, so that the protein is released outside the cell and binds to the corresponding chip.

[0101] For secretory protein assays, single-cell droplet suspensions are temporarily incubated for 0-4 hours under different conditions according to cell culture requirements or assay needs. This step primarily allows secretory proteins to be released extracellularly and bind to the microarray.

[0102] The single cell containing droplet suspension is prepared according to the single cell preparation instrument of each brand. The single cell containing droplet suspension is incubated for 0-4 hours under different conditions according to the requirements of cell culture or test, which is mainly to release the metabolic products outside the cells and combine with the chip.

[0103] S105: Add antigen antibody binding inhibitors and membrane breakers to the single cell droplet mixture; break the cell droplets, release the single cell chip into the liquid; and transfer the lysed liquid to a centrifuge tube for centrifugal precipitation and impurity removal operation;

[0104] Add immune binding inhibitors and membrane breakers to the single cell droplet mixture (which can be designed according to different experimental requirements). Break the cell droplets and release the single cell chip into the liquid.

[0105] Chip cleaning: transfer the lysed liquid to a 1.5ml centrifuge tube, centrifuge at 100g for 1min to precipitate the chip. Carefully remove the supernatant to remove cell debris, reagents from the previous step, proteins after cell lysis and impurities, etc. Resuspend the chip with 1ml PBS and centrifuge at 100g for 1min to remove the supernatant. Repeat this step 3 times to thoroughly remove impurities.

[0106] S106: Use multiple antibody premixes to label proteins and incubate at room temperature for a predetermined time, and sequentially perform primary antibody washing, secondary antibody incubation and secondary antibody washing.

[0107] Add anti-detected protein primary antibody: preferably from the same species, such as human-derived samples, use Mouse Anti-Human-Protein. Multiple antibodies can be premixed "cocktail" to label proteins. Incubate at room temperature for 1 hour or at 4 degrees for 12 hours.

[0108] Primary antibody washing: PBS is washed 3 times according to the steps of 5.1.8;

[0109] Secondary antibody incubation: use fluorescently labeled, HRP or AP labeled Anti-Mouse IgG antibody to stain the chip. Incubate at room temperature for 1 hour or at 4 degrees for 12 hours.

[0110] Secondary antibody washing: PBS is washed 3 times according to the steps of 5.1.8.

[0111] S107: Color development and imaging of the antibody-labeled chip.

[0112] More preferably, the color development and imaging of the antibody-labeled chip comprises:

[0113] Resuspend the washed chip with 1ml PBS; and add the resuspended liquid evenly to a 12-well or 6-well cell culture plate, if the liquid is not enough to cover the whole plate, add PBS to evenly cover the plate;

[0114] Let it stand for 5 minutes to make the chip evenly precipitate at the bottom of the cell culture plate, and use a bottom-reading scanner or a microscopic imaging system to take panoramic images of the chip under bright field imaging conditions.

[0115] Specifically, the color development of the fluorescent antibody-labeled chip: resuspend the washed chip with 1ml PBS, and evenly add it to a 12-well or 6-well cell culture plate (if the liquid is not enough to cover the whole plate, you can add PBS to evenly cover the surface). Let it stand for 5 minutes to make the chip evenly precipitate at the bottom of the plate. Use a bottom-reading scanner or a microscopic imaging system to take panoramic images of the chip according to the excitation and emission wavelengths of the labeled fluorescent dye.

[0116] Color development of HRP or AP antibody-labeled chip: resuspend the washed chip with 1ml PBS, and evenly add it to a 12-well or 6-well cell culture plate (if the liquid is not enough to cover the whole plate, you can add PBS to evenly cover the surface). Add appropriate color developing solution for staining. After staining, wash the chip 3 times with PBS according to the steps in 5.1.8. Resuspend the washed chip with 1ml PBS, and evenly add it to a 12-well or 6-well cell culture plate (if the liquid is not enough to cover the whole plate, you can add PBS to evenly cover the surface). Let it stand for 5 minutes to make the chip evenly precipitate at the bottom of the plate. Use a bottom-reading scanner or a microscopic imaging system to take panoramic images of the chip under bright field imaging conditions.

[0117] Example Three

[0118] Please refer to Figure 5 , Figure 5 is a flowchart of the image analysis method of the single-cell chip disclosed in the embodiments of the present application. The execution subject of the method described in the embodiments of the present application is composed of software or / and hardware, which can receive relevant information through wired or / and wireless means and can send certain instructions. Of course, it can also have certain processing and storage functions. The execution subject can control multiple devices, such as remote physical servers or cloud servers and related software, or it can be a local host or server and related software that performs related operations on a device placed somewhere. In some scenarios, it can also control multiple storage devices, which can be placed in the same place or different places. As Figure 5 shown, the image analysis method based on the single-cell chip includes the following steps:

[0119] S201: obtaining each initial image information of the antibody chip in the suspension through a display imaging system, and performing image rearrangement on the initial image information to obtain chip image information in a unified direction; the chip image information includes a biological recognition area for capturing and detecting the to-be-detected substance and a physical function area for marking the chip image information;

[0120] In this step, the chip image is mainly obtained; the chips are separated one by one according to the imaging image and recognized by software; and then the biological recognition area and the physical function area can be recognized according to the imaging result, and the functions of different function areas are also different.

[0121] More preferably, Figure 6 is a flowchart of the image positioning adjustment disclosed in the embodiment of the present application, as Figure 6 shown, the image rearrangement on the initial image information to obtain chip image information in a unified direction includes:

[0122] S2011: identifying a positioning core point of the physical function area in the initial image information to determine position information and shape information of the positioning core point;

[0123] S2012: determining positive-negative state information of the initial image information according to the shape information;

[0124] S2013: determining direction state information of the initial image information according to the position information;

[0125] S2014: performing image flipping and image rotation on the corresponding initial image information according to a preset image direction, the positive-negative state information and the direction state information to obtain chip image information in a unified direction.

[0126] Figure 7 is a schematic diagram of the image presentation effect disclosed in the embodiment of the present application; Figure 8 is a flowchart of the image flipping and rotation disclosed in the embodiment of the present application; as Figure 7 and Figure 8 shown, Figure 7 is a front of the chip and different rotation angles of the front, and a back of the chip and different rotation angles. In actual testing, the image rotation and flipping are performed on all chips by taking the “positioning core point” of the two chip function areas as a reference. After the image is translated, rotated and flipped, the image is uniformly arranged to be “positioning core point L” located at the lower left and “positioning core point R” located at the lower right, and the image is unified. The normalized image further performs signal recognition and signal intensity recognition on the core point of the “biological recognition area”.

[0127] S202: normalizing the chip image information according to the quantitative core point array of the physical function area, so as to modulate all chip image information into images with the same intensity or the same color;

[0128] More preferably, the normalization of the chip image information according to the quantitative core point array of the physical function area, so as to modulate all chip image information into images with the same intensity or the same color, comprises:

[0129] S2021: acquiring the signal intensity of the quantitative core point array of the physical function area of all chip image information, and constructing quantitative standard information according to the signal intensity;

[0130] S2022: normalizing the brightness of the fluorescent core point or the dyed core point of all biological recognition areas according to the quantitative standard information.

[0131] Figure 9 It is a different exposure schematic diagram disclosed by the embodiment of the present application; as shown in the figure, Figure 9 After the chip is combined with the target protein and labeled by immunolabeling or probe labeling, panoramic imaging is performed by a panoramic scanner, and whether it is bright field (optical density) imaging or dark field (fluorescence) imaging, image exposure problems are involved. For the same sample, insufficient exposure will cause low signal, and overexposure will cause the signal to exceed the upper limit, resulting in reduced data accuracy. Especially for fluorescence imaging, exposure time, filter configuration and machine settings will cause certain brightness deviation of the image, resulting in poor repeatability of experimental data between different batches.

[0132] Specifically, the quantitative core point array provides an external "quantitative standard" that can be used as a standard for the image. By adjusting the brightness and contrast of all images, the brightness of all fluorescent core points or dyed core points is normalized, so that errors caused by different imaging steps, machine settings or different hardware can be excluded. A "uniform" standard is provided for the image.

[0133] By comparing the light intensity or optical density of the biological recognition area core point with the standard core point, all core points on the chip in the same group of experiments or different groups of experiments can also be quantified by a "standard curve". Therefore, the quantitative core point array of the physical function area can not only serve as a "uniform" standard for all chip images, but also as a "standard curve" standard for the quantification of core points on the chip.

[0134] S203: performing fluorescent signal intensity analysis or dyed optical density analysis on each recognition core point in the biological recognition area of the modulated chip image information to obtain signal intensity data of each recognition core point;

[0135] Specifically, this involves core dot reading, which involves analyzing the fluorescence signal intensity or staining optical density of each core dot in the biometric region array to obtain the signal intensity and analyzing the signal strength.

[0136] S204: Perform bioinformatics analysis on the obtained signal strength data to obtain the core point analysis results.

[0137] Specifically, this refers to bioinformatics analysis operations: data is exported as CSV or array files, and data analysis is performed using any dimensionality reduction software.

[0138] The chip mentioned in this invention possesses orientation recognition core points, a quantitative core point array, and an n*m multi-biometric matrix. Therefore, it allows the chip to perform all functions, including localization, quantification, and multiplex analysis, using only a single color of optical markers such as fluorescence, luminescence, or optical density. Furthermore, the single-color optical markers make imaging more convenient and faster. The requirements for scanners are also relatively simpler, making it more suitable for the application and promotion of this microchip in various detection devices. At the same time, it retains the upgrade potential for multi-color core point marking. In addition to multi-color marking, multi-color fluorescent marking is also allowed to achieve more functions.

[0139] Example 4

[0140] Please see Figure 11 , Figure 11 This is a schematic diagram of the structure of the single-cell chip image analysis device disclosed in an embodiment of the present invention. Figure 11 As shown, the image analysis device for this single-cell chip may include:

[0141] Image acquisition module 21: used to acquire initial image information of antibody chips in suspension through a display imaging system, and rearrange the initial image information to obtain chip image information with a unified orientation; the chip image information includes a biometric region for capturing and detecting the substance to be detected and a physical functional region for marking the chip image information;

[0142] Normalization processing module 22: used to normalize the chip image information according to the quantitative core dot array of the physical functional area, so as to modulate all the chip image information into images with the same intensity or the same color;

[0143] Signal analysis module 23: used to perform fluorescence signal intensity analysis or staining optical density analysis on each recognition core point in the biometric recognition area of ​​the modulated chip image information to obtain the signal intensity data of each recognition core point;

[0144] Bioinformatics analysis module 24: used to perform bioinformatics analysis on the obtained signal strength data to obtain core point analysis results.

[0145] The chip mentioned in the embodiments of the present application has a direction recognition core point, a quantitative core point array, and an n*m multiple biological recognition matrix. Therefore, the chip can complete positioning, quantification, multiple analysis and other functions by using only one color of optical markers such as fluorescence, luminescence, and optical density. At the same time, the single color of optical markers makes imaging more convenient and fast. The requirements for the scanner are relatively simpler, and the microchip is more suitable for application and promotion on various detection devices. At the same time, the core point has an upgrade space for multiple colors. In addition to multi-color markers, multi-color fluorescent markers are also allowed to achieve more functions.

[0146] Embodiment five

[0147] Please refer to Figure 12 , Figure 12 is a structural schematic diagram of an electronic device disclosed by the embodiments of the present application. The electronic device can be a computer, a server, and the like. Of course, in certain cases, it can also be a smart device such as a mobile phone, a tablet computer, and a monitoring terminal, and an image acquisition device with processing function. As shown in Figure 12 , the electronic device can include:

[0148] a memory 510 storing executable program codes;

[0149] a processor 520 coupled with the memory 510;

[0150] The processor 520 calls the executable program codes stored in the memory 510 to execute part or all of the steps of the image analysis method of the single cell chip in embodiment one.

[0151] The embodiments of the present application disclose a computer readable storage medium storing a computer program, wherein the computer program causes a computer to execute part or all of the steps of the image analysis method of the single cell chip in embodiment one.

[0152] The embodiments of the present application also disclose a computer program product, wherein when the computer program product runs on a computer, the computer program product causes the computer to execute part or all of the steps of the image analysis method of the single cell chip in embodiment one.

[0153] The embodiments of the present application also disclose an application publishing platform, wherein the application publishing platform is used to publish a computer program product, and when the computer program product runs on a computer, the computer program product causes the computer to execute part or all of the steps of the image analysis method of the single cell chip in embodiment one.

[0154] In various embodiments of the present application, it should be understood that the size of the sequence number of the processes does not mean the inevitable sequence of execution, and the execution sequence of the processes should be determined according to their functions and internal logic, and should not constitute any limitation on the implementation process of the embodiments of the present application.

[0155] The units described as separate components can or can not be physically separated, and the components displayed as units can or can not be physical units, that is, they can be located in one place, or they can be distributed to multiple network units. According to actual needs, part or all of the units can be selected to achieve the purpose of the embodiment scheme.

[0156] In addition, each functional unit in each embodiment of the present application can be integrated in one processing unit, or each unit can exist physically, or two or more units can be integrated in one unit. The integrated unit can be realized in the form of hardware or in the form of a software functional unit.

[0157] The integrated unit, if realized in the form of a software functional unit and sold or used as an independent product, can be stored in a computer-accessible memory. Based on such understanding, the technical solutions of the present application, essentially or the part that contributes to the prior art, or all or part of the technical solutions, can be embodied in the form of a software product. The computer software product is stored in a storage medium, and includes a number of steps for causing a computer device (which can be a personal computer, a server or a network device, etc., and specifically can be a processor in the computer device) to execute the methods of the various embodiments of the present application. Part or all of the steps.

[0158] In the embodiments provided by the present application, it should be understood that "B corresponding to A" means that B is associated with A, and B can be determined according to A. However, it should also be understood that determining B according to A does not mean that B is determined only according to A, but B can also be determined according to A and / or other information.

[0159] Those skilled in the art can understand that part or all of the steps in the various methods of the embodiments can be completed by instructing the relevant hardware by a program, and the program can be stored in a computer readable storage medium, including Read-Only Memory (ROM), Random Access Memory (RAM), Programmable Read-Only Memory (PROM), Erasable Programmable Read-Only Memory (EPROM), One-time Programmable Read-Only Memory (OTPROM), Electrically-Erasable Programmable Read-Only Memory (EEPROM), Compact Disc Read-Only Memory (CD-ROM) or other optical disk memories, magnetic disk memories, magnetic tape memories, or any other computer readable medium capable of carrying or storing data.

[0160] The image analysis method, device, electronic equipment and storage medium of the single-cell chip disclosed in the embodiments of the present application are described in detail above, and the principles and implementation manners of the present application are described by applying specific examples in this paper. The above description of the embodiments is only used to help understand the method of the present application and its core idea; at the same time, for those skilled in the art, according to the idea of the present application, the specific implementation manner and application range will be changed, and the above description should not be understood as a limitation of the present application.

Claims

1. A single-cell antibody chip, characterized in that, include: A chip substrate, on which a biometric area and a physical functional area are disposed; a first core dot group is disposed in the biometric area, the first core dot group including multiple biometric core dots, and each biometric core dot is pre-coated with a different marker for capturing and detecting the substance to be detected; A second core dot group is provided in the physical functional area. The second core dot group includes multiple physical identification core dots, including a first positioning core dot and a second positioning core dot. The first positioning core dot and the second positioning core dot have different display states. The first positioning core dot and the second positioning core dot are used to identify the orientation of the chip substrate. The physical functional area also includes a quantitative core dot array. Standard fluorescent substances of equal concentration are added sequentially at each concentration core dot of the quantitative core dot array. The quantitative core dot array is used to normalize the relative expression level of proteins.

2. The single-cell antibody chip as described in claim 1, characterized in that, The marker is any one of a protein antibody, a nucleic acid probe, or a fluorescent spot, and the different display states are different in shape, size, or color.

3. The single-cell antibody chip as described in claim 1, characterized in that, The chip substrate is a 1*1um microarray, and the thickness of the chip substrate is any value between 1 and 5um; the first positioning core point and the second positioning core point are located on both sides of the same edge row of the chip substrate, and the chip is a liquid-phase suspended chip.

4. A method for preparing a single-cell chip, characterized in that, include: Single-cell suspensions were prepared according to laboratory standard operating procedures, and the concentration of the single-cell suspensions was determined by using a cell counter or hemocytometer. Aspirate a predetermined number of cells from the cell suspension according to the cell density; The method involves preparing single-cell droplets by adding a pre-coated antibody combination to a single-cell antibody chip as described in any one of claims 1 to 3 during the preparation process, and adjusting the single-cell droplet parameters so that each single-cell droplet carries a single-cell antibody chip; and the number of single-cell antibody chips is greater than the number of cells. Liquid suspensions containing single cells are temporarily incubated under different conditions for a set time according to cell culture requirements or testing needs, so that proteins are released extracellularly and bind to the corresponding chip. An antigen-antibody binding inhibitor and a membrane-breaking agent are added to a single-cell droplet mixture; the cell droplets are then broken up, releasing the single-cell chip into the liquid. The pyrolyzed liquid was then transferred to centrifuge tubes for centrifugation, sedimentation, and impurity removal. Multiple antibody premixes were used to label the proteins, and the mixtures were incubated at room temperature for a preset time. Then, the primary antibody washing, secondary antibody incubation, and secondary antibody washing were performed sequentially. Colorimetric imaging was performed on the antibody-labeled chip.

5. The method for preparing a single-cell chip as described in claim 4, characterized in that, The process of performing colorimetric imaging on the antibody-labeled chip includes: Resuspend the cleaned chip in 1 ml PBS; and add the resuspended liquid evenly to a 12-well or 6-well cell culture plate. If the liquid is not enough to cover the entire plate, add PBS until it evenly covers the plate. Let it stand for 5 minutes to allow the chip to settle evenly at the bottom of the cell culture plate. Then, use a bottom-reading scanner or microscopic imaging system to perform panoramic imaging of the chip under bright-field imaging conditions. The impurity removal process includes: Remove the supernatant to remove cell debris, reagents from the previous step, proteins and impurities after cell lysis; The chip was resuspended by pipetting with 1 ml of PBS and centrifuged at 100 g for 1 minute to remove the supernatant; this step was repeated 3 times to remove impurities.

6. An image analysis method for a single-cell chip, characterized in that, include: The initial image information of the antibody chip in the suspension is acquired by a display imaging system, and the initial image information is rearranged to obtain chip image information with a unified orientation; the chip image information includes a biometric region for capturing and detecting the substance to be detected and a physical functional region for marking the chip image information; the antibody chip is a single-cell antibody chip as described in any one of claims 1 to 3. The chip image information is normalized according to the quantitative core dot array of the physical functional area so as to modulate all chip image information into images with the same intensity or the same color. Fluorescence signal intensity analysis or staining optical density analysis is performed on each recognition core point in the biometric recognition area of ​​the modulated chip image information to obtain the signal intensity data of each recognition core point; Bioinformatics analysis is performed on the obtained signal strength data to obtain the core point analysis results.

7. The image analysis method for a single-cell chip as described in claim 6, characterized in that, The step of rearranging the initial image information to obtain chip image information with a unified orientation includes: The location and shape information of the positioning core points of the physical functional areas in the initial image information are determined by identifying the positioning core points. The positive and negative state information of the initial image information is determined based on the morphological information; The orientation state information of the initial image information is determined based on the location information; Based on the preset image orientation, the positive / negative state information, and the orientation state information, the corresponding initial image information is flipped and rotated to obtain chip image information with a unified orientation.

8. The image analysis method for a single-cell chip as described in claim 6, characterized in that, The step of normalizing the chip image information based on the quantitative chip dot array of the physical functional area to modulate all chip image information into images with the same intensity or color includes: The signal strength of the quantitative chip dot array at the physical functional area of ​​all chip image information is obtained, and quantitative standard information is constructed based on the signal strength. The brightness of fluorescent or stained core dots in all biometric regions is normalized based on the aforementioned quantification standard information.

9. A computer-readable storage medium, characterized in that, The computer-readable storage medium stores a computer program, wherein the computer program causes a computer to perform the image analysis method for a single-cell chip according to any one of claims 6 to 8.

Images