A kit and method for in situ sequential quantitative detection of the same single cell secreted protein and intracellular protein

Abstract

This invention relates to the field of single-cell functional analysis and microfluidic chip technology, and discloses a kit and method for in-situ sequential quantitative detection of secreted and intracellular proteins in the same single cell. The kit includes: a microcavity array chip; a secreted protein capture antibody barcode chip and an intracellular protein capture antibody barcode chip; a plastic clamp; a bioaffinity modification reagent; secreted protein standards, intracellular protein standards, secreted protein detection antibody stock solution, and intracellular protein detection antibody stock solution; a fluorescent conjugate, antibody buffer, antibody blocking solution, cell lysis buffer, and cell washing buffer. The method of this invention, through a sequential detection process of first capturing and incubating secreted proteins, followed by in-situ lysis to capture intracellular proteins, achieves high-throughput, high-sensitivity, and high-specificity in-situ sequential quantitative detection of secreted and intracellular proteins in the same single cell without cell transfer or loss of spatial location information. It can establish a correlation analysis of "intracellular signaling pathway activation - external functional output" in single cells, providing a powerful tool for disease mechanism research, drug screening, and immunotherapy evaluation.

Description

Technical Field

[0001] This invention relates to the field of single-cell functional analysis and microfluidic chip technology, and in particular to a kit and method for in situ sequential quantitative detection of secreted proteins and intracellular proteins in the same single cell. Background Technology

[0002] In research on major diseases such as inflammation and infection, tumor drug resistance, and immunotherapy, a small subset of highly functional cells often determines the overall disease progression and treatment response. This reflects the significant heterogeneity among individual cells even within the same microenvironment. Single-cell protein detection can directly reflect the external secretory function state or internal signaling pathway activation status of a single cell, which is of great significance for a deeper understanding of cellular heterogeneity, identifying key functional cell subsets, and elucidating the mechanisms of cell-cell interactions.

[0003] Currently, common protein detection methods mainly include enzyme-linked immunosorbent assay (ELISA), Western blotting (WB), and flow cytometry (FC), but each of these methods has its own limitations. (1) ELISA and Western Blot can only detect the average protein expression level of cell populations, and cannot distinguish the differences between individual cells, making it difficult to identify high-functioning cell subpopulations that determine the course of disease. (2) Although flow cytometry can achieve high-throughput detection at the single-cell level, it usually requires cell suspension and fluorescent labeling, and relies on expensive and complex instruments and equipment. The operation process is complicated and affects cell viability, and it cannot support in-situ dynamic monitoring of cells, especially for adherent cells. In addition, this method cannot observe the adherent proliferation status of single cells in real time, has zero time resolution, and requires a large minimum sample volume (from several hundred microliters to several milliliters), making it difficult to apply to rare sample analysis. The system cost is high and it is not suitable for point-of-care detection scenarios.

[0004] In recent years, single-cell protein detection technologies based on microcavity array chips and antibody barcode chips have developed rapidly. For example, microimprinting technology captures single-cell secretions in a micro-well array and forms protein "blots" on the chip, enabling high-throughput analysis of secreted proteins. Single-cell barcode chips isolate single cells in microcavities and prepare parallel antibody band arrays on a glass slide for the quantitative detection of multiple indicators of secreted proteins or intracellular signaling proteins. Most of these chips are made of polydimethylsiloxane, which has good optical properties, thermal stability, and biocompatibility, and has become an important tool for low-cost, portable biochemical micro-detection.

[0005] However, existing technologies still have the following shortcomings: (1) When only secreted proteins are detected, the activation status of signaling pathways inside the cell cannot be obtained simultaneously, making it difficult to establish the relationship between function and signal regulation; (2) When only intracellular proteins are detected, the true secretory function output of the cell cannot be obtained, making it difficult to assess its effector function; (3) Even if the two types of proteins are detected separately, cell transfer or repeated loading is often required, which cannot guarantee that the secreted protein and intracellular protein data come from the same cell, thus limiting the functional association analysis. (4) Some barcode preparation methods are complex and have high barriers to entry, which is not conducive to universal promotion and large-scale preparation.

[0006] Therefore, there is an urgent need to develop a simple, low-cost method and kit for in-situ sequential capture and quantitative detection of secretory and intracellular proteins in the same single cell, which can achieve high-throughput, high-sensitivity, and high-specificity single-cell protein functional analysis without transferring cells or losing spatial location information. Summary of the Invention

[0007] To address the aforementioned technical problems, this invention provides a kit and method for in-situ sequential quantitative detection of secretory and intracellular proteins in the same single cell. This achieves rapid, accurate, high-throughput, and low-cost in-situ sequential capture and quantitative detection of secretory and intracellular proteins in the same single cell without cell transfer or loss of spatial location information. Furthermore, it enables the establishment of a correspondence between intracellular pathway activation and secretory function output in the same single cell during subsequent functional assessment analysis at the single-cell level.

[0008] To achieve the above objectives, the technical solution of the present invention is as follows: A kit for in situ sequential quantitative detection of secreted and intracellular proteins from the same single cell, comprising: Microcavity array chip; Two types of antibody barcode chips are used: a secretory protein capture antibody barcode chip and an intracellular protein capture antibody barcode chip. A plastic clamp is used to clamp the microcavity array chip and the antibody barcode chip to form a closed microcavity; Bioaffinity modifying agents are used to modify the surface of microcavity array chips; Secretory protein standards, intracellular protein standards, secretory protein detection antibody stock solution, intracellular protein detection antibody stock solution; Fluorescent conjugates, antibody buffers, antibody blocking solutions, cell lysis buffers, and cell washing solutions.

[0009] In the above scheme, the microcavity array chip is made of polydimethylsiloxane and has multiple microcavities on its surface for single-cell capture and culture.

[0010] In the above scheme, the antibody barcode chip is provided with multiple parallel antibody channels, each channel being 5-200 μm wide and spaced 10-100 μm apart, for capturing different types of proteins.

[0011] In the above scheme, the bioaffinity modification reagent is a BSA solution with a mass concentration of 1-5% prepared by sterile phosphate buffered saline (PBS), sterile BSA solution, and sterile deionized water.

[0012] In the above scheme, the working concentration dilution factor of the fluorescent conjugate is 1:100-1:500, and the final concentration is not less than 100 μg / ml.

[0013] In the above scheme, the antibody buffer is a BSA solution with a mass concentration of 0.5-2% prepared by sterile phosphate buffer PBS, sterile BSA solution and sterile cation solution, which is used to dilute the original antibody solution for secretory protein detection or intracellular protein detection, so that the concentration of the diluted detection antibody is greater than 100 μg / ml.

[0014] In the above scheme, the antibody blocking solution is a 3-6% BSA solution prepared by mixing sterile phosphate buffer PBS, sterile BSA solution and sterile deionized water in a certain proportion.

[0015] A method for in situ sequential quantitative detection of secreted and intracellular proteins from the same single cell includes the following steps: Step 1: Perform bio-affinity pretreatment on the microcavity array chip; Step 2: Block the antibody barcode chip using antibody blocking solution; Step 3: Extract and resuspend cells, control cell density, and load them into a microcavity array chip; Step 4: Assemble the microcavity array chip and the secretory protein capture antibody barcode chip, clamp them with plastic clamps, and incubate to capture secretory proteins; Step 5: Disassemble the chip, perform antibody incubation and fluorescent labeling on the secretory protein capture antibody barcode chip, and scan to obtain secretory protein data; Step 6: Add cell lysis buffer to the microcavity array chip, assemble the microcavity array chip and the intracellular protein capture antibody barcode chip, and incubate to capture intracellular proteins; Step 7: Disassemble the chip, perform antibody incubation and fluorescent labeling on the intracellular protein capture antibody barcode chip, and scan to obtain intracellular protein data; Step 8: Convert the fluorescence value into protein concentration according to the standard curve to achieve quantitative analysis of secreted proteins and intracellular proteins in the same single cell.

[0016] In the above scheme: the microcavity array chip is plasma cleaned, then the surface of the microcavity is bio-affinity modified with a bio-affinity modification reagent, and then dried; before use, the microcavity is wetted with cell washing solution to remove air bubbles inside the microcavity.

[0017] In the above scheme, the fluorescent labeling in steps 5 and 7 uses APC-fluorescent conjugate, and fluorescence scanning is performed after incubation in the dark.

[0018] Through the above technical solutions, the kit and method provided by the present invention for in situ sequential quantitative detection of secreted proteins and intracellular proteins in the same single cell have the following beneficial effects: 1. Achieve paired detection of secreted and intracellular proteins from the same single cell. This invention employs a sequential detection process of "first capturing and incubating secreted proteins, then lysing and capturing intracellular proteins in situ," utilizing the same microcavity array chip for in situ fixation and localization of cells. This ensures that the data on secreted proteins and intracellular proteins originate from the same single cell, avoiding the data pairing loss problem caused by cell transfer or repeated sample loading in traditional methods. This provides a reliable data foundation for establishing the correlation analysis of "intracellular signaling pathway activation - external functional output" in single cells.

[0019] 2. In-situ detection, maintaining cell viability and functional status. After being modified for biocompatibility, the microcavity array chip exhibits excellent cell adhesion and biocompatibility, without affecting cell activity and normal secretory function. The entire detection process is carried out in a closed microcavity, eliminating the need for cell transfer or suspension treatment, thus maintaining the cells' natural state and allowing the detection results to more accurately reflect the functional characteristics of the cells.

[0020] 3. High-throughput detection capability The microcavity array chip designed in this invention contains tens of thousands of microcavities (55,968 microcavities in the example), and can obtain more than 20,000 pairs of secreted protein and intracellular protein data of single cells in a single experiment; the antibody barcode chip can simultaneously detect multiple protein indicators (at least 4 and up to 64), realizing high-throughput single-cell multi-parameter functional analysis.

[0021] 4. High detection sensitivity Single cells can be cultured and lysed independently in nL-sized microcavities. The extremely small reaction volume significantly increases the local concentration of secreted and intracellular proteins. Combined with high-sensitivity fluorescence detection technology, the protein detection sensitivity can reach 0.1 pg / ml, enabling the detection of low-abundance protein expression and meeting the analytical needs of rare samples and trace proteins.

[0022] 5. High detection specificity The closed microcavity structure avoids cell cross-contamination and protein crosstalk between different microcavities; the antibody barcode chip adopts a dual-sandwich detection mode of specific capture antibody and detection antibody, which ensures high specificity of the detection results and no cross-positive signals.

[0023] 6. Simple operation and short testing cycle The kit of this invention has a clear operating procedure. Excluding cell incubation time, the overall detection time does not exceed 3 hours. No complicated instruments or equipment are required. Quantitative results can be obtained after fluorescence scanning. It is suitable for routine applications in clinical testing and research laboratories.

[0024] 7. Low cost and wide applicability The chip is fabricated using PDMS material, which has a simple processing technology and low cost. The kit components are standardized, and the antibody combination can be changed according to different research objectives. It is suitable for the detection of secreted and intracellular proteins in various cell types (such as esophageal cancer, gastric cancer, colon cancer cells, and normal intestinal epithelial cells), and has strong versatility and scalability.

[0025] 8. Small sample size required Each microcavity chip requires only about 80,000 cells to complete single-cell loading, making it suitable for the analysis of rare cell samples (such as circulating tumor cells, stem cells, clinical puncture samples, etc.), thus broadening the application scenarios of single-cell functional analysis.

[0026] 9. Rich data information, supporting multi-dimensional analysis By acquiring expression data of multiple secreted and intracellular proteins from the same single cell, multi-dimensional data mining can be performed, including cluster analysis, correlation analysis, and nuclear density analysis. This reveals cellular heterogeneity, signaling pathway activation status, and their association with secretory function, providing a powerful tool for disease mechanism research, drug screening, and immunotherapy evaluation. Attached Figure Description

[0027] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the description of the embodiments or the prior art will be briefly introduced below.

[0028] Figure 1 This is a flowchart illustrating the in situ sequential quantitative detection of single-cell secreted proteins and intracellular proteins in Example 1 of the present invention. Figure 2 This is a schematic diagram of the microcavity array chip and antibody barcode chip in Embodiment 1 of the present invention; Figure 3 This is a graph showing the survival rate assessment results of cells under different culture conditions in Example 1 of the present invention; Figure 4The following are sensitivity maps of protein concentration gradient detection in the kit of Example 1 of the present invention: (a) is the sensitivity map of IL6 protein concentration gradient detection, (b) is the sensitivity map of IL8 protein concentration gradient detection, (c) is the sensitivity map of p-p65 protein concentration gradient detection, and (d) is the sensitivity map of p-p38 protein concentration gradient detection. Figure 5 The images show the protein specificity of the kit in Example 1 of this invention, where (a) is the specificity of IL6 protein detection at 1 μg / ml, (b) is the specificity of IL8 protein detection at 1 μg / ml, (c) is the specificity of p-p65 protein detection at 1 μg / ml, and (d) is the specificity of p-p38 protein detection at 1 μg / ml. Figure 6 Clustering diagram of the four cell types; Figure 7 Four-dimensional distribution maps of four secretory proteins and four intracellular proteins in single colon cancer cells; where (a) is the four-dimensional distribution map of secretory proteins and (b) is the four-dimensional distribution map of intracellular proteins. Figure 8 This is a graph showing the correlation between proteins in single cells of colon cancer. Figure 9 The images show the differential nuclear density maps of IL6+p-p65 and IL8+p-p38 between single cells of colon cancer and single cells of normal intestinal epithelium. (a) shows the differential nuclear density map of IL6+p-p65 between single cells of colon cancer and single cells of normal intestinal epithelium, and (b) shows the differential nuclear density map of IL8+p-p38 between single cells of colon cancer and single cells of normal intestinal epithelium. In the diagram, 1 is a microcavity array chip; 2 is a microcavity; 3 is an antibody barcode chip; and 4 is a barcode. Detailed Implementation

[0029] The technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention.

[0030] Example 1: This embodiment provides a kit for in situ sequential quantitative detection of secreted proteins and intracellular proteins in the same single cell. The kit targets esophageal cancer, gastric cancer, colon cancer and normal intestinal epithelial cells, and selects four secreted proteins (IL6, IL8, TGFβ, VEGF) and four intracellular proteins (p-p65, p-p38, p-ERK, p-AKT) as detection indicators.

[0031] 1. Kit Components The kit in this embodiment includes the components shown in Table 1.

[0032] Table 1. Kit Components 2. Chip Structure and Fabrication 2.1 Microcavity Array Chip The microcavity array chip 1 used in this embodiment is prepared by mixing polydimethylsiloxane and curing agent at a mass ratio of 10:1, baking at 80°C for 2 hours for curing, and then demolding. The chip has a single-layer structure, with the center point as the origin, the chip surface is divided into the first quadrant, the second quadrant, the third quadrant, and the fourth quadrant. Each quadrant contains one group of microcavity arrays, and each group contains 13,992 microcavities 2. The entire chip contains a total of 55,968 microcavity reaction chambers.

[0033] Based on cell adhesion and growth characteristics, the microcavity dimensions were designed to be 200 μm long, 50 μm wide, and 30 μm deep. After treatment with a bioaffinity-modified reagent, the microcavity walls promote cell adhesion and growth, ensuring cell viability during the detection process.

[0034] 2.2 Antibody Barcode Chip The antibody barcode chip 3 is also divided into four quadrants with the center point as the origin. Each quadrant contains one set of barcodes 4, and each set of barcodes contains four channels. The entire chip contains four sets of barcodes and 16 channels. The barcode channels are 20μm wide and spaced 30μm apart. Each set of barcodes corresponds one-to-one with each microcavity array, ensuring that each microcavity can correspond to four different antibody channels.

[0035] Among them: secretory protein capture antibody barcode chip: each group of barcode channels is printed with IL6, IL8, TGFβ, and VEGF capture antibodies respectively; intracellular protein capture antibody barcode chip: each group of barcode channels is printed with p-p65, p-p38, p-ERK, and p-AKT capture antibodies respectively.

[0036] 3. Reagent preparation 3.1 Bioaffinity Modifying Agents Prepare a 5% (w / w) BSA solution using sterile phosphate buffer, sterile BSA solution, and sterile deionized water.

[0037] 3.2 Antibody Buffer A 1% (w / w) BSA solution was prepared using sterile phosphate buffer, sterile BSA solution, and sterile cation exchange solution.

[0038] 3.3 Antibody blocking solution Prepare a 3% (w / w) BSA solution using sterile phosphate buffer, sterile BSA solution, and sterile deionized water.

[0039] 3.4 Fluorescent Conjugate Working Solution The APC-fluorescent conjugate was diluted 1:400 with antibody buffer to a final concentration of 100 μg / ml.

[0040] 3.5 Cell lysate Using mild RIPA lysis stock solution, add a 1:1000 diluted protease inhibitor and a 1:1000 diluted phosphatase inhibitor, and mix thoroughly.

[0041] 3.6 Cell washing solution Prepare a solution containing 2% FBS using sterile PBS and sterile FBS.

[0042] 4. Detection Method In this embodiment, the above-mentioned kit was used to perform in situ sequential quantitative detection of the same single-cell secreted and intracellular proteins in esophageal cancer, gastric cancer, colon cancer, and normal intestinal epithelial cells. The specific operation steps are as follows: Step 1: Bioaffinity Preprocessing of Microcavity Array Chip The microcavity array chip was removed from its vacuum packaging and placed in a plasma cleaner for hydroxylation treatment. Immediately after removal, the microcavity surface was treated with a bioaffinity modification agent and then dried at room temperature. Before use, an appropriate amount of cell washing buffer was used to wet the microcavities, and the microcavities were gently agitated to remove air bubbles and maintain a moist state.

[0043] Step 2: Antibody barcode chip preprocessing Remove the secretory protein capture antibody barcode chip and the intracellular protein capture antibody barcode chip from the dry vacuum packaging, soak them in antibody blocking solution for 10 minutes, and then remove them and spin dry or blow dry for later use.

[0044] Step 3: Cell extraction and resuspension For adherent cells (such as esophageal cancer, gastric cancer, colon cancer, and normal intestinal epithelial cells), digest the cells with trypsin for 1 min, add an equal volume of FBS to terminate the digestion, and collect the cell suspension in a centrifuge tube. Centrifuge at 1000 rpm for 5 min, discard the supernatant, and a white cell pellet will be visible at the bottom of the tube. Resuspend the cell pellet in cell washing buffer, centrifuge again at 1000 rpm for 5 min, and discard the supernatant. Resuspend the cell pellet in cell-appropriate culture medium, perform cell counting, and adjust the cell density to 8 × 10⁶ cells / mL. 4 Approximately 1 per ml.

[0045] Step 4: Single-cell loading Place the pre-treated microcavity array chip horizontally on the operating platform with the microcavities facing upwards. Use a pipette to draw 1 ml of cell suspension and spread it evenly on the surface of the microcavity array chip, ensuring complete coverage of the microcavity area. Allow it to settle for 5 minutes, allowing the cells to naturally settle into the microcavities due to gravity. Gently rinse the chip surface with cell washing buffer to remove any excess cells that have not yet entered the microcavities.

[0046] Step 5: Secretory protein capture With the barcode side down, gently place the secretory protein capture antibody barcode chip onto the surface of the microcavity array chip containing single cells. Clamp the two chips together using plastic clamps to ensure tight contact at the interface and a sealed microcavity. Place the assembled chip in an incubator at 37°C and 5% CO2 for 12 hours to allow the proteins secreted by the single cells to be captured by the antibody barcode chip.

[0047] Step 6: Secretory protein detection Remove the chip assembly from the incubator, gently disassemble the plastic clamp, and remove the secretory protein capture antibody barcode chip. Rinse the chip surface twice with antibody buffer to remove unbound impurities. Take 400 μl of the secretory protein detection antibody working solution diluted with antibody buffer, spread it evenly on the antibody barcode chip, and incubate at room temperature for 45 min. Rinse the chip surface twice with antibody buffer to remove unbound detection antibody. Take 400 μl of APC-fluorescent conjugate working solution, spread it evenly on the antibody barcode chip, and incubate at room temperature in the dark for 30 min. Rinse the chip surface twice with antibody buffer, shake or blow dry the chip, and then scan it in a fluorescence scanner to acquire secretory protein fluorescence signal data.

[0048] Step 7: Intracellular protein capture The microcavity array chip, after completing the capture of secreted proteins, was placed in an environment of 4°C. 50 μl of cell lysis buffer was added to the edge of the chip surface on the non-microcavity side. The intracellular protein capture antibody barcode chip, with its barcode side down, was gently placed over the microcavity array chip surface from the non-microcavity side to the microcavity side, ensuring the lysis buffer evenly covers all microcavities. The two chips were clamped together using plastic clamps and incubated at 4°C for 2 hours to allow the intracellular proteins released by lysis to be captured by the antibody barcode chip.

[0049] Step 8: Intracellular protein detection Remove the chip assembly from the 4°C environment, gently disassemble the plastic clamp, and remove the intracellular protein capture antibody barcode chip. Rinse the chip surface twice with antibody buffer to remove unbound impurities. Take 400 μl of the intracellular protein detection antibody working solution diluted with antibody buffer, spread it evenly on the antibody barcode chip, and incubate at room temperature for 45 min. Rinse the chip surface twice with antibody buffer to remove unbound detection antibody. Take 400 μl of APC-fluorescent conjugate working solution, spread it evenly on the antibody barcode chip, and incubate at room temperature in the dark for 30 min. Rinse the chip surface twice with antibody buffer, dry at room temperature in the dark, and then scan using a fluorescence scanner to acquire intracellular protein fluorescence signal data.

[0050] Step 9: Quantitative Calculation By detecting the concentration gradients of pre-established protein standards, fluorescence intensity-concentration standard curves for each protein are obtained. Substituting the fluorescence signal values ​​obtained in steps 6 and 8 into the corresponding standard curve equations, the actual concentrations of four secretory proteins (IL6, IL8, TGFβ, and VEGF) and four intracellular proteins (p-p65, p-p38, p-ERK, and p-AKT) in each single cell are calculated, enabling in-situ sequential quantitative detection of secretory and intracellular proteins in the same single cell.

[0051] 5. Analysis of Test Results 5.1 Evaluation of reagent kit biocompatibility Cells were cultured for 24 h in culture dishes (normal control group), microarrays modified with 5% BSA bioaffinity, and microarrays without bioaffinity modification, respectively. Cell viability was assessed after staining with a live / dead staining agent. Results are as follows: Figure 3 As shown, compared with the normal control group, there was no significant difference in cell viability in the microcavity chip modified with 5% BSA bioaffinity, indicating that the microcavity environment of the kit of the present invention has good biocompatibility and does not affect cell activity and normal physiological function.

[0052] 5.2 Evaluation of reagent kit detection sensitivity Protein standards such as IL6, IL8, p-p65, and p-p38 were serially diluted 10-fold from 1000 μg / ml to obtain 100 μg / ml (10... 1 (10 times dilution), 10 μg / ml (10 2 (10 times dilution), 1 μg / ml (10 3 (diluted multiple times), 100ng / ml (10 times) 4 (diluted multiple times), 10 ng / ml (10 5 (diluted multiple times), 1ng / ml (10 times) 6 (diluted multiple times), 100 pg / ml (10 times) 7 (diluted multiple times), 10 pg / ml (10 8 (diluted multiple times), 1 pg / ml (10 times) 9 (diluted multiple times), 0.1 pg / ml (10 times) 10 Ten protein samples of different concentrations (diluted several times) were analyzed. The results are as follows: Figure 4 As shown, the fluorescence intensity of each protein exhibits a good linear relationship with the logarithm of its concentration, with a correlation coefficient R0. 2 All values ​​were greater than 0.98, with a minimum detection sensitivity of 0.1 pg / ml, demonstrating the high sensitivity of this kit for protein detection. The quantitative conversion formulas for the eight proteins were then obtained, as follows: IL6: Y = 204.13lgX + 366.23; IL8: Y = 200.37lgX + 451.82; p-p65: Y = 145.66lgX + 164.13; p-p38: Y = 144.25lgX + 180.60.

[0053] 5.3 Evaluation of reagent kit specificity IL6, IL8, p-p65, and p-p38 protein standards (1 μg / ml each) were taken and detected using the kit of this invention to analyze the cross-reactivity between antibodies. Results are as follows: Figure 5 As shown, each protein detection showed a positive signal only in its corresponding antibody channel, with no cross-positive signals, indicating that the kit of the present invention has good detection specificity.

[0054] 5.4 Evaluation of reagent kit detection throughput In this embodiment, the microcavity array chip contains a total of 55,968 microcavities. After loading a single cell, approximately 20,708 effective single-cell microcavities can be obtained, enabling the simultaneous acquisition of expression data for eight proteins from tens of thousands of single cells, thus achieving high-throughput multi-parameter functional analysis of single cells.

[0055] 5.5 Analysis of the association between single-cell function and signaling pathways Multidimensional bioinformatics analysis was performed on the expression data of four secreted proteins and four intracellular proteins from four cell types (esophageal cancer, gastric cancer, colon cancer, and normal intestinal epithelial cells). The results are as follows: Figure 6 As shown: (1) Cluster analysis Figure 6 As shown in the figure): the four cell types were clustered into multiple subpopulations based on the differential expression of eight proteins, reflecting significant heterogeneity among cells; (2) Four-dimensional distribution analysis Figure 7 As shown in the figure): the distribution of secretory and intracellular proteins in single colon cancer cells exhibits significant heterogeneity. (3) Correlation analysis Figure 8 As shown in the figure): In single colon cancer cells, IL6 expression was highly correlated with p-p65 expression, and IL8 expression was highly correlated with p-p38 expression, suggesting that there may be a specific signaling pathway regulatory relationship. (4) Nuclear density analysis Figure 9 As shown in the figure): Compared with normal intestinal epithelial single cells, the proportion of p-p65+IL6 and p-p38+IL8 double-positive cells in colon cancer single cells was significantly increased, indicating that these two signaling pathways may be activated in colon cancer.

[0056] The above results fully demonstrate that the present invention can achieve paired detection of secreted proteins and intracellular proteins in the same single cell, and can be used to establish a correlation analysis of "intracellular signaling pathway activation-external functional output" in single cells.

[0057] Example 2: Detection Condition Optimization Experiment To further optimize the detection conditions, this embodiment conducted optimization experiments on different key parameters.

[0058] 1. Optimization of antibody incubation time The antibody incubation times were set to 10 min, 20 min, 30 min, 45 min, 60 min, and 90 min, and the same concentration of protein standard was tested. The results showed that the fluorescence signal reached a plateau at 45 min, therefore the optimal incubation time was determined to be 45 min.

[0059] 2. Optimization of fluorescent conjugate concentration APC-fluorescent conjugate was diluted at 1:100, 1:200, 1:400, 1:800, and 1:1600 to detect protein standards of the same concentration. The results showed that a dilution of 1:400 yielded the best signal-to-noise ratio and a final concentration not lower than 100 μg / ml; therefore, the optimal dilution was determined to be 1:400.

[0060] 3. Optimization of cell lysis time Cell lysis times were set at 0.5 h, 1 h, 2 h, 3 h, and 4 h to detect intracellular protein capture efficiency. The results showed that intracellular protein signals reached a plateau at 2 h of lysis, and the cell nucleus remained relatively intact. Therefore, the optimal lysis time was determined to be 2 h.

[0061] Example 3: Applicability verification for different cell types To verify the versatility of the kit, esophageal cancer cells (KYSE-30), gastric cancer cells (MGC-803), colon cancer cells (HCT-116), and normal intestinal epithelial cells (FHC) were used for detection. The results showed that all four cell types could be successfully loaded into the microcavity array chip, and the sequential detection of secreted and intracellular proteins was completed, obtaining paired functional data. The cell viability of each cell type was >90%, and the protein detection positivity rate met expectations, demonstrating that the kit is suitable for single-cell functional analysis of multiple cell types.

[0062] Example 4: Validation of the repeatability of test results The same batch of colon cancer cells was tested using three different batches of reagent kits to evaluate the inter-batch reproducibility of the kits. Simultaneously, the same batch of kits was used to test three batches of colon cancer cells cultured at different time points to evaluate the intra-batch reproducibility of the kits. The results showed that the coefficients of variation for each protein detection were all less than 15%, indicating that the kits of this invention have good detection reproducibility and stability.

[0063] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A kit for in situ sequential quantitative detection of secreted and intracellular proteins from the same single cell, characterized in that, include: Microcavity array chip; Two types of antibody barcode chips are used: a secretory protein capture antibody barcode chip and an intracellular protein capture antibody barcode chip. A plastic clamp is used to clamp the microcavity array chip and the antibody barcode chip to form a closed microcavity; Bioaffinity modifying agents are used to modify the surface of microcavity array chips; Secretory protein standards, intracellular protein standards, secretory protein detection antibody stock solution, intracellular protein detection antibody stock solution; Fluorescent conjugates, antibody buffers, antibody blocking solutions, cell lysis buffers, and cell washing solutions.

2. The reagent kit according to claim 1, characterized in that, The microcavity array chip is made of polydimethylsiloxane and has multiple microcavities on its surface for single-cell capture and culture.

3. The reagent kit according to claim 1, characterized in that, The antibody barcode chip has multiple parallel antibody channels, each 5-200 μm wide and 10-100 μm apart, for capturing different types of proteins.

4. The reagent kit according to claim 1, characterized in that, The bioaffinity modification reagent is a BSA solution with a mass concentration of 1-5% prepared by sterile phosphate-buffered saline (PBS), sterile BSA solution, and sterile deionized water.

5. The reagent kit according to claim 1, characterized in that, The working concentration dilution factor of the fluorescent conjugate is 1:100-1:500, and the final concentration is not less than 100 μg / ml.

6. The reagent kit according to claim 1, characterized in that, The antibody buffer is a 0.5-2% BSA solution prepared with sterile phosphate-buffered saline (PBS), sterile BSA solution, and sterile cation exchange solution. It is used to dilute the stock antibody solution for secretory protein detection or intracellular protein detection, so that the concentration of the diluted detection antibody is greater than 100 μg / ml.

7. The kit according to claim 1, characterized in that, The antibody blocking solution is a 3-6% BSA solution prepared by mixing sterile phosphate-buffered saline (PBS), sterile BSA solution, and sterile deionized water in a certain proportion.

8. A method for in situ sequential quantitative detection of secreted proteins and intracellular proteins from the same single cell, characterized in that, Includes the following steps: Step 1: Perform bio-affinity pretreatment on the microcavity array chip; Step 2: Block the antibody barcode chip using antibody blocking solution; Step 3: Extract and resuspend cells, control cell density, and load them into a microcavity array chip; Step 4: Assemble the microcavity array chip and the secretory protein capture antibody barcode chip, clamp them with plastic clamps, and incubate to capture secretory proteins; Step 5: Disassemble the chip, perform antibody incubation and fluorescent labeling on the secretory protein capture antibody barcode chip, and scan to obtain secretory protein data; Step 6: Add cell lysis buffer to the microcavity array chip, assemble the microcavity array chip and the intracellular protein capture antibody barcode chip, and incubate to capture intracellular proteins; Step 7: Disassemble the chip, perform antibody incubation and fluorescent labeling on the intracellular protein capture antibody barcode chip, and scan to obtain intracellular protein data; Step 8: Convert the fluorescence value into protein concentration according to the standard curve to achieve quantitative analysis of secreted proteins and intracellular proteins in the same single cell.

9. The method according to claim 8, characterized in that, Step 1 is as follows: the microcavity array chip is plasma cleaned, then the surface of the microcavity is bio-affinity modified with a bio-affinity modification reagent, and then dried; before use, the microcavity is wetted with cell washing solution to remove air bubbles inside the microcavity.

10. The method according to claim 8, characterized in that, The fluorescent labeling in steps 5 and 7 uses APC-fluorescent conjugate, which is incubated in the dark before fluorescence scanning.

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