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Updated: Jul 8, 2025
Author Spotlight: Unveiling the Polyfunctionality and Heterogeneity in Immune Responses
Published on: March 8, 2024
Single-Cell Secretion Analysis via Microfluidic Cell Membrane Immunosorbent Assay for Immune Profiling
Ying Xu1,2, Ka-Chun Wu3,4, Wenxin Jiang1,2
1Department of Biomedical Engineering, City University of Hong Kong, 83 Tat Chee Avenue, Kowloon Tong, Hong Kong SAR 999077, China.
Insights
A new assay enables high-throughput single-cell analysis of immune cell secretions and surface proteins. This method advances precision medicine by identifying distinct immune cell profiles in healthy donors and nasopharyngeal carcinoma patients.
Area of Science:
- Biomedical Engineering
- Immunology
- Analytical Chemistry
Background:
- Single-cell phenotypic analysis is crucial for precision medicine, but screening cell secretions remains difficult.
- Existing methods struggle with high-throughput, multiplexed analysis of individual cell functions.
- Cell secretions are key indicators of immune cell activity and function.
Purpose of the Study:
- To develop a novel, high-throughput single-cell secretion assay for multiplexed phenotypic analysis.
- To integrate immunosorbent assays and droplet microfluidics for enhanced single-cell screening.
- To validate the assay's capability in analyzing immune cell differentiation and activation.
Main Methods:
- Development of a cell membrane-based assay using cholesterol-linked antibodies (CLAbs).
- Encapsulation of CLAb-grafted single cells in droplets for secretion capture.
- Extraction, fluorescence labeling, and multiplexed measurement of secretions and surface proteins via flow cytometry.
Main Results:
- The assay successfully analyzed THP-1 cells, M1 macrophages, and dendritic cells, indicating differentiation efficiency.
- Peripheral blood mononuclear cells (PBMCs) from healthy donors showed varied active immune cell populations (6.62-47.14%) under stimuli.
- PBMCs from nasopharyngeal carcinoma patients exhibited a higher percentage of actively cytokine-secreting cells in basal state (2.82 ± 1.48%) compared to healthy donors (0.70 ± 0.29%).
Conclusions:
- The developed assay provides a flexible and high-throughput platform for single-cell secretion analysis.
- This method enables precise phenotyping and identification of distinct immune cell populations.
- The findings highlight potential diagnostic biomarkers in nasopharyngeal carcinoma patients based on single-cell immune activity.
Abstract:
Single-cell multiplexed phenotypic analysis expands the biomarkers for diagnosis, heralding a new era of precision medicine. Cell secretions are the primary measures of immune function, but single-cell screening remains challenging. Here, a novel cell membrane-based assay was developed using cholesterol-linked antibodies (CLAbs), integrating immunosorbent assays and droplet microfluidics to develop a flexible high-throughput single-cell secretion assay for multiplexed phenotyping. CLAb-grafted single cells were encapsulated in water-in-oil droplets to capture their own secretions. Subsequently, the cells were extracted from droplets for fluorescence labeling and screening. Multiple secretions and surface proteins were simultaneously measured from single cells by flow cytometry. To validate the approach, THP-1 cells, THP-1-derived M1 macrophages, and dendritic cells were assayed, indicating the differentiation efficiency of THP-1 cells under different chemical stimulations. Moreover, peripheral blood mononuclear cells from healthy donors under various stimuli showed varied active immune cell populations (6.62-47.14%). The peripheral blood mononuclear cells (PBMCs) of nasopharyngeal carcinoma patients were analyzed to identify a higher percentage of actively cytokine-secreted single cells in the basal state (2.82 ± 1.48%), compared with that in the health donors (0.70 ± 0.29%).
