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Updated: Sep 26, 2025
Author Spotlight: Investigating Angiogenesis Through Challenges and Innovations in Assay Development
Published on: May 31, 2024
Single-Cell VEGF Analysis by Fluorescence Imaging-Microfluidic Droplet Platform: An Immunosandwich Strategy on the
Lili Cong1, Yu Tian1,2, Zepeng Huo1
1State Key Laboratory of Supramolecular Structure and Materials, College of Chemistry, Jilin University, Changchun 130012, P. R. China.
Insights
This study introduces a novel microfluidic platform for analyzing single-cell cytokine secretion, specifically vascular endothelial growth factor (VEGF). The method reveals cellular heterogeneity in VEGF secretion without needing extra carriers, advancing single-cell analysis.
Area of Science:
- Biotechnology
- Cell Biology
- Microfluidics
Background:
- Current single-cell analysis platforms have limitations in tracking cytokine-secreting cells.
- Understanding single-cell cytokine secretion is crucial for physiological and pathological studies, particularly in angiogenesis.
Purpose of the Study:
- To develop a microfluidic droplet-based fluorescence imaging platform for analyzing single-cell secreted vascular endothelial growth factor (VEGF).
- To explore cellular physiological clues at the single-cell level by quantifying VEGF secretion.
- To demonstrate the platform's ability to reveal cellular heterogeneity in cytokine secretion.
Main Methods:
- Developed silica nanoparticle (NP)-based immunoprobes for VEGF detection.
- Utilized a microfluidic droplet system to isolate single cells.
- Implemented an immunosandwich assay on the cell surface within droplets, detected via fluorescence imaging.
Main Results:
- Successfully analyzed single-cell VEGF secretion from three different cell lines (MCF-7, HeLa, H8).
- Demonstrated significant cellular heterogeneity in VEGF secretion capabilities among the tested cell lines.
- The assay was performed directly above the cell of interest without additional carriers, enhancing microdroplet utility.
Conclusions:
- The developed platform enables precise analysis of single-cell VEGF secretion, highlighting cellular heterogeneity.
- This method offers a unique approach for single-cell cytokine analysis directly above the cell, improving efficiency.
- The platform is adaptable for analyzing other secreted cytokines, serving as a valuable tool for single-cell metabonomics.
Abstract:
Despite recent advances in single-cell analysis techniques, the ability of single-cell analysis platforms to track specific cells that secreted cytokines remains limited. Here, we report a microfluidic droplet-based fluorescence imaging platform that can analyze single cell-secreted vascular endothelial growth factor (VEGF), an important regulator of physiological and pathological angiogenesis, to explore cellular physiological clues at the single-cell level. Two kinds of silica nanoparticle (NP)-based immunoprobes were developed, and they were bioconjugated to the membrane proteins of the probed cell surface via the bridging of secreted VEGF. Thus, an immunosandwich assay was built above the probed cell via fluorescence imaging analysis of each cell in isolated droplets. This analytical platform was used to compare the single-cell VEGF secretion ability of three cell lines (MCF-7, HeLa, and H8), which experimentally demonstrates the cellular heterogeneity of cells in secreting cytokines. The uniqueness of this method is that the single-cell assay is carried out above the cell of interest, and no additional carriers (beads or reporter cells) for capturing analytes are needed, which dramatically improves the availability of microdroplets. This single-cell analytical platform can be applied for determining other secreted cytokines at the single-cell level by changing other immune pairs, which will be an available tool for exploring single-cell metabonomics.
