Single-Cell VEGF Analysis by Fluorescence Imaging-Microfluidic Droplet Platform: An Immunosandwich Strategy on the Cell Surface

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 singlecell level by changing other immune pairs, which will be an available tool for exploring single-cell metabonomics.

Automated summary

This study introduces a microfluidic droplet-based fluorescence imaging platform for analyzing single cell-secreted VEGF, exploring cellular physiological clues at the single-cell level. By developing silica nanoparticle-based immunoprobes and conducting immunosandwich assays, the method demonstrates cellular heterogeneity in cytokine secretion.