In-droplet multiplex immunoassays for hypoxia-induced single-cell cytokines

Cytokine expression is an important biomarker in understanding hypoxia microenvironments in tumor growth and metastasis. In-droplet-based immunoassays performed above the target cell membrane were employed to track the cytokines of single cells with the aid of three types of immuno-nanoprobes (one capture nanoprobe and two reporter nanoprobes). Single cells and nanoprobes were co-packaged in water-in-oil microdroplets (about 100 μm in diameter) using a cross-shaped microfluidic chip. In each droplet, capture nanoprobes would be first fixed to the cell surface by linking to membrane proteins that have been streptavidinized. Then, the capture nanoprobes can collect cell-secreted cytokines (VEGF and IL-8) by the antibodies, followed by two reporter nanoprobes that emit distinguishable fluorescence. Fluorescence imaging was utilized to record the signal outputs of two reporter probes, which reflect cytokine expressions secreted by a single tumor cell. The cytokine levels at different degrees of hypoxia induction were assessed. Multiple chemometric methods were adopted to distinguish differences in the secretion of two cytokines and the results demonstrated a positive correlation. This study developed an in-droplet, dual-target, simultaneous biosensing strategy for a single cell, which is helpful for understanding the impacts of hypoxia microenvironments on cell cytokines that are vital for assessing early cancer diagnosis and prognosis.