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Updated: Jun 21, 2025
Author Spotlight: Unveiling the Polyfunctionality and Heterogeneity in Immune Responses
Published on: March 8, 2024
In-droplet multiplex immunoassays for hypoxia-induced single-cell cytokines
Lili Cong1, Xiaolei Guo2, Jiaqi Wang1
1State Key Laboratory of Supramolecular Structure and Materials, College of Chemistry, Jilin University, Changchun, 130012, PR China.
Insights
This study developed a microdroplet immunoassay to track single tumor cell cytokine expression, revealing insights into hypoxia
Area of Science:
- Biomedical Engineering
- Cancer Research
- Analytical Chemistry
Background:
- Hypoxia microenvironments are crucial in tumor growth and metastasis.
- Cytokine expression serves as a key biomarker for these processes.
- Understanding single-cell cytokine dynamics is vital for cancer diagnosis and prognosis.
Purpose of the Study:
- To develop a novel in-droplet biosensing strategy for simultaneous, single-cell cytokine analysis.
- To investigate the impact of hypoxia on tumor cell cytokine secretion (VEGF and IL-8).
- To establish a method for assessing early cancer diagnosis and prognosis through cytokine profiling.
Main Methods:
- Utilized in-droplet-based immunoassays with immuno-nanoprobes for cytokine tracking.
- Co-packaged single cells and nanoprobes in microdroplets using a microfluidic chip.
- Employed fluorescence imaging and chemometric methods to quantify dual-cytokine expression.
Main Results:
- Successfully tracked cytokines (VEGF and IL-8) secreted by single tumor cells within microdroplets.
- Demonstrated a positive correlation in the secretion of these two cytokines under varying hypoxia levels.
- Validated the effectiveness of the dual-target, simultaneous biosensing strategy.
Conclusions:
- The developed in-droplet biosensing strategy enables precise analysis of single-cell cytokine expression.
- This method provides valuable insights into the role of hypoxia in tumor progression.
- The findings support the potential for early cancer diagnosis and prognosis assessment.
Abstract:
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.
