Highly Multiplexed Single-Cell Protein Profiling with Large-Scale Convertible DNA-Antibody Barcoded Arrays

Area of Science:

• Biotechnology
• Molecular Biology
• Immunology

Background:

• Detecting proteins from single cells at high multiplexity is a significant challenge in biological research.
• Existing methods often lack the throughput and sensitivity required for detailed cellular analysis.

Purpose of the Study:

• To develop a highly multiplexed in situ tagging technique for assaying single-cell secreted proteins.
• To enhance the capability for high-throughput and high-sensitivity detection of cellular protein secretion.

Main Methods:

• Utilized single-stranded DNA (ssDNA) encoded microbead arrays for multiplexed detection.
• Employed multicolor successive imaging for protein quantification.
• Integrated the system with polydimethylsiloxane (PDMS) microwells for single-cell analysis.

Main Results:

• Demonstrated exponential increase in multiplexing capability.
• Successfully detected ten immune effector proteins from single macrophages stimulated with lipopolysaccharide (LPS).
• Observed significant heterogeneity in protein secretion among differentiated human primary macrophages.

Conclusions:

• The developed technology offers a versatile platform for high-throughput, high-sensitivity single-cell protein analysis.
• This approach is expected to advance systems biology, immune regulation studies, signaling analysis, and molecular diagnostics.
• Revealed heterogeneity in immune effector protein secretion from macrophages, providing insights into cellular responses.