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A Microfluidic Chip for the Versatile Chemical Analysis of Single Cells
Published on: October 16, 2013
Single-cell immunosensors for protein detection
Rebecca J Whelan1, Richard N Zare
1Department of Chemistry, Stanford University, Stanford, CA 94305-5080, USA.
Insights
This study presents a novel single-cell detector using immune cells and calcium signaling for protein detection. This biosensor offers a sensitive and specific method for identifying target analytes.
Area of Science:
- Biotechnology
- Immunology
- Cellular Biology
Background:
- Whole-cell biosensors offer natural signal amplification.
- Immunological recognition provides flexibility and specificity.
- Immune cells expressing immunoglobulin G (IgG) receptors can be utilized for detection.
Purpose of the Study:
- To develop a single-cell detector combining biosensing with immunological specificity.
- To utilize the calcium (Ca2+) signaling pathway in immune cells for analyte detection.
- To optimize the U-937 monocytic cell line for use as an immunity-based detector.
Main Methods:
- Loading immune cells with Ca(2+)-indicating dye and specific antibodies.
- Inducing receptor cross-linking with multivalent protein antigens.
- Utilizing the human monocytic cell line U-937, treated with interferon-gamma, for Ca(2+) signal detection.
Main Results:
- Cross-linking of IgG receptors on U-937 cells triggers a detectable increase in cytosolic Ca(2+).
- The U-937 cell line produces a large, transient Ca(2+) signal upon stimulation.
- The detector demonstrated sensitivity to ovalbumin in micromolar concentrations and responded to repeated stimulation.
Conclusions:
- The developed U-937-based detector effectively combines immune recognition with Ca(2+) signaling for analyte detection.
- The system shows potential for detecting specific protein antigens with high sensitivity.
- Further research may explore constraints on analyte size and optimize detection limits.
Abstract:
A single-cell detector is described that combines the natural signal amplification of whole-cell biosensors with the flexibility and specificity of immunological recognition. An immune cell that expresses receptors for the constant region of immunoglobulin G (IgG) is loaded with a Ca(2+)-indicating dye and with antibodies directed against the protein of interest. Introduction of a multivalent protein antigen causes cross-linking of the receptors, which results in a detectable increase in the concentration of cytosolic Ca(2+). Some immune cell lines respond to stimulation with oscillations in their cytosolic Ca(2+) levels that complicate their use as detectors. The human monocytic cell line U-937, when treated with the cytokine interferon-gamma, produces a large, short-lived Ca(2+) signal in response to cross-linking of its high-affinity IgG receptors. U-937 was therefore chosen for development as an immunity-based detector. Human and rabbit antibodies are found to effectively stimulate the cell, causing a prompt and transient response. The cell is able to respond to repeated stimulation, though the response diminishes during rapid stimulation. Ovalbumin can be detected in micromolar concentrations. Possible fundamental constraints on the size of a detectable analyte are discussed.
