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Related Experiment Videos

High-throughput single-cell analysis for enzyme activity without cytolysis.

Ning Gao1, Wenlei Wang, Xiaoli Zhang

  • 1School of Chemistry and Chemical Engineering, Shandong University, Jinan 250100, China.

Analytical Chemistry
|April 29, 2006
PubMed
Summary

A new method allows enzyme activity measurement in single cells without cell destruction. This high-throughput technique uses chemical perforation and detects peroxidase activity in neutrophils at over one cell per minute.

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Area of Science:

  • Biochemistry
  • Cell Biology
  • Analytical Chemistry

Background:

  • Measuring intracellular enzyme activity is crucial for understanding cellular function and disease.
  • Existing methods often require cell lysis, which destroys cellular integrity and prevents further analysis.
  • A need exists for high-throughput, non-destructive methods to assess enzyme activity within intact single cells.

Purpose of the Study:

  • To develop and validate a novel, high-throughput method for determining intracellular enzyme activity without causing cell lysis.
  • To demonstrate the method's efficacy using peroxidase (PO) activity in human neutrophils as a model system.

Main Methods:

  • Chemical cell perforation using digitonin to create transient micropores in the cell membrane.
  • Utilizing a capillary-based microsampler and microreactor for continuous cell processing.

Related Experiment Videos

  • Employing an intracellular enzyme-catalyzed reaction (PO converting hydroquinone to benzoquinone) within perforated cells.
  • Detecting the product (benzoquinone) diffusing out of the cells and forming a detectable zone around them.

    • Main Results:

    • Successful development of a non-lytic, high-throughput method for single-cell enzyme activity determination.
    • Demonstrated detection of intracellular peroxidase activity in human neutrophils.
    • Achieved an average detection rate exceeding one cell per minute, indicating high throughput.
    • The method preserves cellular integrity, allowing for potential downstream analyses.

    Conclusions:

    • The developed method offers a significant advancement for intracellular enzyme activity analysis.
    • This technique enables high-throughput, non-destructive assessment of enzyme function in single cells.
    • The approach has broad applicability for studying various intracellular enzymes and cellular processes.