Integrated microfluidic system with chemiluminescence detection for single cell analysis after intracellular labeling

Area of Science:

• Analytical Chemistry
• Biochemistry
• Cell Biology

Background:

• Single cell analysis requires sensitive and precise methods for intracellular component quantification.
• Glutathione (GSH) is a critical antioxidant, and its intracellular levels are vital indicators of cellular health.
• Existing methods for intracellular GSH analysis in single cells have limitations in sensitivity and complexity.

Purpose of the Study:

• To develop and validate a novel microchip electrophoresis with chemiluminescence detection (MCE-CL) assay for the quantitative analysis of intracellular glutathione (GSH) in single human red blood cells.
• To establish the sensitivity, linearity, and reliability of the MCE-CL method for GSH determination at the single-cell level.
• To compare the performance of the MCE-CL assay with existing microchip electrophoresis with laser-induced fluorescence detection (MCE-LIF) methods.

Main Methods:

• Human red blood cells were incubated with diazo-luminol for intracellular GSH labeling.
• Individual cells were injected into a microchip electrophoresis system, followed by in-line lysis.
• Separation was achieved via microchip electrophoresis, with detection using chemiluminescence based on luminol-labeled GSH oxidation.
• Calibration curves were generated using known amounts of GSH.

Main Results:

• The MCE-CL assay demonstrated a linear calibration curve for GSH over a range of 0.2-90 amol (R² = 0.9991).
• A highly sensitive detection limit of 50 zmol (3.6 x 10⁻⁹ M) was achieved.
• The average intracellular GSH content in individual human red blood cells was determined to be 64.9 amol (n=17).
• The MCE-CL method proved to be approximately 100 times more sensitive than previously reported MCE-LIF methods for single-cell GSH analysis.

Conclusions:

• Microchip electrophoresis with chemiluminescence detection (MCE-CL) is a powerful and sensitive technique for single-cell intracellular glutathione (GSH) analysis.
• The developed MCE-CL assay offers a simple, rapid, and highly sensitive alternative for quantifying GSH in individual cells.
• This method has significant potential for applications in cell biology, diagnostics, and drug discovery where precise intracellular analyte quantification is crucial.