Single-cell enzyme concentrations, kinetics, and inhibition analysis using high-density hydrodynamic cell isolation...

Area of Science:

• Quantitative Systems Biology
• Cellular Biology
• Biotechnology

Background:

• High-quality single-cell data are crucial for quantitative systems biology but challenging to obtain with traditional methods.
• Existing techniques for single-cell analysis are often time-consuming and reagent-intensive.

Purpose of the Study:

• To develop a robust and simple microfluidic method for high-density single-cell trapping and analysis.
• To apply this method for determining single-cell enzyme kinetics and characterizing enzyme inhibitors.

Main Methods:

• Development of a microfluidic device for ordered single-cell isolation in large arrays.
• On-chip sample preparation including fluorescent labeling and washing.
• Application of the method to analyze carboxylesterase enzyme kinetics in HeLa, 293T, and Jurkat cells.
• Characterization of Nordihydroguaiaretic acid (NDGA) as a carboxylesterase inhibitor.

Main Results:

• The microfluidic method allows for high-density microscopic analysis with simplified image processing.
• Novel single-cell enzyme kinetics were determined for three different cell types.
• A kinetic model indicated cell-type-specific variations in carboxylesterase concentration.
• Nordihydroguaiaretic acid (NDGA) was characterized as an inhibitor, with specific effects noted in HeLa cells.

Conclusions:

• The developed microfluidic technology offers a time- and reagent-saving approach for single-cell analysis.
• This method enables the quantification of intracellular enzyme kinetics and inhibitor effects at the single-cell level.
• The technology is adaptable for analyzing a wide range of intracellular enzymes with available fluorogenic substrates.