A microfluidic flow cytometer enabling absolute quantification of single-cell intracellular proteins

Area of Science:

• Biotechnology
• Analytical Chemistry
• Cell Biology

Background:

• Conventional flow cytometry lacks absolute quantification of intracellular proteins in single cells.
• Cellular heterogeneity insights are limited by current quantification methods.
• Calibration approaches are needed for accurate single-cell protein measurement.

Purpose of the Study:

• To develop a microfluidic flow cytometer for absolute quantification of intracellular proteins.
• To enable high-throughput analysis of single-cell protein copy numbers.
• To overcome limitations of conventional flow cytometry in single-cell proteomics.

Main Methods:

• Utilizing a microfluidic device with a constriction channel smaller than cells.
• Forcing single cells through the channel and quantifying fluorescence intensities.
• Developing calibration curves using fluorescence-labelled antibodies within the channel.

Main Results:

• Achieved absolute quantification of intracellular proteins, demonstrated by beta-actin measurements in tumor cells.
• Quantified beta-actin copy numbers in A549, MCF 10A, and Hep G2 cells.
• Demonstrated a high throughput of 100 cells/s with a cell transit time of approximately 10 ms.

Conclusions:

• The developed microfluidic system enables absolute quantification of intracellular proteins in single cells.
• This technique provides a high-throughput solution for single-cell proteomics.
• The platform serves as an enabling technology for deeper cellular heterogeneity studies.