Microfluidic three-dimensional hydrodynamic flow focusing for the rapid protein concentration analysis

Area of Science:

• Biotechnology
• Analytical Chemistry
• Microfluidics

Background:

• Accurate protein quantification is crucial for biological research and diagnostics.
• Existing methods can be time-consuming or require large sample volumes.
• Single-molecule detection offers high sensitivity but often lacks rapid quantification capabilities.

Purpose of the Study:

• To develop a simple microfluidic device for rapid and quantitative protein concentration analysis.
• To demonstrate the device's capability for detecting low concentrations of specific proteins.
• To establish a new method for protein quantification using single-molecule detection.

Main Methods:

• Development of a 3D hydrodynamic flow focusing microfluidic device with a step-depth cross-junction.
• Formation of a triangular analyte stream cross-section for precise laser focusing.
• Utilizing photon burst histograms with Gaussian distribution for quantitative analysis.
• Detection of Quantum Dot 525 with antibody (QD525-antibody) and hemagglutinin epitope tagged MAX (HA-MAX) protein.

Main Results:

• Achieved linear concentration determination for QD525-antibody down to 10 pM.
• Quantified HA-MAX protein concentration in HEK293 cell lysate at 0.283 ± 0.015 nM.
• Demonstrated protein concentration determination in as little as 1 minute.
• Successfully employed single-molecule detection for quantitative protein analysis.

Conclusions:

• The developed microfluidic device provides a simple, rapid, and sensitive method for protein quantification.
• This approach represents the first application of single-molecule detection techniques for routine protein concentration determination.
• The technology holds promise for applications in biological research, diagnostics, and drug discovery.