Protein-protein interaction analysis in single microfluidic droplets using FRET and fluorescence lifetime detection

Area of Science:

• Biochemistry
• Analytical Chemistry
• Microfluidics

Background:

• Protein-protein interactions are crucial in biological systems.
• Microfluidic devices offer miniaturized platforms for biological assays.
• Förster Resonance Energy Transfer (FRET) and fluorescence lifetime measurements are powerful analytical tools.

Purpose of the Study:

• To demonstrate the feasibility of protein-protein interaction analysis in microfluidic droplets.
• To monitor reaction progress using FRET and fluorescence lifetime measurements.
• To develop a quantitative assay for protein detection in microdroplets.

Main Methods:

• Fabrication of microfluidic chips using soft- and photolithography.
• Generation of microdroplets (630 pL and 6.71 nL) at high frequencies.
• Utilizing FRET and microscopic fluorescence lifetime measurements for detection.
• Employing a model system with labeled BSA, streptavidin, and avidin.

Main Results:

• Reliable generation of microdroplets with controlled volumes.
• Quantitative detection of avidin and streptavidin in direct and competitive assays.
• Achieved nanomolar detection limits, corresponding to attomole protein amounts.
• Demonstrated the efficiency of fluorescence lifetime over intensity ratio for FRET determination.
• Completed a competitive on-chip binding assay for avidin in 135 seconds.

Conclusions:

• Microfluidic droplets combined with FRET and fluorescence lifetime measurements are effective for protein analysis.
• The developed microchips enable rapid and sensitive quantitative protein detection.
• This platform holds potential for various biochemical assays and diagnostics.