Subsecond electrophoretic separations from droplet samples for screening of enzyme modulators.

Area of Science:

• Biochemistry
• Analytical Chemistry
• Microfluidics

Background:

• High-throughput screening (HTS) is crucial for drug discovery, with multiwell plates and fluorescence readers being common tools.
• Electrophoresis-based screening offers an alternative to fluorescence assays, potentially avoiding issues like fluorogenic reaction engineering and false positives.
• Limitations in current HTS methods necessitate the development of more robust and accurate screening platforms.

Purpose of the Study:

• To develop and validate a novel screening method combining droplet microfluidics with microchip electrophoresis (MCE).
• To enable rapid and efficient screening of enzyme modulators from multiwell plate-based assays.
• To address challenges associated with fluorescence-based HTS, such as false positives and complex assay development.

Main Methods:

• Coupling multiwell plate assays to microchip electrophoresis (MCE) using droplet microfluidics.
• Reformatting samples into 8 nL plugs segmented by oil within a microfluidic device.
• Utilizing a hybrid polydimethylsiloxane-glass device for sample extraction and analysis via MCE with laser-induced fluorescence detection.
• Screening a library of 140 compounds against protein kinase A.

Main Results:

• Successfully screened 140 compounds against protein kinase A using the developed droplet microfluidics-MCE system.
• Achieved rapid analysis of 96 samples in 12 minutes with 1-second separations.
• Demonstrated high separation resolution (1.2) and efficiency (16,000 plates/s) with real samples.
• Identified 25 potential enzyme modulators during primary screening, subsequently verified by dose-response curves.

Conclusions:

• The droplet microfluidics-MCE system provides a powerful and efficient platform for high-throughput screening of enzyme modulators.
• This method overcomes limitations of traditional fluorescence assays, offering improved accuracy and reduced complexity.
• The validated system demonstrates significant potential for accelerating drug discovery and evaluation processes.