LEGO®-inspired electrically-actuated microfluidics for on-chip protein crystallization and in situ X-ray...

Area of Science:

• Structural Biology
• Biophysics
• Materials Science

Background:

• X-ray crystallography is vital for determining 3D protein structures, essential for understanding protein function.
• Protein crystallization is a critical bottleneck in the X-ray crystallography workflow.
• Existing methods often require complex fluid handling and separate steps for crystallization and analysis.

Purpose of the Study:

• To develop an innovative microfluidic device for efficient protein crystallization and in situ X-ray crystallography.
• To create a streamlined platform for protein structure determination, reducing experimental complexity and time.
• To demonstrate the utility of electrically-actuated fluid control for on-demand crystallization reagent mixing.

Main Methods:

• Fabrication of a microfluidic device using photolithography and nanoimprinting with a LEGO®-inspired architecture.
• Integration of gold-LEGO®-electrodes (GLEs) for electrically-actuated fluid handling and on-demand counter-diffusive mixing.
• Utilizing an X-ray transparent polymer for direct in situ X-ray crystallography without crystal extraction.

Main Results:

• Successful demonstration of the LEAP-X platform with model proteins (lysozyme, thaumatin, proteinase K).
• Achieved crystallization and room-temperature in situ structural analysis of the metalloprotein rubrerythrin.
• The device operates at low voltages (<1 V) and is fabricated into a 150 μm thin format.

Conclusions:

• The LEAP-X platform offers a novel, efficient, and streamlined approach to protein crystallization and structure determination.
• Electrically-actuated fluid control and in situ analysis significantly simplify the crystallography workflow.
• The platform holds potential for advanced applications, including time-resolved crystallography with chemical triggers.