Automatic and integrated micro-enzyme assay (AIμEA) platform for highly sensitive thrombin analysis via an engineered...

Area of Science:

• Biochemistry
• Analytical Chemistry
• Biotechnology

Background:

• High-throughput enzyme activity assays are crucial for enzyme discovery, engineering, and drug screening.
• Existing platforms often lack integration, speed, sensitivity, or high-throughput capabilities.

Purpose of the Study:

• To develop a novel automatic and integrated micro-enzyme assay (AIμEA) platform for enzyme activity detection.
• To demonstrate the platform's capability using thrombin as a model enzyme.

Main Methods:

• Fabrication of a microreaction system using a green fluorescence protein (GFP)-functionalized monolithic capillary column.
• Engineering a recombinant GFP probe with a His-tag and thrombin substrate sequence for immobilization and release.
• Integration of thrombin injection, online enzymatic digestion, and label-free GFP detection using capillary electrophoresis-laser-induced fluorescence (CE-LIF).

Main Results:

• The AIμEA platform achieved a 30-fold higher catalytic efficiency compared to bulk reactions.
• Demonstrated high sensitivity with a limit of detection as low as 1 pM for thrombin.
• Successfully detected thrombin in human serum and its inhibition by hirudin, showing robustness and reliability.

Conclusions:

• The AIμEA platform provides a sensitive, robust, and integrated solution for enzyme activity assays.
• The platform exhibits significant potential for high-throughput biological applications, disease diagnostics, and drug screening.