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A high-throughput, low-volume enzyme assay on solid support.
Peter Babiak1, Jean-Louis Reymond
1Department of Chemistry and Biochemistry, University of Berne, Freiestrasse 3, CH-3012 Berne, Switzerland.
Analytical Chemistry
|January 15, 2005
Summary
A novel high-throughput enzyme assay uses robotic handling for over 1000 tests/h on silica gel plates. This method enables efficient screening of enzyme activity and inhibition using fluorogenic substrates.
Area of Science:
- Biochemistry
- Enzymology
- Assay Development
Background:
- Enzyme activity screening is crucial for drug discovery and biotechnology.
- Existing high-throughput methods often require large sample volumes or lack versatility.
Purpose of the Study:
- To develop a miniaturized, high-throughput enzyme assay compatible with diverse substrates.
- To establish a robust and reproducible platform for enzyme activity and inhibition measurements.
Main Methods:
- Enzyme solutions (≤1 μL) are robotically deposited onto silica gel plates pre-impregnated with fluorogenic substrates.
- Reactions are quantified using fluorescence detection.
- Assay compatibility was tested with lipases, glycosidases, and proteases using various substrates, including umbelliferyl esters.
Main Results:
- Achieved throughput exceeding 1000 tests/h with minimal enzyme solution per test.
- Demonstrated compatibility with water-insoluble, water-soluble, and whole-protein substrates.
- Successfully performed enzyme inhibition measurements and lipase assays without background hydrolysis using umbelliferyl esters.
- Fingerprint analysis confirmed high throughput and reproducibility across 37 fluorogenic ester substrates.
Conclusions:
- The developed high-throughput enzyme assay is versatile, reproducible, and efficient.
- It enables rapid screening of enzyme activity and inhibition for various enzyme classes.
- A selected set of eight fluorogenic umbelliferyl esters is optimal for lipase and esterase activity screening on silica gel plates.