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In vivo Dual Substrate Bioluminescent Imaging
Published on: October 11, 2011
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5,5-Dialkylluciferins are thermal stable substrates for bioluminescence-based detection systems
Ce Shi1, Michael P Killoran2, Mary P Hall2
1Promega Biosciences, Inc., San Luis Obispo, California, United States of America.
Plos One
|December 14, 2020
Summary
Researchers developed a novel luciferase/luciferin system to improve the stability of bioluminescence ATP detection reagents. This innovation addresses the instability of current hygiene monitoring solutions, enabling more reliable and convenient ATP detection.
Area of Science:
- Biochemistry
- Enzyme kinetics
- Analytical chemistry
Background:
- Firefly luciferase assays are vital for hygiene monitoring but suffer from unstable reagents.
- Current detection reagents have limited shelf life, necessitating frequent manual preparation.
- Engineered Ultra-Glo™ luciferase offers thermal stability but reagent solutions degrade over time.
Purpose of the Study:
- To identify the cause of instability in Ultra-Glo™ luciferase and luciferin detection reagents.
- To develop a more stable luciferin substrate and optimize the luciferase enzyme for improved reagent shelf life.
- To create a next-generation bioluminescence ATP detection system with enhanced reagent stability.
Main Methods:
- Investigated the inhibitory effects of luciferin breakdown products on Ultra-Glo™ luciferase.
- Synthesized and tested 5,5-dialkyl luciferin analogs for improved thermostability.
- Employed protein engineering to enhance Ultra-Glo™ luciferase compatibility with modified substrates.
- Developed and validated a novel luciferase/luciferin pair for stable ATP detection.
Main Results:
- Dehydroluciferin, a luciferin oxidation product, was identified as a potent inhibitor of Ultra-Glo™ luciferase.
- 5,5-dialkyl luciferins showed improved thermostability but poor substrate utilization by the enzyme.
- A novel engineered luciferase/luciferin system demonstrated significantly improved reagent stability at ambient temperatures.
- The new system provides a foundation for more stable bioluminescence ATP detection assays.
Conclusions:
- The instability of ATP detection reagents is primarily due to dehydroluciferin inhibition of Ultra-Glo™ luciferase.
- Structural modification of luciferin and protein engineering of luciferase are key to enhancing reagent stability.
- The developed novel luciferase/luciferin system offers a promising solution for next-generation, stable bioluminescence ATP detection assays.
