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Updated: May 8, 2026
A High-throughput-compatible FRET-based Platform for Identification and Characterization of Botulinum Neurotoxin Light Chain Modulators
Published on: December 27, 2013
EGFP/RFP-based FRET sensors for botulinum neurotoxin A biological activity detection and methodological validation
Yu Wang1, Bingbing Zhang2, Ruohui Zhang2
1Joint Drug Development and Innovation Centre for Neurological Disorders of Lanzhou University-China National Biotec Group-Lanzhou Biotechnology Development Co., School of Pharmacy, Lanzhou University, Lanzhou, Gansu, 730000, PR China; MOE Frontiers Science Center for Rare Isotopes, Lanzhou University, Lanzhou, Gansu, 730000, PR China.
Background:
Botulinum neurotoxin type A (BoNT/A) is the most potent and prevalent neurotoxin known to cause botulism, and is also widely used in medical and cosmetic applications. The detection of BoNT/A is of great significance for botulism diagnosis and drug potency determination. Currently, the mouse bioassay (MBA) has long been the gold standard method but has disadvantages of ethical concerns, long testing duration, and high costs. CFP/YFP-based FRET sensors for detecting BoNT/A biological activity require complicated experimental control due to crosstalk, as well as inconvenient operation, intricate data analysis and UV-induced phototoxicity.
Results:
In present work, a FRET-based EGFP-SNAP25(141-206)-DsRED molecular sensor was developed to analyze the endopeptidase activity of BoNT/A, and methodological validation was performed. The results showed that the sensor has a high sensitivity of 4 U, with a linear range from 7.8 U to 125 U, a recovery rate of 96.8 % to 122.7 %, and a precision within 20 %. Furthermore, a cellular sensor for FRET-based BoNT/A biological activity detection was successfully constructed with a Neuro-2a cell line stably expressing the EGFP-SNAP25-tDimer2 variant, enabling the monitoring of the entire process of BoNT/A action. The cell-based assay exhibited a sensitivity of up to 100 pM of BoNT/A with a linear range from 3.125 nM to 50 nM and a recovery rate of 86.3 % to 117.2 %.
Significance:
The FRET-based molecular and cellular sensors provided a convenient and rapid method for measuring BoNT/A biological activity, making them suitable for assessing BoNT/A raw material biological activities in clinical and environmental samples, as well as batches during pharmaceutical production processes.
