EGFP/RFP-based FRET sensors for botulinum neurotoxin A biological activity detection and methodological validation

Area of Science:

• Biochemistry
• Molecular Biology
• Toxicology

Background:

• Botulinum neurotoxin type A (BoNT/A) is a potent neurotoxin with significant medical and cosmetic applications.
• Current detection methods like the mouse bioassay (MBA) are time-consuming, costly, and ethically concerning.
• Existing FRET sensors for BoNT/A activity face challenges including experimental complexity and phototoxicity.

Purpose of the Study:

• To develop and validate novel FRET-based molecular and cellular sensors for detecting BoNT/A endopeptidase activity.
• To establish a more efficient and reliable method for BoNT/A detection compared to existing techniques.

Main Methods:

• Development of a FRET-based molecular sensor using EGFP-SNAP25(141-206)-DsRED.
• Construction of a cell-based sensor using a Neuro-2a cell line stably expressing EGFP-SNAP25-tDimer2.
• Methodological validation of both sensor types for BoNT/A activity analysis.

Main Results:

• The molecular sensor demonstrated high sensitivity (4 U) with a linear range of 7.8–125 U, and good recovery (96.8–122.7%) and precision (<20%).
• The cell-based sensor achieved high sensitivity (100 pM) with a linear range of 3.125 nM–50 nM and acceptable recovery (86.3–117.2%).

Conclusions:

• The developed FRET-based molecular and cellular sensors provide a convenient and rapid method for BoNT/A biological activity measurement.
• These sensors are suitable for assessing BoNT/A in clinical samples, environmental monitoring, and pharmaceutical production quality control.