A graphene oxide based fluorescence resonance energy transfer (FRET) biosensor for ultrasensitive detection of botulinum neurotoxin A (BoNT/A) enzymatic activity

• A graphene oxide (GO) based FRET biosensor was developed for ultrasensitive detection of BoNT/A light chain enzymatic activity.
• This FRET biosensor platform was realized by covalently immobilization of GFP modified SNAP-25 peptide substrate on GO surface.
• Target BoNT/A light chain can specifically cleave SNAP-25-GFP substrate for fluorescence signal recovery.
• The linear detection range for this biosensor is from 1 fg/mL to 1 pg/mL.
• An ultra-low detection limit of 1 fg/mL was achieved for botulinum neurotoxin A (BoNT/A) light chain detection.

Botulinum neurotoxins (BoNTs) are among the most potent toxic bacterial proteins for humans, which make them potential agents for bioterrorism. Therefore, an ultrasensitive detection of BoNTs and their active states is in great need as field-deployable systems for anti-terrorism applications. We report the construction of a novel graphene oxide (GO)-peptide based fluorescence resonance energy transfer (FRET) biosensor for ultrasensitive detection of the BoNT serotype A light chain (BoNT-LcA) protease activity. A green fluorescence protein (GFP) modified SNAP-25 peptide substrate (SNAP-25-GFP) was optimally designed and synthesized with the centralized recognition/cleavage sites. This FRET platform was constructed by covalent immobilization of peptide substrate on GO with BSA passivation which have advantages of low non-specific adsorption and high stability in protein abundant solution. BoNT-LcA can specifically cleave SNAP-25-GFP substrate covalently immobilized on GO to release the fragment with GFP. Based on fluorescence signal recovery measurement, the target BoNT-LcA was detected sensitively and selectively with the linear detection range from 1 fg/mL to 1 pg/mL. The limit of detection (LOD) for BoNT-LcA is around 1 fg/mL.