|Title||Functional fluorescence assay of botulinum neurotoxin A in complex matrices using magnetic beads|
|Author(s)||Klisara, Nevena; Peters, Jeroen; Haasnoot, Willem; Nielen, Michel W.F.; Palaniappan, Alagappan; Liedberg, Bo|
|Source||Sensors and Actuators B: Chemical 281 (2019). - ISSN 0925-4005 - p. 912 - 919.|
BU Authenticity & Bioassays
Monsteradministratie & Coördinatie
|Publication type||Refereed Article in a scientific journal|
|Keyword(s)||Botulinum neurotoxin A - Fluorescence detection - Peptide - Superparamagnetic beads|
The extremely toxic botulinum neurotoxin poses a threat for health and food safety, requiring rapid and easy-to-use detection platforms. To meet these requirements, we have explored a novel functional assay format for detection of botulinum neurotoxin serotype A light chain (BoNT/A LC) in complex matrices. The proposed assay utilizes a synthetic peptide designed to mimic the SNAP-25 protein (synaptosomal-associated protein 25) as substrate bound to a superparamagnetic bead and a fluorescent dye. Cleavage of the peptide by BoNT/A LC yields a reduction in fluorescence signal revealing the presence of the BoNT/A LC in the sample matrices tested. The superparamagnetic beads enable efficient separation of the cleaved peptides from food matrices, thereby improving the reliability of responses. Herein, we demonstrate a protocol offering an assay time of 6 h and a LOD of 0.5 nM (25 ng/ml). The proposed protocol is validated using carrot juice and diluted milk pending further improvements in sensitivity and overall assay time. Robustness, cost-effectiveness, low sample volume requirements in conjunction with the possibility of multiplexing for other proteolytic enzymes makes the proposed protocol competitive in comparison with conventional BoNT assays reported elsewhere.