Abstract
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.
| Language | English |
|---|---|
| Pages | 912-919 |
| Journal | Sensors and Actuators, B: Chemical |
| Volume | 281 |
| DOIs | |
| Publication status | Published - 15 Feb 2019 |
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Keywords
- Botulinum neurotoxin A
- Fluorescence detection
- Peptide
- Superparamagnetic beads
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Functional fluorescence assay of botulinum neurotoxin A in complex matrices using magnetic beads. / Klisara, Nevena; Peters, Jeroen; Haasnoot, Willem; Nielen, Michel W.F.; Palaniappan, Alagappan; Liedberg, Bo.
In: Sensors and Actuators, B: Chemical, Vol. 281, 15.02.2019, p. 912-919.Research output: Contribution to journal › Article › Academic › peer-review
TY - JOUR
T1 - Functional fluorescence assay of botulinum neurotoxin A in complex matrices using magnetic beads
AU - Klisara, Nevena
AU - Peters, Jeroen
AU - Haasnoot, Willem
AU - Nielen, Michel W.F.
AU - Palaniappan, Alagappan
AU - Liedberg, Bo
PY - 2019/2/15
Y1 - 2019/2/15
N2 - 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.
AB - 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.
KW - Botulinum neurotoxin A
KW - Fluorescence detection
KW - Peptide
KW - Superparamagnetic beads
U2 - 10.1016/j.snb.2018.10.100
DO - 10.1016/j.snb.2018.10.100
M3 - Article
VL - 281
SP - 912
EP - 919
JO - Sensors and Actuators B: Chemical
T2 - Sensors and Actuators B: Chemical
JF - Sensors and Actuators B: Chemical
SN - 0925-4005
ER -