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Detection and Quantification of Biologically Active Botulinum Neurotoxin Serotypes A and B Using a Förster Resonance Energy Transfer-Based Quantum Dot Nanobiosensor
journal contribution
posted on 2017-08-25, 00:00 authored by Yun Wang, H. Christopher Fry, Guy E. Skinner, Kristin M. Schill, Timothy V. DuncanBotulinum neurotoxin
(BoNT) is the most potent toxin known. The ingestion of food contaminated
with biologically active BoNT causes foodborne botulism, which can
lead to respiratory paralysis, coma, and death after ingestion of
as little as 70 μg for a 70 kg human. Because of its lethality
and challenges associated with current detection methods, there is
an urgent need for highly sensitive rapid screening techniques capable
of detecting biologically active BoNT. Here, we describe a Förster
resonance energy transfer-based nanobiosensor that uses quantum dots
(QDs) and two specific quencher-labeled peptide probes to detect and
differentiate two biologically active forms of BoNT, serotypes A and
B, which were responsible for 80% of human foodborne botulism cases
in the U.S. from 2012 to 2015. Each peptide probe contains an enzymatic
cleavage site specific to only one serotype. QDs were selected based
on the spectral overlap with the quenchers. In the presence of the
target BoNT serotype, the peptide probe is cleaved and the quenching
of QD photoluminescence (PL) is reduced, giving a signal that is easily
detected by a PL spectrophotometer. This sensor performance was evaluated
with light chains of BoNT/A and BoNT/B (LcA and LcB), catalytic domains
of the respective serotypes. LcA and LcB were detected in 3 h with
limits of detection of 0.2 and 2 ng/mL, respectively. The specificity
of the sensor was evaluated, and no cross-reactivity from nontarget
serotypes was observed with 2 h of incubation. Because each serotype-specific
peptide is conjugated to a QD with a unique emission wavelength, multiple
biologically active BoNT serotypes could be detected in one PL spectrum.
The sensor was also shown to be responsive to BoNT/A and BoNT/B holotoxins.
Good performance of this sensor implies its potential application
as a rapid screening method for biologically active BoNT/A and BoNT/B
in the laboratory and in the field.