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Title: Detection and Quantification of Biologically Active Botulinum Neurotoxin Serotypes A and B Using a Förster Resonance Energy Transfer-Based Quantum Dot Nanobiosensor

Abstract

Botulinum 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 mu g 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 Forster 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 respectivemore » 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.« less

Authors:
 [1]; ORCiD logo [2];  [1];  [1]; ORCiD logo [1]
  1. Center for Food Safety and Applied Nutrition, U.S. Food and Drug Administration, 6502 S. Archer Road, Bedford Park, Cook County, Illinois 60501, United States
  2. Center for Nanoscale Materials, Argonne National Laboratory, 9700 S. Cass Avenue, Lemont, DuPage County, Illinois 60439, United States
Publication Date:
Research Org.:
Argonne National Lab. (ANL), Argonne, IL (United States)
Sponsoring Org.:
USDOE Office of Science - Office of Basic Energy Sciences - Scientific User Facilities Division
OSTI Identifier:
1413497
DOE Contract Number:
AC02-06CH11357
Resource Type:
Journal Article
Resource Relation:
Journal Name: ACS Applied Materials and Interfaces; Journal Volume: 9; Journal Issue: 37
Country of Publication:
United States
Language:
English
Subject:
Förster resonance energy transfer (FRET); botulinum neurotoxin; food safety; foods; nanobiosensor; nanotechnology; quantum dot

Citation Formats

Wang, Yun, Fry, H. Christopher, Skinner, Guy E., Schill, Kristin M., and Duncan, Timothy V. Detection and Quantification of Biologically Active Botulinum Neurotoxin Serotypes A and B Using a Förster Resonance Energy Transfer-Based Quantum Dot Nanobiosensor. United States: N. p., 2017. Web. doi:10.1021/acsami.7b08736.
Wang, Yun, Fry, H. Christopher, Skinner, Guy E., Schill, Kristin M., & Duncan, Timothy V. Detection and Quantification of Biologically Active Botulinum Neurotoxin Serotypes A and B Using a Förster Resonance Energy Transfer-Based Quantum Dot Nanobiosensor. United States. doi:10.1021/acsami.7b08736.
Wang, Yun, Fry, H. Christopher, Skinner, Guy E., Schill, Kristin M., and Duncan, Timothy V. Mon . "Detection and Quantification of Biologically Active Botulinum Neurotoxin Serotypes A and B Using a Förster Resonance Energy Transfer-Based Quantum Dot Nanobiosensor". United States. doi:10.1021/acsami.7b08736.
@article{osti_1413497,
title = {Detection and Quantification of Biologically Active Botulinum Neurotoxin Serotypes A and B Using a Förster Resonance Energy Transfer-Based Quantum Dot Nanobiosensor},
author = {Wang, Yun and Fry, H. Christopher and Skinner, Guy E. and Schill, Kristin M. and Duncan, Timothy V.},
abstractNote = {Botulinum 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 mu g 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 Forster 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.},
doi = {10.1021/acsami.7b08736},
journal = {ACS Applied Materials and Interfaces},
number = 37,
volume = 9,
place = {United States},
year = {Mon Mar 20 00:00:00 EDT 2017},
month = {Mon Mar 20 00:00:00 EDT 2017}
}
  • Botulinum neurotoxins (BoNTs), produced by Clostridium botulinum, are a group of seven (A-G) immunologically distinct proteins and cause the paralytic disease botulism. These toxins are the most poisonous substances known to humans and are potential bioweapon agents. Therefore, it is necessary to develop highly sensitive assays for the detection of BoNTs in both clinical and environmental samples. In the present study, we have developed an ELISA-based protein antibody microarray for the sensitive and simultaneous detection of BoNT serotype A, B, C, D, E and F. With engineered high-affinity antibodies, the assays have sensitivities in buffer of 8 fM (1.2 pg/mL)more » for serotypes A and B, and 32 fM (4.9 pg/mL) for serotypes C, D, E, and F. Using clinical and environmental samples (serum and milk), the microarray is capable of detecting BoNT/A-F to the same levels as in standard buffer. Cross reactivity between assays for individual serotype was also analyzed. These simultaneous, rapid, and sensitive assays have the potential to measure botulinum toxins in a high-throughput manner in complex clinical or environmental samples.« less
  • A fluorescence sandwich immunoassay using high affinity antibodies and quantum dot (QD) reporters has been developed for detection of botulinum toxin serotype A (BoNT/A). For the development of the assay, a nontoxic recombinant fragment of the holotoxin (BoNT/A-HC-fragment) has been used as a structurally valid simulant for the full toxin molecule. The antibodies used, AR4 and RAZ1, bind to nonoverlapping epitopes present on both the full toxin and on the recombinant fragment. In one format, the immunoassay is carried out in a 96-well plate with detection in a standard plate reader. Detection down to 31 pM of the BoNT/Hc-fragment wasmore » demonstrated with a total incubation time of 3 hours, using AR4 as the capture antibody and QD-coupled RAZ1 as the reporter. In a second format, the AR4 capture antibody was coupled to Sepharose beads, and the immunochemical reactions were carried out in microcentrifuge tubes with an incubation time of 1 hour. These beads were subsequently captured and concentrated in a rotating rod “renewable surface” flow cell as part of a sequential injection fluidic system. This flow cell was equipped with a fiber optic system for fluorescence measurements. In PBS buffer solution matrix, the BoNT/A-HC-fragment was detected to concentrations as low as 5 pM using the fluidic measurement approach.« less
  • In this work, we report enhanced power conversion efficiency (PCE) of bulk heterojunction polymer solar cells by Förster resonance energy transfer (FRET) from samarium-doped luminescent gadolinium orthovanadate (GdVO{sub 4}:Sm{sup 3+}) quantum dots (QDs) to polythieno[3,4-b]-thiophene-co-benzodithiophene (PTB7) polymer. The photoluminescence emission spectrum of GdVO{sub 4}:Sm{sup 3+} QDs overlaps with the absorption spectrum of PTB7, leading to FRET from GdVO{sub 4}:Sm{sup 3+} to PTB7, and significant enhancements in the charge-carrier density of excited and polaronic states of PTB7 are observed. This was confirmed by means of femtosecond transient absorption spectroscopy. The FRET from GdVO{sub 4}:Sm{sup 3+} QDs to PTB7 led to amore » remarkable increase in the power conversion efficiency (PCE) of PTB7:GdVO{sub 4}:Sm{sup 3+}:PC{sub 71}BM ([6,6]-phenyl-C{sub 71}-butyric acid methyl ester) polymer solar cells. The PCE in optimized ternary blend PTB7:GdVO{sub 4}:Sm{sup 3+}:PC{sub 71}BM (1:0.1:1.5) is increased to 8.8% from 7.2% in PTB7:PC{sub 71}BM. This work demonstrates the potential of rare-earth based luminescent QDs in enhancing the PCE of polymer solar cells.« less
  • Clostridium botulinum neurotoxins (BoNTs) released by the bacterium Clostridium botulinum are the most potent toxins causing the fatal disease called botulism. There are seven distinct serotypes of BoNTs (A to G) released by various strains of botulinum. They all have high sequence homology and similar three-dimensional structure. The toxicity of BoNT follows a four-step process – binding, internalization, translocation, and cleavage of its target protein, one of the three components of the SNARE complex (Soluble N-ethylmaleimde-sensitive factor attachment protein receptor) required for membrane docking and neurotransmitter release. Cleavage of one of the three proteins causes blockage of neurotransmitter release leadingmore » to flaccid paralysis. Though anyone of the above four steps could be a target for developing antidotes for botulism, the catalytic domain is the most suitable target for post exposure treatment. Of the seven serotypes BoNT/A, B, E and probably F affect humans, with BoNT/A considered to be the most potent. Development of drugs for botulism is focused on serotype specific inhibitors, but a pan-active inhibitor acting on several serotypes is preferable since it is difficult to identify the serotype before the treatment, especially since there is at least a 36-hour window before botulism can be diagnosed. Using structure-based drug discovery, we have developed three heptapeptides based on the SNARE proteins which inhibit BoNT/A, B and E equally well. Probable reasons for pan-activity of these peptides are discussed.« less