{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,6,29]],"date-time":"2026-06-29T06:32:51Z","timestamp":1782714771911,"version":"3.54.5"},"reference-count":39,"publisher":"MDPI AG","issue":"11","license":[{"start":{"date-parts":[[2018,11,15]],"date-time":"2018-11-15T00:00:00Z","timestamp":1542240000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/100000199","name":"U.S. Department of Agriculture","doi-asserted-by":"publisher","award":["NP108 CRIS 5325-42000-049-00D"],"award-info":[{"award-number":["NP108 CRIS 5325-42000-049-00D"]}],"id":[{"id":"10.13039\/100000199","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Toxins"],"abstract":"<jats:p>Botulinum neurotoxin (BoNT) intoxication can lead to the disease botulism, characterized by flaccid muscle paralysis that can cause respiratory failure and death. Due to the significant morbidity and mortality costs associated with BoNTs high toxicity, developing highly sensitive, rapid, and field-deployable assays are critically important to protect the nation\u2019s food supply against either accidental or intentional contamination. We report here that the B-cell based biosensor assay CANARY\u00ae (Cellular Analysis and Notification of Antigen Risks and Yields) Zephyr detects BoNT\/A holotoxin at limits of detection (LOD) of 10.0 \u00b1 2.5 ng\/mL in assay buffer. Milk matrices (whole milk, 2% milk and non-fat milk) with BoNT\/A holotoxin were detected at similar levels (7.4\u20137.9 ng\/mL). BoNT\/A complex was positive in carrot, orange, and apple juices at LODs of 32.5\u201375.0 ng\/mL. The detection of BoNT\/A complex in solid complex foods (ground beef, smoked salmon, green bean baby puree) ranged from 14.8 ng\/mL to 62.5 ng\/mL. Detection of BoNT\/A complex in the viscous liquid egg matrix required dilution in assay buffer and gave a LOD of 171.9 \u00b1 64.7 ng\/mL. These results show that the CANARY\u00ae Zephyr assay can be a highly useful qualitative tool in environmental and food safety surveillance programs.<\/jats:p>","DOI":"10.3390\/toxins10110476","type":"journal-article","created":{"date-parts":[[2018,11,15]],"date-time":"2018-11-15T11:32:47Z","timestamp":1542281567000},"page":"476","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":17,"title":["A Rapid, Sensitive, and Portable Biosensor Assay for the Detection of Botulinum Neurotoxin Serotype A in Complex Food Matrices"],"prefix":"10.3390","volume":"10","author":[{"given":"Christina C.","family":"Tam","sequence":"first","affiliation":[{"name":"Foodborne Toxin Detection and Prevention Research Unit, Western Regional Research Center, Agricultural Research Services, United States Department of Agriculture, 800 Buchanan Street, Albany, CA 94710, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Andrew R.","family":"Flannery","sequence":"additional","affiliation":[{"name":"PathSensors, Inc. 701 East Pratt Street, Baltimore, MD 21202, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Luisa W.","family":"Cheng","sequence":"additional","affiliation":[{"name":"Foodborne Toxin Detection and Prevention Research Unit, Western Regional Research Center, Agricultural Research Services, United States Department of Agriculture, 800 Buchanan Street, Albany, CA 94710, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2018,11,15]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"1059","DOI":"10.1001\/jama.285.8.1059","article-title":"Botulinum toxin as a biological weapon: Medical and public health management","volume":"285","author":"Arnon","year":"2001","journal-title":"JAMA"},{"key":"ref_2","unstructured":"Center for Disease Control and Prevention (2016). 2015 Annual Report of the Federal Select Agent Program."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"535","DOI":"10.1038\/nrmicro3295","article-title":"Botulinum neurotoxins: Genetic, structural and mechanistic insights","volume":"12","author":"Rossetto","year":"2014","journal-title":"Nat. Rev. Microbiol."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"9","DOI":"10.1016\/j.toxicon.2015.09.009","article-title":"The long journey of botulinum neurotoxins into the synapse","volume":"107","author":"Rummel","year":"2015","journal-title":"Toxicon"},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"87","DOI":"10.1016\/j.toxicon.2012.11.011","article-title":"Botulinum neurotoxins: Mechanism of action","volume":"67","author":"Tighe","year":"2013","journal-title":"Toxicon"},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"2","DOI":"10.1016\/j.toxicon.2015.09.011","article-title":"Genetic diversity within the botulinum neurotoxin-producing bacteria and their neurotoxins","volume":"107","author":"Hill","year":"2015","journal-title":"Toxicon"},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"183","DOI":"10.1093\/infdis\/jit449","article-title":"A novel strain of Clostridium botulinum that produces type B and type H botulinum toxins","volume":"209","author":"Barash","year":"2014","journal-title":"J. Infect. Dis."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"203","DOI":"10.4315\/0362-028X-67.1.203","article-title":"Comparison of the mouse bioassay and enzyme-linked immunosorbent assay procedures for the detection of type a botulinal toxin in food","volume":"67","author":"Ferreira","year":"2004","journal-title":"J. Food Prot."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"3787","DOI":"10.1128\/AEM.65.9.3787-3792.1999","article-title":"Development of an in vitro bioassay for Clostridium botulinum type B neurotoxin in foods that is more sensitive than the mouse bioassay","volume":"65","author":"Wictome","year":"1999","journal-title":"Appl. Environ. Microbiol."},{"key":"ref_10","first-page":"257","article-title":"Progress in cell based assays for botulinum neurotoxin detection","volume":"364","author":"Pellett","year":"2013","journal-title":"Curr. Top. Microbiol. Immunol."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"80","DOI":"10.3389\/fbioe.2018.00080","article-title":"Botulinum neurotoxin detection methods for public health response and surveillance","volume":"6","author":"Thirunavukkarasu","year":"2018","journal-title":"Front. Bioeng.Biotechnol."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"1231","DOI":"10.1128\/AEM.72.2.1231-1238.2006","article-title":"Detection of type A, B, E, and F Clostridium botulinum neurotoxins in foods by using an amplified enzyme-linked immunosorbent assay with digoxigenin-labeled antibodies","volume":"72","author":"Sharma","year":"2006","journal-title":"Appl. Environ. Microbiol."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"23","DOI":"10.1016\/j.jim.2012.03.008","article-title":"Rapid and selective detection of botulinum neurotoxin serotype-A and -B with a single immunochromatographic test strip","volume":"380","author":"Ching","year":"2012","journal-title":"J. Immunol. Methods"},{"key":"ref_14","doi-asserted-by":"crossref","unstructured":"Babrak, L., Lin, A., Stanker, L.H., McGarvey, J., and Hnasko, R. (2016). Rapid microfluidic assay for the detection of botulinum neurotoxin in animal sera. Toxins, 8.","DOI":"10.3390\/toxins8010013"},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"922","DOI":"10.1021\/ac504054u","article-title":"Centrifugal microfluidic platform for ultrasensitive detection of botulinum toxin","volume":"87","author":"Koh","year":"2015","journal-title":"Anal. Chem."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"135","DOI":"10.1016\/j.ijfoodmicro.2008.05.012","article-title":"Detection of botulinum neurotoxin-A activity in food by peptide cleavage assay","volume":"126","author":"Rasooly","year":"2008","journal-title":"Int. J. Food Microbiol."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"5569","DOI":"10.1021\/ac4008418","article-title":"Substrates and controls for the quantitative detection of active botulinum neurotoxin in protease-containing samples","volume":"85","author":"Bagramyan","year":"2013","journal-title":"Anal. Chem."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"4305","DOI":"10.1128\/IAI.00405-09","article-title":"Antibody protection against botulinum neurotoxin intoxication in mice","volume":"77","author":"Cheng","year":"2009","journal-title":"Infect. Immun."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"755","DOI":"10.1021\/jf3041963","article-title":"Detection of botulinum neurotoxin serotypes A and B using a chemiluminescent versus electrochemiluminescent immunoassay in food and serum","volume":"61","author":"Cheng","year":"2013","journal-title":"J. Agric. Food Chem."},{"key":"ref_20","doi-asserted-by":"crossref","unstructured":"Scotcher, M.C., Cheng, L.W., and Stanker, L.H. (2010). Detection of botulinum neurotoxin serotype B at sub mouse LD(50) levels by a sandwich immunoassay and its application to toxin detection in milk. PLoS ONE, 5.","DOI":"10.1371\/journal.pone.0011047"},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1016\/j.jim.2008.03.003","article-title":"Development and partial characterization of high-affinity monoclonal antibodies for botulinum toxin type A and their use in analysis of milk by sandwich elisa","volume":"336","author":"Stanker","year":"2008","journal-title":"J. Immunol. Methods"},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"2212","DOI":"10.3390\/toxins5112212","article-title":"A monoclonal antibody based capture elisa for botulinum neurotoxin serotype B: Toxin detection in food","volume":"5","author":"Stanker","year":"2013","journal-title":"Toxins"},{"key":"ref_23","doi-asserted-by":"crossref","unstructured":"Bagramyan, K., Barash, J.R., Arnon, S.S., and Kalkum, M. (2008). Attomolar detection of botulinum toxin type A in complex biological matrices. PLoS ONE, 3.","DOI":"10.1371\/journal.pone.0002041"},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"23","DOI":"10.1007\/978-1-61779-102-4_3","article-title":"Ultrasensitive detection of botulinum neurotoxins and anthrax lethal factor in biological samples by alissa","volume":"739","author":"Bagramyan","year":"2011","journal-title":"Methods Mol. Biol."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"1578","DOI":"10.3201\/eid1110.041279","article-title":"Botulinum neurotoxin detection and differentiation by mass spectrometry","volume":"11","author":"Barr","year":"2005","journal-title":"Emerg. Infect. Dis."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"4779","DOI":"10.1007\/s00216-017-0421-8","article-title":"Further optimization of peptide substrate enhanced assay performance for BoNT\/A detection by MALDI-TOF mass spectrometry","volume":"409","author":"Wang","year":"2017","journal-title":"Anal. Bioanal. Chem."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"1765","DOI":"10.3390\/toxins7051765","article-title":"Recommended mass spectrometry-based strategies to identify botulinum neurotoxin-containing samples","volume":"7","author":"Kalb","year":"2015","journal-title":"Toxins"},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"227","DOI":"10.1186\/s11671-017-1944-9","article-title":"An ultrasensitive gold nanoparticle-based lateral flow test for the detection of active botulinum neurotoxin type A","volume":"12","author":"Liu","year":"2017","journal-title":"Nanoscale Res. Lett."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"10419","DOI":"10.1021\/acs.analchem.6b02066","article-title":"Multiplex biosensing based on highly sensitive magnetic nanolabel quantification: Rapid detection of botulinum neurotoxins A, B, and E in liquids","volume":"88","author":"Orlov","year":"2016","journal-title":"Anal. Chem."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"7687","DOI":"10.1128\/AEM.01664-12","article-title":"Detection of botulinum neurotoxin serotype A, B, and F proteolytic activity in complex matrices with picomolar to femtomolar sensitivity","volume":"78","author":"Dunning","year":"2012","journal-title":"Appl. Environ. Microbiol."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"4935","DOI":"10.3390\/toxins7124857","article-title":"Qualitative and quantitative detection of botulinum neurotoxins from complex matrices: Results of the first international proficiency test","volume":"7","author":"Worbs","year":"2015","journal-title":"Toxins"},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"213","DOI":"10.1126\/science.1084920","article-title":"A B cell-based sensor for rapid identification of pathogens","volume":"301","author":"Rider","year":"2003","journal-title":"Science"},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"81","DOI":"10.1089\/hs.2016.0044","article-title":"Evaluation of immunoassays and general biological indicator tests for field screening of Bacillus anthracis and ricin","volume":"15","author":"Bartholomew","year":"2017","journal-title":"Health Secur."},{"key":"ref_34","unstructured":"Center for Food Safety and Applied Nutrition, U.S. Food and Drug adminstration (FDA) (2001). Bacteriological Analytical Manual."},{"key":"ref_35","unstructured":"Doores, S., Salfinger, Y., and Tortorello, M.L. (2013). Clostridium botulinum and its toxins. Compendium of Methods for the Microbiologial Examination of Foods, American Public Health Association."},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"1490","DOI":"10.1086\/379326","article-title":"An outbreak of foodborne botulism associated with food sold at a salvage store in Texas","volume":"37","author":"Kalluri","year":"2003","journal-title":"Clin. Infect. Dis."},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"172","DOI":"10.1086\/515615","article-title":"A large outbreak of botulism: The hazardous baked potato","volume":"178","author":"Angulo","year":"1998","journal-title":"J. Infect. Dis."},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"376","DOI":"10.1093\/cid\/cis901","article-title":"National outbreak of type A foodborne botulism associated with a widely distributed commercially canned hot dog chili sauce","volume":"56","author":"Juliao","year":"2013","journal-title":"Clin. Infect. Dis."},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"1245","DOI":"10.1086\/592574","article-title":"International outbreak of severe botulism with prolonged toxemia caused by commercial carrot juice","volume":"47","author":"Sheth","year":"2008","journal-title":"Clin. Infect. Dis."}],"container-title":["Toxins"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2072-6651\/10\/11\/476\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,11]],"date-time":"2025-10-11T15:29:51Z","timestamp":1760196591000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2072-6651\/10\/11\/476"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2018,11,15]]},"references-count":39,"journal-issue":{"issue":"11","published-online":{"date-parts":[[2018,11]]}},"alternative-id":["toxins10110476"],"URL":"https:\/\/doi.org\/10.3390\/toxins10110476","relation":{"has-preprint":[{"id-type":"doi","id":"10.20944\/preprints201808.0390.v1","asserted-by":"object"}]},"ISSN":["2072-6651"],"issn-type":[{"value":"2072-6651","type":"electronic"}],"subject":[],"published":{"date-parts":[[2018,11,15]]}}}