title: Multi-detection method for five common microalgal toxins based on the use of microspheres coupled to a flow-cytometry system.
abstract: Freshwater and brackish microalgal toxins, such as microcystins, cylindrospermopsins, paralytic toxins, anatoxins or other neurotoxins are produced during the overgrowth of certain phytoplankton and benthic cyanobacteria, which includes either prokaryotic or eukaryotic microalgae. Although, further studies are necessary to define the biological role of these toxins, at least some of them are known to be poisonous to humans and wildlife due to their occurrence in these aquatic systems. The World Health Organization (WHO) has established as provisional recommended limit 1μg of microcystin-LR per liter of drinking water. In this work we present a microsphere-based multi-detection method for five classes of freshwater and brackish toxins: microcystin-LR (MC-LR), cylindrospermopsin (CYN), anatoxin-a (ANA-a), saxitoxin (STX) and domoic acid (DA). Five inhibition assays were developed using different binding proteins and microsphere classes coupled to a flow-cytometry Luminex system. Then, assays were combined in one method for the simultaneous detection of the toxins. The IC50's using this method were 1.9±0.1μg L(-1) MC-LR, 1.3±0.1μg L(-1) CYN, 61±4μg L(-1) ANA-a, 5.4±0.4μg L(-1) STX and 4.9±0.9μg L(-1) DA. Lyophilized cyanobacterial culture samples were extracted using a simple procedure and analyzed by the Luminex method and by UPLC-IT-TOF-MS. Similar quantification was obtained by both methods for all toxins except for ANA-a, whereby the estimated content was lower when using UPLC-IT-TOF-MS. Therefore, this newly developed multiplexed detection method provides a rapid, simple, semi-quantitative screening tool for the simultaneous detection of five environmentally important freshwater and brackish toxins, in buffer and cyanobacterial extracts.
authors.affiliation: Departamento de Farmacología, Facultad de Veterinaria, Universidad de Santiago de Compostela, 27002 Lugo, Spain.
authors.forename: María
authors.initials: M
authors.lastname: Fraga
authors.affiliation: Departamento de Farmacología, Facultad de Veterinaria, Universidad de Santiago de Compostela, 27002 Lugo, Spain. Electronic address: natalia.vilarino@usc.es.
authors.forename: Natalia
authors.initials: N
authors.lastname: Vilariño
authors.affiliation: Departamento de Farmacología, Facultad de Veterinaria, Universidad de Santiago de Compostela, 27002 Lugo, Spain.
authors.forename: M Carmen
authors.initials: MC
authors.lastname: Louzao
authors.affiliation: Departamento de Farmacología, Facultad de Veterinaria, Universidad de Santiago de Compostela, 27002 Lugo, Spain.
authors.forename: Laura P
authors.initials: LP
authors.lastname: Rodríguez
authors.affiliation: Departamento de Farmacología, Facultad de Veterinaria, Universidad de Santiago de Compostela, 27002 Lugo, Spain.
authors.forename: Amparo
authors.initials: A
authors.lastname: Alfonso
authors.affiliation: Institute for Global Food Security (IGFS), School of Biological Sciences, Queen's University Belfast, David Keir Building, Stranmillis Road, Belfast BT9 5AG, Northern Ireland, UK.
authors.forename: Katrina
authors.initials: K
authors.lastname: Campbell
authors.affiliation: Institute for Global Food Security (IGFS), School of Biological Sciences, Queen's University Belfast, David Keir Building, Stranmillis Road, Belfast BT9 5AG, Northern Ireland, UK.
authors.forename: Christopher T
authors.initials: CT
authors.lastname: Elliott
authors.affiliation: Department of Pharmacology, Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California at San Diego, La Jolla, CA 92093-0657, United States.
authors.forename: Palmer
authors.initials: P
authors.lastname: Taylor
authors.affiliation: Interdisciplinary Centre of Marine and Environmental Research, CIIMAR, and Faculty of Sciences, University of Porto, Rua dos Bragas 289, Porto 4050-123, Portugal.
authors.forename: Vítor
authors.initials: V
authors.lastname: Ramos
authors.affiliation: Interdisciplinary Centre of Marine and Environmental Research, CIIMAR, and Faculty of Sciences, University of Porto, Rua dos Bragas 289, Porto 4050-123, Portugal.
authors.forename: Vítor
authors.initials: V
authors.lastname: Vasconcelos
authors.affiliation: Departamento de Farmacología, Facultad de Veterinaria, Universidad de Santiago de Compostela, 27002 Lugo, Spain. Electronic address: luis.botana@usc.es.
authors.forename: Luis M
authors.initials: LM
authors.lastname: Botana
mesh_terms: D000470:Alkaloids; D001427:Bacterial Toxins; D000458:Cyanobacteria / Q000737:chemistry; D000087522:Cyanobacteria Toxins; D005434:Flow Cytometry / Q000379:methods*; D005618:Fresh Water / Q000032:analysis; D007608:Kainic Acid / Q000031:analogs & derivatives* / Q000032:analysis / Q000302:isolation & purification; D008387:Marine Toxins; D058086:Microalgae / Q000737:chemistry*; D052998:Microcystins / Q000032:analysis* / Q000302:isolation & purification; D008863:Microspheres; D012530:Saxitoxin / Q000032:analysis* / Q000302:isolation & purification; D014326:Tropanes / Q000032:analysis* / Q000302:isolation & purification; D014498:Uracil / Q000031:analogs & derivatives* / Q000032:analysis / Q000302:isolation & purification
chemical_list: D000470:Alkaloids; D001427:Bacterial Toxins; D000087522:Cyanobacteria Toxins; D008387:Marine Toxins; D052998:Microcystins; D014326:Tropanes; C089595:cylindrospermopsin; D012530:Saxitoxin; D014498:Uracil; C509783:anatoxin a; C057862:cyanoginosin LR; C012301:domoic acid; D007608:Kainic Acid
keywords: Aquatic toxins; Flow-cytometry system; Microalgal toxins; Microsphere-based array; Multi-detection; Screening method