title: Aqueous two-phase systems for enhancing immunoassay sensitivity: simultaneous concentration of mycotoxins and neutralization of matrix interference.
abstract: Immunoassays have a broad application range, from environmental and food toxicology to biomedical analysis, providing rapid and simple methods for analyte quantification. Immunoassays, however, are often challenging at nM and sub nM concentrations and are affected by detrimental matrix interference effects, as is the case of the detection of ochratoxin A (OTA) and Aflatoxin B1 (AFB1). These are widespread mycotoxins found in food and feed, with serious potential implications for human health. This work demonstrates the use of polymer-salt aqueous two phase systems (ATPSs) for the simultaneous concentration of mycotoxins and neutralization of matrix interference. In particular, polyethylene glycol (PEG)-phosphate salt ATPSs were used to enhance the detection sensitivity of OTA and AFB1 in wines and beer by an indirect competitive ELISA. Using this methodology it was possible to quantify both analytes spiked in red wine with limits-of-detection (LoD) down to 0.19 ng/mL and 0.035 ng/mL, respectively, with results comparable to those obtained using solutions of toxins in phosphate buffered saline (PBS) buffer (0.7 ng/mL and 0.009 ng/mL, respectively). Furthermore, a very low matrix-to matrix variability was observed, with LoD and half inhibitory concentration (IC50) values of 5.17 ± 1.08 and 33.2 ± 3.5 ng/mL (±SD) obtained in the detection of OTA spiked in red and white wines, beer or PBS buffer. These results indicate the potential of ATPS as a fast and simple concentration step and in providing matrix-independent analyte quantification for enhanced immunoassay sensitivity below regulatory levels.
authors.affiliation: INESC Microsistemas e Nanotecnologias and IN-Institute of Nanoscience and Nanotechnology, Lisbon, Portugal; IBB-Institute for Biotechnology and Bioengineering, Centre for Biological and Chemical Engineering, Instituto Superior Técnico, Universidade de Lisboa, Lisbon, Portugal.
authors.forename: R R G
authors.initials: RR
authors.lastname: Soares
authors.affiliation: INESC Microsistemas e Nanotecnologias and IN-Institute of Nanoscience and Nanotechnology, Lisbon, Portugal; Leibniz-Institut für Analytische Wissenschaften - ISAS - e.V., Otto-Hahn-Str. 6b, 44227 Dortmund, Germany(†).
authors.forename: P
authors.initials: P
authors.lastname: Novo
authors.affiliation: Department of Bioengineering, Instituto Superior Técnico, Universidade de Lisboa, Lisbon, Portugal; IBB-Institute for Biotechnology and Bioengineering, Centre for Biological and Chemical Engineering, Instituto Superior Técnico, Universidade de Lisboa, Lisbon, Portugal.
authors.forename: A M
authors.initials: AM
authors.lastname: Azevedo
authors.affiliation: Department of Bioengineering, Instituto Superior Técnico, Universidade de Lisboa, Lisbon, Portugal; IBB-Institute for Biotechnology and Bioengineering, Centre for Biological and Chemical Engineering, Instituto Superior Técnico, Universidade de Lisboa, Lisbon, Portugal.
authors.forename: P
authors.initials: P
authors.lastname: Fernandes
authors.affiliation: INESC Microsistemas e Nanotecnologias and IN-Institute of Nanoscience and Nanotechnology, Lisbon, Portugal.
authors.forename: V
authors.initials: V
authors.lastname: Chu
authors.affiliation: INESC Microsistemas e Nanotecnologias and IN-Institute of Nanoscience and Nanotechnology, Lisbon, Portugal; Department of Bioengineering, Instituto Superior Técnico, Universidade de Lisboa, Lisbon, Portugal.
authors.forename: J P
authors.initials: JP
authors.lastname: Conde
authors.affiliation: Department of Bioengineering, Instituto Superior Técnico, Universidade de Lisboa, Lisbon, Portugal; IBB-Institute for Biotechnology and Bioengineering, Centre for Biological and Chemical Engineering, Instituto Superior Técnico, Universidade de Lisboa, Lisbon, Portugal. Electronic address: rabarros@tecnico.ulisboa.pt.
authors.forename: M R
authors.initials: MR
authors.lastname: Aires-Barros
mesh_terms: D016604:Aflatoxin B1 / Q000032:analysis*; D001515:Beer / Q000032:analysis; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D006863:Hydrogen-Ion Concentration; D057230:Limit of Detection; D009793:Ochratoxins / Q000032:analysis*; D014920:Wine / Q000032:analysis
chemical_list: D009793:Ochratoxins; C025589:ochratoxin A; D016604:Aflatoxin B1
keywords: Aflatoxin B1; Aqueous two-phase system; Concentration; Immunoassay; Matrix; Ochratoxin A