title: Use of anchor protein modules in fluorescence polarisation aptamer assay for ochratoxin A determination.
abstract: A new strategy for sensitive fluorescence polarisation (FP) analysis is proposed which uses aptamer as the receptor and anchor protein modules as the enhancers by including the aptamers in complexes with protein modules. This approach is based on increasing the size differences of bound and unbound fluorophores. The strategy was applied in an ochratoxin A (ОТА) assay with the competitive binding of fluorophore-labelled and free OTA with aptamer-based receptors. We showed that the binding of labelled OTA with aptamer included in complexes with anchors led to higher a FP than binding with free aptamer. This allowed the aptamer concentration to be reduced, thus lowering the limit of detection by a factor of 40, down to 3.6 nM. The assay time was 15 min. To evaluate the applicability of the FP assay with aptamer-anchor complex to real samples, we conducted OTA measurements in spiked white wine. The OTA limit of detection in wine was 2.8 nM (1.1 μg/kg), and the recoveries ranged from 83% to 113%. The study shows that the proposed anchor strategy is efficient for increasing the sensitivity of FP-based aptamer assays.
authors.affiliation: A.N. Bach Institute of Biochemistry, Research Center of Biotechnology of the Russian Academy of Sciences, Moscow 119071, Russia.
authors.forename: Alexey V
authors.initials: AV
authors.lastname: Samokhvalov
authors.affiliation: A.N. Bach Institute of Biochemistry, Research Center of Biotechnology of the Russian Academy of Sciences, Moscow 119071, Russia.
authors.forename: Irina V
authors.initials: IV
authors.lastname: Safenkova
authors.affiliation: Faculty of Chemistry, M.V. Lomonosov Moscow State University, Moscow 119991, Russia.
authors.forename: Sergei A
authors.initials: SA
authors.lastname: Eremin
authors.affiliation: A.N. Bach Institute of Biochemistry, Research Center of Biotechnology of the Russian Academy of Sciences, Moscow 119071, Russia.
authors.forename: Anatoly V
authors.initials: AV
authors.lastname: Zherdev
authors.affiliation: A.N. Bach Institute of Biochemistry, Research Center of Biotechnology of the Russian Academy of Sciences, Moscow 119071, Russia. Electronic address: dzantiev@inbi.ras.ru.
authors.forename: Boris B
authors.initials: BB
authors.lastname: Dzantiev
mesh_terms: D052157:Aptamers, Nucleotide / Q000378:metabolism*; D015374:Biosensing Techniques / Q000379:methods*; D005454:Fluorescence Polarization / Q000379:methods*; D007074:Immunoglobulin G / Q000378:metabolism*; D057230:Limit of Detection; D009793:Ochratoxins / Q000032:analysis*; D019809:Streptavidin / Q000378:metabolism*
chemical_list: D052157:Aptamers, Nucleotide; D007074:Immunoglobulin G; D009793:Ochratoxins; C025589:ochratoxin A; D019809:Streptavidin
keywords: Aptamers; Fluorescence polarisation; Ochratoxin A; Protein modules