ИСТИНА

Aptamers are single strand oligonucleotide which can bind different compounds with high affinity and selectivity. They are promising receptors for detection of contaminants in food. The aim of the study was to develop amplification strategy for fluorescence polarisation (FP) aptamer assays, based on anchor attaching to aptamer receptor and increasing differences of sizes for bound and unbound labeled antigens. The strategy was realized for FP assays that used either native aptamers or their complexes with anchor construction. Ochratoxin A (OTA) was chosen as target analyte. ОТА is an ubiquitous toxic contaminant of various crops and food stuffs of plant origin, such as wheat, rye, oats, nuts, dried fruits, wine etc. Thus, there is a great demand for simple and rapid methods to detect OTA. The principal feature of the study is the application of aptamer complexes with proteins and gold nanoparticles (GNPs) to enhance the FP signal of the OTAfluorophore conjugate being bound with the aptameric receptor. 3´-biotinylated derivative of a OTA-specific aptamer with the sequence 5´-GAT-CGGGTG- TGG-GTG-GCG-TAA-AGG-GAG-CAT-CGG-ACA-3’(11.7 kDa) being proposed by Cruz- Aguado A. et al. (J. Agric. Food Chem. 2008, 56, 10456) was used in the study. Antiparallel quadruplex structure (necessary for OTA binding) of the biotinylated aptamer was confirmed by circular dichroism spectroscopy. The proposed FP assay is based on the competition between free OTA (contained in sample) and fluorescently labeled OTA for limited binding sites of aptamer receptor. The fluorophorelabeled OTA derivative was synthesized by carbodiimide coupling with 4´- (aminomethyl)fluorescein and purified by thin layer chromatography. The mixture of the FP assay was irradiated by plane-polarized light with λex 480 nm, and the emission was measured at λem 535 nm. The depolarization degree of registered fluorescence depends of free/bound state ratio of labeled OTA which allows determining OTA concentration in the tested sample. Use of proteins (streptavidin and streptavidin – IgG complex) as anchors in FP assay was studied. The FP assays of OTA based on free aptamer and aptamer included in the protein complexes were compared. Thus, the substitution of free aptamer by aptamer – streptavidin – IgG complex allows 40-fold reducing the limit of detection from 130 (53.1 μg/kg) to 3 nM (1.2 μg/kg). The aptamer – streptavidin complex causes only 3-fold reduction of the limit of detection as compared with free aptamer only three times. The assay time is 15 min. As the second variant of the anchor the GNPs were used. GNPs with diameter of 7.5 nm (data of transmission electron microscopy) were synthesized. To obtain the aptamer – GNP conjugate, the GNP surface was modified with streptavidin by physical adsorption, and then the biotinylated aptamer was bound. The use of the aptamer – GNP conjugate leads to reaching the detection limit equal to 5 nM (2 μg/kg). This is 26 times lower than in the FP assay without the anchor. The assay time is 5 min. - 195 - Comparison of the anchors shows that FP assay with GNPs is faster but FP assay with streptavidin – IgG is more sensitive. The system based on aptamer – streptavidin – IgG receptor was chosen for OTA detection in white wine, taking into consideration the maximal residue limits for OTA in wine in European Union and Russia Federation (2–5 μg/kg). The limit of OTA detection in white wine for the developed assay is 2.8 nM (1.1 μg/kg). Thus, the proposed technique is rapid, sensitive and easy to use which make it a promising tool for food safety control. The study shows enhanced assay sensitivity as a result of the increased size of aptamer receptors due to significant decrease of rotation mobility of the analyte–fluorophore conjugate bound by aptamer. The suggested anchor strategy is efficient for application in FP assay for detection of different low-molecular weight compounds.