Ochratoxin B

The aim of this work was to optimize the preparation of an anti-ochratoxin A (OTA) oligosorbent (OS), a solid-phase extraction sorbent based on OTA aptamers covalently immobilized on sepharose. Different syntheses were carried out by modifying the side of the oligonucleotide chain bound to the sepharose, the length of the spacer arm between the aptamer and the sepharose and the amount of the aptamers introduced during the covalent grafting. Indeed, the capacity of OSs prepared using 3'- or 5'-amino-modified sequences with a C6 or a C12 was studied. In the best conditions, the concentration of aptamers sequence used during their grafting was increased and a capacity close to 40 nmol g(-1) of OS was reached. The potential of the resulting OSs was also studied in pure media. For this, their selectivity was checked by comparing them to a control sorbent prepared without immobilizing aptamers. Extraction recoveries close to 100% were obtained on all OSs, while no retention was observed on the control sorbent.

The natural occurrence of ochratoxin A, ochratoxin B, aflatoxin B1, aflatoxin B2, aflatoxin G1 and aflatoxin G2 (OTA, OTB, AFB1, AFB2, AFG1, AFG2) in red wines was investigated by HPLC/FLD after immunoaffinity column clean-up in 57 market samples produced in Sicily (Italy). The results showed a very low incidence of these mycotoxins in analysed samples, confirming the high degree of quality and safety of Sicilian red wines. The results indicated 71.9% and 64.9% positive samples for OTA and OTB respectively, with an average level of 0.13 μg l(-1), well below the European maximum permitted levels (MLs). The aflatoxin most frequently detected in the samples was AFG1, present in 57.9% of samples, while the other aflatoxins were rarely present. Recovery experiments were carried out on eight mycotoxin-free red wines spiked with OTA, OTB, AFB1, AFB2, AFG1 and AFG2 at two different levels. The limits of detection (LODs) in wines were 0.02 µg l(-1) for OTA, 0.

A complete characterization of a novel target-specific DNA aptamer-based miniaturized solid phase extraction (SPE)-sorbent coupled on-line to nanoLC is presented. A miniaturized oligosorbent (mOS) was prepared via the in situ sol-gel synthesis of a hybrid organic-inorganic monolith in 100 μm i.d. capillary columns using tetraethoxysilane and 3-aminopropyltriethoxysilane as precursors, followed by covalent binding of a 5'-amino-modified DNA aptamer with a C12 spacer arm specific for a molecule of small molecular weight. Ochratoxin A (OTA), one of the most abundant naturally occurring mycotoxins, was chosen as model analyte to demonstrate the principle of such an approach. The mOS was coupled on-line to RP-nanoLC-LIF. Selective extraction of OTA on several mOSs was demonstrated with an average extraction recovery above 80 % when percolating spiked binding buffer and a low recovery on control monoliths grafted with a non-specific aptamer. Reproducibility of mOSs preparation was highlighted by comparing extraction yields.

An impedimetric aptasensor for ochratoxin A (OTA) detection has been developed on the base of a gold electrode covered with a new modifier consisting of electropolymerized Neutral Red and a mixture of Au nanoparticles suspended in the dendrimeric polymer Botlorn H30®. Thiolated aptamer specific to OTA was covalently attached to Au nanoparticles via Au-S bonding. The interaction of the aptamer with OTA induced the conformational switch of the aptamer from linear to guanine quadruplex form followed by consolidation of the surface layer and an increase of the charge transfer resistance. The aptasensor makes it possible to detect from 0.1 to 100 nM of OTA (limit of detection: 0.02 nM) in the presence of at least 50 fold excess of ochratoxin B. The applicability of the aptasensor for real sample assay was confirmed by testing spiked beer samples. The recovery of 2 nM OTA was found to be 70% for light beer and 78% for dark beer.