Characterization of oligosorbents and application to the purification of ochratoxin A from wheat extracts

Abstract

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. OS does not demonstrate any cross-reactivity towards OTA metabolites, i.e., ochratoxin B and ochratoxin hydroquinone. The oligosorbent was finally applied to the clean-up of OTA from wheat sample extracts. Extraction recoveries were not affected by matrix interferences and the resulting chromatogram clearly highlights the selectivity of the sorbent that allows the removal of matrix components thus improving the reliability of the quantitation of OTA in real samples.

Recent Methods for Purification and Structure Determination of Oligonucleotides

“…Oligonucleotides of 2–30 m could be separated and purified successfully by anion-exchange chromatography based of the number of charged phosphate groups in the oligonucleotide backbone [10,14,26,27]. Purification of target oligonucleotides depends on their length, while yields of the oligonucleotides are usually sacrificed to obtaining better purity by heart-cutting the component of interest.…”

“…Three 2′-fluoro-modified RNAs they used were F-U 57-nt, F-U 58-nt, and F-UC 58-nt. Their work also made it possible to separate RNA aptamers of similar sizes, but with different chemical modifications, such as uridine isomerization, nucleobase methylations, and hypermodified nucleotides in complex RNA maturation processes [10,43]. …”

“…Additionally, some modifications have been introduced, such as phosphorothioate linkages, incorporating modified nucleosides as 2′-OMe, 2′-OMOE, or 2′-F in the sequence, and attaching polyethylene glycol at the terminus to the structures of oligonucleotides in order to increase stability and bioavailability. However, these modifications also enhanced the challenges for analysis and purification [10,11,12]. …”

Selective tools for the solid-phase extraction of Ochratoxin A from various complex samples: immunosorbents, oligosorbents, and molecularly imprinted polymers

“…This was shown for the extraction of OTA from wine whose ethanol content caused a loss of biotinylated-aptamers, thus forbidding the reusability of the OS [46]. Concerning the covalent immobilization of aptamers, a few sorbents widely used for the covalent grafting of antibodies have been used for the immobilization of OTA aptamers such as CNBr-activated sepharose [46,47] or NHS-activated sepharose [35]. The immobilization of OTA aptamers on diaminodipropylamine agarose was also described [44,48].…”

“…As for ISs, non-specific interactions can occur between OTA and the solid-phase used for the immobilization of aptamers but also with the nucleotide sequence. In order to investigate the retention mechanism of OTA on the OS and to evaluate the risk of nonspecific interactions it is interesting to compare its retention in parallel on a non-bonded sorbent [46,47,50,51], on the sorbent bonded with non-specific aptamers [47] and on the sorbent bonded with a scrambled sequence that contains the same nucleotides than in the OTA aptamer but in a random position (thus preventing the formation of the specific complex with the target analyte) [50]. These types of experiment that are similar to those previously mentioned for ISs (part 1-3) give an indication whether nonselective interactions are mainly due to the solid-phase, to the nucleotides, to the spacer or to the coupling groups.…”