DNA-aptamers: innovative binding materials for mycotoxins analysis.

Aptamers are synthetic oligonucleotides that are mainly produced using a procedure known as SELEX (Systematic Evolution of Ligands by EXponential enrichment) which does not require the use of animals and offers a greater degree of control with respect to binding conditions. Their ability to fold into distinct tertiary structures forms the basis for target recognition, even in the case of very closely related structures. Based on their characteristics, aptamer-based technologies are becoming a promising and convenient alternative to antibodies for the detection of contaminants in foods. Aptamers have been produced for several targets, including mycotoxins, e.g. ochratoxin A (OTA), fumonisin B1 and aflatoxin B1. One of the reported DNA aptamers with high affinity and specificity to OTA was used as oligosorbent for the preparation of aptamer-based solid phase extraction (SPE) columns. Once standardising the procedure for their preparations, SPE columns were successfully used in combination with high performance liquid chromatography and fluorescence detection (HPLC-FLD) for the analysis of OTA in wheat. Columns showed equivalent performance to relevant immunoaffinity columns (IACs). Average recoveries from wheat samples spiked at levels of 0.5-50 µg/kg ranged from 74% to 88% (relative standard deviation <6%). The quantification limit was 77 pg/g, far below the EU regulatory limits for OTA in unprocessed wheat (5.0 µg/kg). These findings were potentially useful to develop optimized DNA-aptamer SPE columns and bring the technology to commercial readiness. As a follow-up, aptamer-SPE columns were used for the first time in combination with a novel DNA-ligand system for high throughput OTA analysis in wheat. The technology was based on the measurement of Time Resolved-Fluorescence Resonance Energy Transfer (TR-FRET) response of OTA-terbium-aptamer interaction. The entire procedure was performed in less than 30 min, including sample preparation, and allowed analysis of several samples simultaneously with a 96-well microplate reader. Average recoveries from wheat samples spiked at 2.5-25 µg/kg OTA ranged from 72% to 81% (relative standard deviation <6%) with a quantification limit of 0.5 µg/kg. Comparative analyses of 29 naturally contaminated (up to 14 µg/kg) wheat samples by aptamer-SPE columns/TR-FRET or IACs/HPLC-FLD showed a good correlation (r = 0.985) in the tested range. The trueness of the aptamer-based method was additionally confirmed by analysis of two quality control wheat materials for OTA. The DNA-ligand system is innovative, simple, and rapid, and can be used to screen large quantities of samples for OTA contamination at levels below the EU regulatory limit with analytical performances satisfying EU criteria for method acceptability.