Abstract

The use of synthetic materials as alternative to antibodies are increasingly being investigated and proposed for use in mycotoxin analysis. Aptamers are single-stranded oligonucleotides that are mainly selected using SELEX (Systematic Evolution of Ligands by EXponential) enrichment for their ability to bind targets with high affinity and specificity. A DNA aptamer with high affinity and specificity towards OTA was produced and preliminarily used as an oligosorbent for the preparation of affinity columns. Recently, an optimised procedure for the preparation of aptamer-SPE (solid phase extraction) columns has been described and successfully applied to the quantitative determination of OTA in unprocessed wheat at levels below the EU maximum permitted level. The effect of different parameters, such as oligosorbent volume, column size and breakthrough volume (maximum eluting solvent) has been tested. The SPE columns, packed with 300 µl oligosorbent (24 nmol aptamer), showed a linearity of the dose-response curve in the range of 0.4-500 ng OTA and were used for the clean-up of durum wheat extracts prior to OTA determination by HPLC-FLD (high performance liquid chromatography and fluorescence detection). OTA mean recoveries (from 74 to 88%) from wheat samples spiked at levels from 0.5 to 50 ng/g fulfilled EU requirements for acceptability of the analytical method. Limit of quantification of the method was 77 pg/g, far below the EU regulatory limits for OTA in unprocessed wheat (5.0 ng/g). The comparative HPLC-FLD analyses of 33 naturally contaminated durum wheat samples cleaned-up on both aptamer-SPE and IMA (immunoaffinity) columns showed a good correlation. Furthermore, aptamer-SPE columns could be re-used up to five times without any loss of performance.Recently, the selection of aptamer sequences binding for fumonisin B1 (FB1) has been also described. Sequences obtained after 18 rounds of SELEX were screened for their ability to bind to FB1. Six unique sequences were obtained, each showing improved binding to FB1 compared to controls. Between them, the sequence containing the lowest guanosine content (only 8%) yielded the highest binding to FB1 with a dissociation constant of 100 ± 30 nM. Experiments to identify the minimal target-binding sequence to shorten the aptamer and improve binding are in progress.The successful outcome of this research provides evidence that DNA aptamers can be used to replace antibodies as binding reagents in diagnostic assays with commercial application for an economically important mycotoxin analysis

Autore Pugliese

• De Girolamo Annalisa , Schena Roberto

Tutti gli autori

• De Girolamo A.; Schena R.; McKeague M.; DeRosa M.C.; Miller J.D.; Visconti A.

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Anno di pubblicazione

2011

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