Nucleic acid aptamers are versatile molecular recognition agents that bind to their targets with high selectivity and affinity. The past few years have seen a dramatic increase in aptamer development and interest for diagnostic and therapeutic applications. As the applications for aptamers expand, the need for a more standardized, stringent, and informative characterization and validation methodology increases. Here we performed a comprehensive analysis of a panel of conventional affinity binding assays using a suite of aptamers for the small molecule target ochratoxin A (OTA). Our results highlight inconsistency between conventional affinity assays and the need for multiple characterization strategies. To mitigate some of the challenges revealed in our head-to-head comparison of aptamer binding assays, we further developed and evaluated a set of novel strategies that facilitate efficient screening and characterization of aptamers in solution. Finally, we provide a workflow that permits rapid and robust screening, characterization, and functional verification of aptamers thus improving their development and integration into novel applications.

Fumonisins are mycotoxins produced by Fusarium verticillioides and F. proliferatum, fungi that are ubiquitous in corn (maize). Insect damage and some other environmental conditions result in the accumulation of fumonisins in corn-based products worldwide. Current methods of fumonisin detection rely on the use of immunoaffinity columns and high-performance liquid chromatography (HPLC). The use of aptamers offers a good alternative to the use of antibodies in fumonisin cleanup and detection due to lower costs and improved stability. Aptamers are single-stranded oligonucleotides that are selected using Systematic Evolution of Ligands by EXponential enrichment (SELEX) for their ability to bind to targets with high affinity and specificity. Sequences obtained after 18 rounds of SELEX were screened for their ability to bind to fumonisin B₁. Six unique sequences were obtained, each showing improved binding to fumonisin B₁ compared to controls. Sequence FB₁ 39 binds to fumonisin with a dissociation constant of 100 ± 30 nM and shows potential for use in fumonisin biosensors and solid phase extraction columns.

A liquid chromatographic (LC) method for the determination of fumonisins B₁ (FB₁) and B₂ (FB₂) in corn and corn flakes was collaboratively studied by 23 laboratories, which analyzed 5 blind duplicate pairs of each matrix to establish the accuracy, repeatability, and reproducibility characteristics of the method. Fumonisin levels in the corn ranged from <0.05 (blank) to 1.41 μg/g for FB₁ and from <0.05 to 0.56 μg/g for FB₂, whereas in the corn flakes they ranged from <0.05 to 1.05 μg/g for FB₁ and from <0.05 to 0.46 μg/g for FB₂. The method involved double extraction with acetonitrile–methanol–water (25 + 25 + 50), cleanup through an immunoaffinity column, and LC determination of the fumonisins after derivatization with o-phthaldialdehyde. Relative standard deviations for the within-laboratory repeatability (RSD_r) of the corn analyses ranged from 19 to 24% for FB₁ and from 19 to 27% for FB₂; for the corn flakes analyses, RSD_r ranged from 9 to 21% for FB₁ and from 8 to 22% for FB₂. Relative standard deviations for the between-laboratories reproducibility (RSD_R) of the corn analyses ranged from 22 to 28% for FB₁ and from 22 to 30% for the FB₂; for corn flakes analyses, RSD_R ranged from 27 to 32% for FB₁ and from 26 to 35% for FB₂. Mean recoveries of FB₁ and FB₂ from corn spiked with FB₁ at 0.80 μg/g and with FB₂ at 0.40 μg/g were 76 and 72%, respectively; for corn flakes spiked at the same levels recoveries were 110 and 97% for FB₁ and FB₂, respectively. HORRAT ratios for the analyses of corn ranged from 1.44 to 1.53 for FB₁ and from 0.96 to 1.48 for FB₂, whereas for corn flakes they ranged from 1.60 to 1.82 for FB₁ and from 1.39 to 1.68 for FB₂.

Natural toxins include a wide range of toxic metabolites also occurring in food and products, thus representing a risk for consumer health. In the last few decades, several robust and sensitive analytical methods able to determine their occurrence in food have been developed. Liquid chromatography mass spectrometry is the most powerful tool for the simultaneous detection of these toxins due to its advantages in terms of sensitivity and selectivity. A comprehensive review on the most relevant papers on methods based on liquid chromatography mass spectrometry for the analysis of mycotoxins, alkaloids, marine toxins, glycoalkaloids, cyanogenic glycosides and furocoumarins in food is reported herein. Specifically, a literature search from 2011 to 2021 was carried out, selecting a total of 96 papers. Different approaches to sample preparation, chromatographic separation and detection mode are discussed. Particular attention is given to the analytical performance characteristics obtained in the validation process and the relevant application to real samples.