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.

Ochratoxin A (OTA) is a mycotoxin produced as a secondary metabolite by several species of Aspergillus and Penicillium and frequently found as a natural contaminant in a wide range of food commodities. Novel and robust biorecognition agents for detecting this molecule are required. Aptamers are artificial nucleic acid ligands able to bind with high affinity and specificity to a given target molecule. In the last few years, three separate research groups have selected aptamers for ochratoxin A. While each of these three families of aptamers have been incorporated into various methods for detecting OTA, it is unclear if each aptamer candidate is better suited for a particular application. Here, we perform the first head-to-head comparison of solution-based binding parameters for these groups of aptamers. Based on our results, we provide recommendations for the appropriate choice of aptamer for incorporation into solution-based biorecognition assays and applications.

Graphical abstract

Aptamers are single-stranded oligonucleotides with the ability to bind tightly and selectively to a target analyte. High-affinity and specific aptamers for a variety of mycotoxins have been reported over the past decade. Increasingly, these molecular recognition elements are finding applications in biosensors and assays for the detection of mycotoxins in a variety of complex matrixes. This review article highlights the mycotoxin aptamers that are available for mycotoxin detection and the array of biosensing platforms into which they have been incorporated. Key advantages that aptamers have over analogous technology, and areas in which these advantages may be applied for the benefit of practical mycotoxin detection, are also discussed.

Both naturally occurring and synthetic small molecules play a significant role in a variety of applications. For example, small molecule food contaminants that occur infield or postharvest are of importance, both agriculturally and economically. Mycotoxins are toxic secondary metabolites produced by filamentous fungi that can potentially contaminate a variety of foodstuffs. Detection of these small molecules is required as mycotoxins pose multiple health-risks and proceed past processing and food safety.Current detection methods are expensive, time consuming and unavailable for on-site detection leaving an unmet need for alternative methods of detection.Aptamers are single stranded oligonucleotides that can bind to specific target molecules with high selectivity and affinity. Emerging as molecular recognition agents, aptamers can be used in a number of novel detection methods for small molecule quantification and analysis. Aptamers are selected through an in vitro process known as Systematic Evolution of Ligands by Exponential Enrichment (SELEX). Aptamers can be adsorbed onto the surface of citrate-capped AuNPs to serve as the molecular recognition element of a rapid colourimetric biosensor assay. Based on this interaction, AuNPs could potentially serve as a novel platform for small molecule SELEX. Although molecular recognition of small molecules is of great interest, selection of aptamers for small molecules has proven to be a challenge. Furthermore, not all of these reported small molecule aptamers can be easily incorporated in the AuNP bioassay. Selecting for aptamers in a manner that will mimic established AuNP biosensor conditions provides a number of advantages compared to traditional SELEX. As a first step towards establishing a AuNP SELEX platform, we evaluated the partitioning of mycotoxin iv aptamers that remain on the AuNP surface from aptamers-target complexes in solution.Having uncovered several challenges associated with AuNPs as a SELEX partitioning strategy, we next synthesized, optimized, and characterized core-shell gold coated magnetic nanoparticles (Fe3O4-AuNPs), which were phase-transferred aqueous solution.We demonstrated that Fe3O4-AuNPs could function as an improved AuNP SELEX platform and provide a novel method to study ssDNA aptamer-AuNP non-specific interactions. Finally, our studies on the adsorption and separation of ssDNA aptamers using Fe3O4-AuNPs highlight future opportunities for novel aptamer biomedical applications and other biosensor development.v