Aptamer Development for mycotoxin detection in grains

Principal Investigator: Richard Manderville

Scientific Summary:

Mycotoxins are a major health threat to humans and animals and cause substantial economic losses. Aptamers are single-stranded DNA or RNA that bind a variety of targets, including small molecules, proteins, and cells, with high affinity and specificity. Aptamers are attractive for developing low-cost, robust sensors for mycotoxin detection. Compared to antibody-based technologies for mycotoxin detection, aptamers are much cheaper to manufacture, are more stable, and can be re-used repeatedly. However, antibodies are comprised of proteins, and have twenty amino acid building blocks, which provide great chemical diversity and specificity for its target; while DNA aptamers have only four building blocks called bases, which limits chemical diversity and specificity for its target. The DNA bases also lack diagnostic features that can be used for detection of target binding. Despite these limitations in DNA bases, DNA aptamers have been used commercially to detect mycotoxins, such as ochratoxin A (OTA) and aflatoxin B1.

A goal of our research was to generate modified DNA bases that can provide a unique florescent signal when the aptamer binds the mycotoxin. Fluorescent aptamers (aptasensors) have been shown to be effective for rapid testing for mycotoxins and show great promise for use commercially in a variety of detection platforms. The Manderville laboratory has synthesized fluorescent DNA bases that can be readily incorporated into DNA aptamers. These bases can be manipulated to provide a fluorescent signal when the mycotoxin binds to the aptasensor and can be used to determine the binding site of the mycotoxin. The change in florescence intensity of the fluorescent probe within the aptamer can be used to detect the amount of mycotoxin in a given sample. Efforts of this project were focused on optimizing aptasensors that bind to OTA and aflatoxin B1 for detection in the field. The modified DNA bases will then serve as new tools for aptasensor development for a wider range of mycotoxins that contaminate grain products.     

The long-range goal of this research is to develop a cheap hand-held device for OTA and aflatoxin detection in the field. Thus, we investigated the development of emissive aptasensor that undergo excitation with visible light. This would significantly reduce the cost of a hand-held device due to the reduced costs of a visible light source compared to a UV light source. In the first year of the project we were focused on the development of BODIPY dyes (BODIPY, abbreviation for boron-dipyrromethene), which is a class of fluorescent dyes, for our intended purpose. These dyes proved to be difficult to synthesize, purify and were not compatible with the DNA synthesis conditions for dye incorporation into the DNA aptamer. At the same time the laboratory was focused on the synthesis of C8-aryl-2’-deoxyguanosine triphosphates for enzymatic incorporation into DNA aptamers. The triphosphates also proved difficult to synthesize and turned out to be poor substrates for enzymatic incorporation into DNA using commercially available DNA polymerases. We then focused on the synthesis of cyanine-type dyes and have enjoyed much success. These dyes turned out to be relatively easy to prepare, purify and incorporate into DNA aptamers and emit light in the visible light range. We have now worked out the chemical synthesis of cyanine-dye analogs and are moving forward with their incorporation into OTA and aflatoxin aptasensor.