Sensitive, Quantitative, and Portable Anatoxin Assay using Aptamers and Quantum Dot Nanoshell Reporting

This proposed project directly addresses an U.S. Environmental Protection Agency (EPA) calling for improved detection and measurement techniques including screening kits for algal neurotoxins (especially cyanobacterial anatoxin) and cytotoxins in drinking water systems. Bloom-forming cyanobacteria have been observed in water bodies, including drinking water reservoirs all over the world. Several strains of these microorganisms have the ability to produce potent toxins as secondary metabolites, the so-called cyanotoxins. These have caused many animal deaths and also have been implicated in cases of human illness in the United States, Australia, China, Brazil, and Great Britain. According to their effects on health, cyanotoxins are divided into hepatotoxins (liver damaging), neurotoxins (nerve damaging; anatoxin-a, saxitoxins), and dermatotoxins (skin damaging). More recent evidence suggests that algal toxins may be linked to diseases similar to Alzheimers even years after exposure, and toxins can be easily aerosolized, increasing their potency and range of effect. In addition to concern over their natural occurrence in water sources as some of the most potent biotoxins known, cyanotoxins have low-level acute toxicity that is comparable to the toxicity levels from conventional chemical weapons and, therefore, warrant consideration as a potential threat from intentional misuse (bioterrorism). Anatoxin-a is a particularly potent, naturally occurring toxin produced by some strains of Anabaena (particularly Anabaena flos-aquae) and at least four other genera of freshwater cyanobacteria (commonly referred to as blue-green algae), including Aphanizomenon, Microcystis, Planktothrix, and Oscillatoria. The focus of this research project is to develop a rapid assay kit for cyanobacterial anatoxins using aptamers as the primary molecular recognition element. The developed sensor chemistries will result in a detectable signal in 20 minutes or less and will require a very inexpensive, portable solid state detector system for quantitative test interpretation (readout). BioTex will first develop aptamers for highly sensitive and specific binding to anatoxins and then optimize the composition of a fluorescence resonance energy transfer (FRET)-based sensor employing quantum dot (QD) fluorescence reporters and aptamers labeled with quenching chromophores. Finally, BioTex will demonstrate the sensitivity and specificity of the novel sensing chemistry for field and laboratory use.