Detection of Anatoxin-a(s) in Environmental Samples of Cyanobacteria by Using a Biosensor with Engineered Acetylcholinesterases

Bioassays are little used to detect individual toxins in the environment because, compared to analytical methods, these assays are still limited by several problems, such as the sensitivity and specificity of detection. We tentatively solved these two drawbacks for detection of anatoxin-a(s) by engineering an acetylcholinesterase to increase its sensitivity and by using a combination of mutants to obtain increased analyte specificity. Anatoxin-a(s), a neurotoxin produced by some freshwater cyanobacteria, was detected by measuring the inhibition of acetylcholinesterase activity. By using mutated enzyme, the sensitivity of detection was brought to below the nanomole-per-liter level. However, anatoxin-a(s) is an organophosphorous compound, as are several synthetic molecules which are widely used as insecticides. The mode of action of these compounds is via inhibition of acetylcholinesterase, which makes the biotest nonspecific. The use of a four-mutant set of acetylcholinesterase variants, two mutants that are sensitive to anatoxin-a(s) and two mutants that are sensitive to the insecticides, allows specific detection of the cyanobacterial neurotoxin.