Novel bioanalytical tests and biosensor systems for detection of cyanide traces in marine fish

Sodium cyanide has been used in the Philippines to collect tropical marine fishes for aquarium and food trades since the early 1960s. Cyanide fishing is a fast method to stun and collect fishes. By some estimates, more than a thousand tonnes of cyanide have been poured into the waters of the Philippines by fishermen. This practice is damaging the coral reefs irreversibly. In most countries cyanide fishing is illegal, but most of the exporting and importing countries do not have test and certificate systems. Many analytical methods are available for the detection of cyanide in environmental and biological samples. However, most of the techniques are time-consuming, and some lack specificity or sensitivity. Besides, an ultra sensitive cyanide detection method is needed due to the rapid detoxification mechanism in fish. This thesis presents a novel approach to detect cyanide in marine fishes. The detection was achieved by incorporating a cyanide-degrading enzyme, cyanide hydratase (E.C. 4.2.1.66), into a multi-purpose biosensor based on cofactor recycling. The principle is that cyanide hydratase catalyzes the hydration of metalcyano complex to form formamide. Then formamide is converted by formamidase (E.C. 3.5.1.49) into formate and ammonia. In the biosensor formate dehydrogenase (E.C.1.2.1.2) converts formate into CO2 using NAD+. Corresponding NADH produced is oxidized back to NAD+ by salicylate hydroxylase (E.C. 1.14.13.1) in the presence of salicylate and oxygen. The oxygen consumption, which is proportional to cyanide concentration, is monitored with a Clark-electrode. It has a linear detection range of 3 - 30 μM cyanide (0.078 - 0.78 ppm). The advantages of this bioanalytical approach are: the cyanide-degrading enzymes convert the toxic cyanide into a non-toxic product which is then detected in the biosensor, the enzymes that have been used are highly specific, no vigorous sample pre-treatment (reflux distillation) is needed, and no hazardous chemical is used during the analyses