Development of a haemolytic–enzymatic assay with mediated amperometric detection for palytoxin analysis: application to mussels

An electrochemical sensor for palytoxin (PlTX) detection, based on a strip of eight screen-printed electrodes connected to a cost-effective and portable apparatus, is reported. Sheep erythrocytes were used to test the palytoxin detector and degree of haemolysis was evaluated by measuring release of the cytosolic lactate dehydrogenase (LDH). Percentage haemolysis and, therefore, the amount of LDH measured, by use of NADH/pyruvate and appropriate electrochemical mediators, was correlated with the concentration of the toxin. Two different electrochemical approaches were investigated for evaluation of LDH release, but only one based on the use of a binary redox mediator sequence (phenazine methosulfate in conjugation with hexacyanoferrate(III)) proved useful for our purpose. After analytical and biochemical characterization, the sensor strip was used to measure palytoxin. Sheep blood and standard solutions of PlTX were left to react for two different incubation times (24 h or 4 h), resulting in working ranges of 7 × 10−3–0.02 ng mL−1 and 0.16–1.3 ng mL−1, respectively. The specificity of the test for palytoxin was evaluated by use of ouabain, which acts in the same way as PlTX on the Na+/K+-ATPase pump. A cross-reactivity study, using high concentrations of other marine biotoxins was also conducted. Experiments to evaluate the matrix effect and recovery from mussels are discussed.