Development of Immunosensor for Early Detection of Aquatic Harmful Algal Blooms (HABs)

Abstract

Blooms of toxic and harmful microalgae, known as "red tides", represent a significant and tremendous threat to human health and fisheries resources throughout the Southeast Asia region and the world. A harmful microalga which produces secondary metabolite known as saxitoxin (STX) is a neurotoxin produced by a variety of genera of dinoflagellates. The ingestion of this toxin through contaminated shellfish will inhibit the neuron depolarizations and action potentials which result in respiratory failure in human and known as PSP (paralytic shellfish poisoning). PSP takes effect as soon as five minutes depending on the species and the concentration consumed. Currently, the identification of dinoflagellates cells is done by taxonomy, which is based on a broad expertise of specially trained staff, expensive equipment like electron microscopes and is very time consuming. In many countries, for the toxin monitoring, they use mouse bio-assay which is bulky, expensive, loss of animal life and high variability and higher limit of detection (40 µg per 100 g meat) and HPLC methods required many pre-treatment steps for the analysis. Therefore, in order to overcome this problem, development of early detection of harmful algae bloom and bio-toxin in aquatic ecosystem is needed. The detection is based on screen-printed carbon working electrode with onboard carbon counter and silver-silver chloride pseudo-reference electrode. A direct ELISA (enzyme-linked immunosorbent assay) format was developed and optimized on the surface of a carbon screen-printed working electrode by immobilizing the capture antibody using electro-deposition of gold nanoparticles conjugated with polyclonal anti-toxin antibodies. The detection reagent, toxin-HRP (horseradish peroxidase) conjugate was used as an enzyme label. Electrochemical detection was then carried out using 3,3',5,5'-tetramethylbenzidine dihydrochloride (TMB)/H 2 O 2 as the enzyme mediator/substrate system and conducted using chronoamperometry at 100 mV vs. onboard screen-printed Ag-AgCl pseudo-reference electrode. The minimum detection limits for harmful micro algae about 1 cell per mL. The immunosensor is very selective which is able to detect Alexandrium minitum with 100% selectivity and minimal cross reaction with others nontoxic algae (< 2%).