All coastal countries are affected by the proliferation of toxic algae or harmful algal blooms (HABs). Some of these microalgae biosynthesize toxins affecting marine organisms and, when accumulated in shellfish such as bivalves, can cause disease and death in humans after their ingestion. Worldwide as well as in Portugal, increase in the number of HABs, toxins and toxic algal species, as well as economic losses associated with HAB events. Marine are generally classified into 3 distinct groups, depending on the symptoms in humans: diarrheic shellfish toxins (DSTs), paralytic shellfish toxins (PSTs) and amnesic shellfish toxins (ASTs). Monitoring of marine toxins in bivalve is necessary in view of the unpredictability of occurrences of HABs. Surveillance program for these groups of toxins have been implemented in the European Union (EU). In Portugal, monitoring program started in 1986 for PSTs, in 1987 for DSTs and in 1996 for ASTs toxins. Although these toxins have been detected along the entire Portuguese coast, the northwest region, Ria de Aveiro and Algarve are the most problematic with frequent occurrence of contamination.

The routine analyzes of marine toxins in bivalves in Portugal are carried out by the National Reference Laboratory – Portuguese Institute of Sea and Atmosphere. Although the occurrence of PSTs is less frequent than the rest, they are of particular interest due to the severity of the neurological symptoms they cause in humans. Until recently, reference method for the detection of marine toxins was a bioassay in rats, which used the death of animals exposed to toxins as the endpoint in determining toxicity. In addition to the ethical implications of this method, the bioassays in rats have been criticized for having insufficient sensitivity and selectivity. Efforts to replace bioassays by an alternative analytical techniques have resulted in EU approval of Liquid Chromatography with Fluorimetric Detection (LC-FLD) as an alternative method for the detection of PSTs. However, LC-FLD is a laboratory technique that requires costly equipment and specialized personnel. Thus, there is a need to develop alternative analytical assays that are inexpensive and can be used as screening and alarm tools. Sensors and immunoassays are the best candidates for the development of such tool, and promising results have been already reported.

This project proposes the development of a bioelectronic language based on chemical sensors and biosensors for the rapid detection of PSTs, focusing exclusively on the toxins detected on the Portuguese coast. The electronic tongue consists of an array of sensors with cross-sensitivity, a measuring device and a data processing tools. Electronic tongue combines the advantages of sensors such as rapid analysis, simple instrumentation and relatively low cost with improved analytical characteristics. Successful applications of the electronic tongues for the quantification of organic and inorganic compounds have been extensively reported by project participants.

Application of the electronic tongue to the detection of marine toxins would improve management of bivalve cultivation and harvesting, mainly in remote and / or offshore areas, reduce costs of negative sample analysis in monitoring and reduce risks of supplying contaminated bivalves for human consumption.

 

Main results

The main objective of the project LinguaTox was development of a prototype of the bioelectronic tongue for the detection of paralytic shellfish toxins (PSTs) in bivalves. Special attention has been paid to the PSTs commonly detected in bivalves from the Portuguese coast such as decarbamoyl and sulfocarbamoyl toxins. The following results were achieved:

1. Set of potentiometric chemical sensors with plasticized polyvinylchloride membranes with sensitivity to PSTs was manufactured. Developed sensors possessed different patterns of sensitivity and selectivity to four studied PSTs and thus could be used in the electronic tongue for toxins’ quantification.

2. Enzymatic assay with potentiometric detection based on carbamoylase extracted from surf clam S. Solida was developed. Enzymatic assay allowed quantification of two PSTs, namely saxitoxin and gonyautoxin V (GTX5).

3. High input impedance digital voltmeter for potentiometric measurements with 16 channels was developed. Measurement controls software based on the WEB browser together with WEB service permitting remote control of the measurements was implemented.

4. Potentiometric sensor array and enzymatic assya have been applied to the quantification of PSTs in the extracts of contaminated musses.Obtained results were in agréments with the reference method – Liquide Chromatography with Fluorimetric Detection.

Dissemination of the project results was done through presentation at sceintific meetings and conferences including:

     - Rudnitskaya, LínguaTox – Bioelectronic tongue for the detection of marine toxins in bivalves, Forum de Mar, 28 a 30 de Maio de 2014, Matosinhos, Portugal.

     - Rudnitskaya, A.M.S. Costa, I. Delgadillo, Calibration update strategies for an array of potentiometric chemical sensors, In the Proc. of the International Symposium on Olfaction and electronic Noses – ISOEN, June 28 – July 1, 2015, Dijon, France, #143.

     - Rudnitskaya, N.S. Ferreira, M.T.S.R. Gomes, Chemical sensor array for the detection of marine toxins. In the Proc. of the Euroanalysis XVIII, September 6-10, 2015, Bordeaux, France.

     - M.I.C. Raposo, M.T.S.R. Gomes, M.J. Botelho, S.T. Costa, C. Vale, A. Rudnitskaya, Carbamoylase-based biosensor for the detection of shellfish poisoning toxins, In the Proc. of Ibersensor – X Congresso Iberamericano de Sensores, October 26-28, 2016, Valparaiso, Chile. 

CESAM members on this project

Alisa Rudnitskaya
Coordinator

João Oliveira
Researcher

Maria Teresa Seabra dos Reis Gomes
Researcher

Mariana Isabel Cordeiro Raposo
Researcher

Newton Carlos Marcial Gomes
Researcher

Ricardo Jorge Guerra Calado
Researcher