The Center for Alternatives to Animal Testing is an academic center affiliated with the Division of Toxicological Sciences in the Department of Environmental Health Sciences of the Johns Hopkins University Bloomberg School of Public Health.

 

Johns Hopkins School of Public Health

Commemorative Booklet for CAAT 20th Anniversary Symposium

Development of Enzymatic Inhibition Bioelectrodes for the Direct Determination of Phycotoxins

Francesco Botré, Cecilia Bartuli, Elisabetta Podestá, and Franco Mazzei
University "La Sapienza", Italy

This research program is aimed at developing biosensor-based analytical methods for the analysis of algal toxins (phycotoxins) in mussels. These new methods may offer an effective alternative to the commonly applied "global toxicity tests" -- i.e., highly invasive bioassays on mammals -- that currently are the only internationally recognized methods for the screening of these toxins in seafood.

Enzymatic inhibition biosensors (EIBs), a form of response-based analytical device, represent a good compromise between purely "biological" and "physico-chemical" techniques. EIBs can be used as "artificial bioindicators," partially combining the flexibility of a biological effect-based test and the selectivity of a physico-chemical assay.

The screening analysis to detect contamination of seafood by phycotoxins is carried out by "feeding the biosensor" and monitoring the alteration of a specific biochemical parameter, rather than by "feeding the mouse" and following the development of generic toxic effects. The biological parameter to be monitored is the rate of an enzyme-catalyzed reaction that takes place at the sensor interface. Different enzymatic systems have been selected preliminarily as potential biological targets of diarrhetic shellfish poisonous (DSP) toxins.

Two families of EIBs already have been realized: traditional, renewable lab-scale sensors, tested as prototypes, and disposable and/or partially disposable screen-printed sensors. The analytical performance of a bienzymatic, lab-scale prototype, set up for the direct determination of algal toxins belonging to the DSP group, has been evaluated on artificially contaminated and naturally contaminated mussels. The reliability of the method also has been verified by comparison with other testing techniques and methods.

A monoenzymatic bioelectrode is being developed, since the presence of only one enzyme on the sensor would drastically simplify the enzyme immobilization process, improve the stability of the sensor and prolong its lifetime of operation. Bienzymatic, disposable screen-printed sensors are under investigation, and their analytical performance is being compared with that of their lab-scale prototypes. The final goal of the research program is to propose the use of screen-printed biosensors for timely monitoring of risky areas and possibly also for ship-board operations.