Advances in the development of biosensors for mycotoxins in Latin-America

H. FERNÁNDEZ

Carlos Paz, Córdoba

Conferencia; Myco-globe Argentina 2006 ? Conference; 2006

International Society of Micotoxins

Mycotoxins are organic compounds produced by fungi as secondarymetabolites. The ingestion of mycotoxins can become a serious human health risk,with carcinogenic, teratogenic and mutagenic effects. The agriculturalcommodities, particularly, a wide variety of crops, both in the field and postharvest,are the main source of production of mycotoxins in the food chains forhumans and animals. Mycotoxins are normally found in low concentrations, whichmake difficult their detection [1, 2].At present, different chromatographic techniques have been used for theirdetermination. However, considering the great worldwide attention that foodindustry is paying to the food quality control, it is very important to develop fast andeconomical new techniques for mycotoxin determinations [2, 3].Although biosensors development for mycotoxin detection has been initiatedin Europe and United Stated of North America some years ago, few studies havebeen done in Latin America so far. Particularly, the Electroanalytical Group of theUniversidad Nacional de Río Cuarto has begun to study the electrochemicalbehaviour of some electroactive mycotoxins of Alternaria alternata, Aspergillus andFusarium genus with the purpose of generating the bases and knowledge for thedevelopment of analytical methodology supported in electroanalytical techniques.Thus, different electroanalytical methodologies have been proposed, mainly thosebased on potential pulse techniques, for the determination of mycotoxins from bulksolution as well as from adsorbed analytes onto the surface of different electrodematerials. Limits of detection in the order of ppb have been found throughadsorptive methods [4 ? 9].Recently, some studies performed in our laboratory have shown that it ispossible to obtain a considerable improvement in the AME electrochemical signalwhen its oxidation is accomplished on dodecanethiol self-assembled monolayerson gold electrodes. Higher sensitivity and lower detection limits are obtainedthrough these nano materials when compared with electrochemistry onconventional electrodes. Analytical values similar to those of the chromatographictechniques were obtained [10].The adsorptive accumulation of a related toxin, cercosporin (CER)phytotoxin, on glassy carbon (GC) electrodes from 1 M HClO4 aqueous solutionshas been used to detect and quantify CER isolated from infected leaf tissuecollected from field-grown peanut in five locations in southern regions of theProvinces of Córdoba and San Luis, Argentina. The adsorptive stripping squarewave voltammetry (ASSWV) is employed to perform the quantitative determinationof CER in naturally infected extracts [11, 12].The application of electroanalytical techniques to detect and quantifyzearalenone (ZEA) mycotoxin that frequently contaminates maize and foodstuffwas also studied. The electro-oxidation of ZEA adsorbed on the surface of glassycarbon (GC) electrodes in 20% acetonitrile (ACN) + 80% 1 M HClO4 (aqueoussolution) reaction medium was studied by using square-wave voltammetry (SWV).The ZEA quantitative determination was performed by SWV combined with thestandard addition method. Detection limit of 30ppb at a signal to noise ratio of 3: 1was obtained [13].On the other hand, we have proved that the affinity of mushroom tyrosinasefor alternariol (AOH) and alternariol monomethyl ether (AME), mycotoxins of theAlternaria alternata genus, is verified both spectroscopically and electrochemically.Commercial enzyme and fresh mushroom tissue were employed forelectrochemical measurements. The results obtained demonstrate, for the firsttime, that both AME and AOH are substrates for mushroom tyrosinase. Theanalytical performance was also evaluated in the detection limits 2.4x10-5 and1.9x10-5 M for AME and AOH, respectively, with a linear range up to 1.8x10-4 and2.0x10-4 M for the same analytes. It was concluded that the enzyme has relativelygood affinities for both substrates, which is a promising result for developing anenzymatic biosensor [14].Very recently, we have also demonstrated that AME and AOH are alsosubstrates of the horse radish peroxidase (HRP) enzyme. UV-vis absorptionspectra of mycotoxins were studied in hydrogen peroxide solution with and withoutthe enzyme. These studies showed that, in effect, the enzyme catalysed theoxidation of both mycotoxins. Actually, studies are directed to find the optimumexperimental conditions for the enzymatic reaction [15].A direct, competitive electrochemical enzyme-linked immunosorbent assay(ELISA) has been developed in cooperation between an Italian Group and anArgentinean researcher for the quantitative determination of ochratoxin A (OTA)using polyclonal antibodies. The assay was carried out on carbon-based screenprinted electrodes (SPE). Optimisation of the ELISA competitive conditions allowedto realise an assay with improved analytical behaviour compared to the classicalspectrophotometric ELISA based assay. The performance was comparable to apublished monoclonal based assay. The method appears to be suitable for OTAcontamination screening in food samples [16].Besides, a methodology for the determination of zearalenone mycotoxinthrough an electrochemical immunosensor (made of a zearalenone rabbitpolyclonal antibody physically adsorbed on the surface of glassy carbonelectrodes) is now under development in pH 7.40 phosphate buffer solutions bysquare wave voltammetry. Electrochemical impedance and cyclic voltammetricexperiments are used to characterise the electrode/electrolyte interface byemploying two molecule probes of opposite charges such as [Fe(CN)6]-3/-4 and[Ru(NH3)6]+3/+2 [17].References1) V. M. Scussel, ?Micotoxinas em alimentos?, N. Rolin de Moura (Ed.), Editora Insular,Florianólopis, Brasil, 1998.2. M. W. Trucksess and A. E. Poland in ?Mycotoxin protocols: Methods in Molecular Biology?,Humana Press, Totowa, New Jersey, Vol. 157, 2001.3. Dorner J. W., ?Chromatographic analysis of mycotoxins? in Chromatographic Analysis ofEnvironmental and Food Toxicants?, T. Shibamoto (Ed.), Marcel Dekker Inc., Vol 77, pp. 113-168, 1998.4. P. G. Molina, Zón M. A. and H. Fernández, ?Electrochemical behaviour of some mycotoxins ofthe Alternaria alternata genus?. Bol. Soc. Chil. Quím., 42 (1997) 465.5. M. A. Zón, Ceballos C. D., Molina P. G., Marchiando N. C., Moressi M. B. and H. Fernández,?Recent Research Developments in Electroanalytical Chemistry?, Vol 1, Transworld ResearchNetwork, Trivandrum, India, pp. 115-136, 1999.6. P. G. Molina, Zón M. A. and H. Fernández, ?Determination of the acid dissociation constants forsome Mycotoxins of the Alternaria alternata genus by spectrophotometric measurements?. Can.J. Chem., 76 (1998) 576.7. P. G. Molina, Zón M. A. and H. Fernández. ?The Redox Kinetics of Adsorbed ATX-I at CarbonElectrodes by Anodic Stripping Square-Wave Voltammetry?. Electroanalysis, 12 (2000) 791.8. P. G. Molina, Zón M. A. and H. Fernández. ?The electrochemical behavior of the Altenuenemycotoxin and its acidic properties?, J. Electroanal. Chem., 520 (2002) 94.9. P. G. Molina, Zón M. A. and H. Fernández. ?Determination of electrochemical properties of theadsorbed zearalenone (ZEA) mycotoxin by using cyclic and square wave voltammetry?. IndianJ. Chem., 42A (2003) 789.10. M. B. Moressi, Andreu R., Calvente J. J., Fernández H. and M. A. Zón. ?Improvement ofalternariol monomethyl ether detection at gold electrodes modified with a dodecanothiol selfassembledmonolayers?. J. Electroanal. Chem., 570 (2004) 209.11. N. C. Marchiando, Zón M. A. and H. Fernández. ?Characterization of the surface redox processof adsorbed cercosporin (CER) at glassy carbon electrodes by anodic stripping square wavevoltammetry?. Electroanalysis, 15 (2003) 40.12. N. C. Marchiando, M. A. Zón and H. Fernández. ?Determination of cercosporin (CER) phytotoxinisolates from infected peanut leaves by using adsorptive stripping square wave voltammetry?.Anal. Chim. Acta, 550 (2005) 199-203.13. E. A. Ramírez, P. G. Molina, M. A. Zón and H. Fernández. ?Development of an electroanalyticalmethod for the quantification of zearalenone (ZEA) in maize samples?. Electroanalysis, 17(2005) 1635-1640.14. M. B. Moressi, M. A. Zón, H. Fernández, G. Rivas and V. M. Solís. ?Amperometric quantificationof Alternaria mycotoxins with a mushroom tyrosinase modified carbon paste electrode?.Electrochemistry Communications 1 (1999) 472.15. V. G. L. Zachetti, H. Fernández and M. A. Zón. "Oxidación de micotoxinas del género Alternariaalternata mediante peroxidasas extraídas de plantas". XXV Congreso Argentino de Química,Olavarría, Province of Buenos Aires, Argentina, September 22 ? 24, 2004.16. S. H. Alarcón, L. Micheli, G. Palleschi and D. Compagnone. "Development of anelectrochemical immunosensor for ochratoxin A?. Anal. Letters, 37 (2004) 1545.17. P. G. Molina, M. A. Zón and H. Fernández. ?Studies about the electrochemical determination ofzearalenone on a rabbit polyclonal antibody adsorbed on glassy carbon electrodes?. Myco-Globe Argentine 2006, Córdoba, Argentina, March 15 to 17, 2006.