Sensitive horseradish peroxidase biosensor based on screen-printed carbon electrodes for ochratoxin A

Abstract

Ochratoxins belong to a family of structurally related, secondary fungal metabolites produced by various Penicillium and Aspergillus strains. Among them, ochratoxin A (OTA) is receiving increasing concern worldwide because of the hazard it poses to human health. It exhibits unusual toxicokinetics, with a half-life in blood of 35 days after oral ingestion. The toxin is potently nephrotoxic, teratogenic and an immuno-suppressive agent as well as mutagenic and carcinogenic [(Alarcon et al., 2006); (Calcutt et al., 2001); (Kaushik et al., 2009); (Oliveira et al., 2007) ;( Radi et al., 2009) and (Turner et al., 2009)]. It has been considered by the International Agency for Research on Cancer to be a potential carcinogen (group 2B) for humans (Turner et al, 2009). OTA has been detected in a variety of food commodities such as cereals, oleaginous seeds, coffee beans, wine, meat, cocoa, spices, beer, wine, etc. Therefore, the European Commission has fixed maximum levels for OTA in foodstuffs, which have been established in 3 g/kg and 5 g/kg for cereal products and roasted coffee, respectively (Commission Regulation No. 1881/2006 of 19 December 2006). Mycotoxin contamination usually occurs in trace amounts ranging from nanograms to micrograms per gram of foodstuff, therefore, sensitive and accurate analytical methods for OTA determination are highly desirable (Radi et al., 2009). Many detection techniques, such as liquid chromatography coupled with immunoaffinity column or solid phase extraction cleanup, have been used for the determination of OTA in different kind of samples [(Prieto-Simon et al., 2008) and (Turner et al., 2009)]. These methods involve expensive and time-consuming steps. In order to developed sensitive, accurate and fast response methods, electrochemical techniques have been commonly used in recent years [(Alarcon et al., 2004); (Alarcon et al., 2006); (Calcutt et al., 2001); ( Fu, 2007); (Kaushik et al., 2008, 2009); (Khan and Dhayal, 2008, 2009); (Oliveira et al., 2007); (Prieto-Simon et al., 2008); (Radi et al., 2009) and (Wang and Wang, 2008)].This work was then completed on intent to find a sensitive and accurate method to determine OTA and apply the proposed method on complex matrices like is beer and roasted coffee. Hence, this work was put in writing on VII chapters. In the CHAPTER I are presented some aspects about mycotoxins and in particular about the most toxic member of this group, Ochratoxin A. The methods usually applied to determine this toxic compound are resumed in CHAPTER II. All the methods and reagents used on this work are specified on CHAPTER III. On CHAPTER IV, it is showed the results from the electrochemical study of OTA redox behaviour at SPCE, SPCE-AuNPs and SPCE-AgNPs using some electrochemical techniques: square-wave voltammetry, cyclic voltammetry and linear sweep voltammetry. CHAPTER V describes an effective electrochemical approach for the selective detection of OTA using HRP-modified screen-printed carbon electrodes (SPCEs). Horseradish peroxidise has been successfully immobilized in a polypyrrole matrix onto disposable screen-printed carbon electrodes for the selective detection of Ochratoxin A. The chronoamperometric determination of this mycotoxin has been optimized by experimental design methodology. The slopes of the calibration curves built under the optimum conditions of the experimental variables have been used to estimate the reproducibility and the repeatability of the developed biosensor for Ochratoxin A. Residual standard deviation values of 0.95 % (n = 5 and = 0.05) and 6.29 % (n = 4 and  = 0.05) were obtained, respectively. From an analytical point of view, the average minimum detectable net concentration for this method was 0.1 ng mL-1 (n = 3 and  =  = 0.05) The viability of the developed biosensor in the determination of Ochratoxin A in spiked beer (CHAPTER VII) and in the one extracted from roasted coffee (CHAPTER VI) has been shown, yielding average recoveries of 103 % and 99 % with residual standard deviation values less than 5 % (n = 3 and  = 0.05). Some considerations and the conclusion from this work are obtainable in CHAPTER VIII.