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      Development of an Electrochemical Biosensor for the Detection of Aflatoxin M 1 in Milk

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          Abstract

          We have developed an electrochemical immunosensor for the detection of ultratrace amounts of aflatoxin M 1 (AFM 1) in food products. The sensor was based on a competitive immunoassay using horseradish peroxidase (HRP) as a tag. Magnetic nanoparticles coated with antibody (anti-AFM 1) were used to separate the bound and unbound fractions. The samples containing AFM 1 were incubated with a fixed amount of antibody and tracer [AFM 1 linked to HRP (conjugate)] until the system reached equilibrium. Competition occurs between the antigen (AFM 1) and the conjugate for the antibody. Then, the mixture was deposited on the surface of screen-printed carbon electrodes, and the mediator [5-methylphenazinium methyl sulphate (MPMS)] was added. The enzymatic response was measured amperometrically. A standard range (0, 0.005, 0.01, 0.025, 0.05, 0.1, 0.25, 0.3, 0.4 and 0.5 ppb) of AFM 1-contaminated milk from the ELISA kit was used to obtain a standard curve for AFM 1. To test the detection sensitivity of our sensor, samples of commercial milk were supplemented at 0.01, 0.025, 0.05 or 0.1 ppb with AFM 1. Our immunosensor has a low detection limit (0.01 ppb), which is under the recommended level of AFM 1 [0.05 μg L-1 (ppb)], and has good reproducibility.

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          Most cited references26

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          Update of survey, regulation and toxic effects of mycotoxins in Europe.

          The most frequent toxigenic fungi in Europe are Aspergillus, Penicillium and Fusarium species. They produce aflatoxin B1 transformed into aflatoxin M1 found in the milk, as well as Ochratoxins and Zearalenone, Fumonisin B1, T-2 toxin, HT-2 toxin and deoxynivalenol (vomitoxin), which are of increasing concern in human health. These mycotoxins are under continuous survey in Europe, but the regulatory aspects still need to be set up and/or harmonised at European level. They are found in foodstuffs and are not destroyed by normal industrial processing or cooking since they are heat-stable. Some of their metabolites are still toxic and may be involved in human diseases. Their toxic effects (liver, kidney and hematopoetic toxicity, immune toxicity, reproduction toxicity, foetal toxicity and teratogenicity, and mainly carcinogenicity) are mostly known in experimental models, the extrapolation to humans being always inaccurate. The inaccuracy of extrapolation to humans may be explained by the lack of adequate food consumption data, lack of knowledge about relative health risks associated with specifically proposed limits and by the possibility of synergism with other mycotoxins present in the same food commodities. Other pathological causes are viral hepatitis, immune or hormonal deficiencies or organ dysfunction. Even when a specific biomarker of a given mycotoxin is identified in humans, it remains difficult to establish the relation with a given illness, because of genetic polymorphism and the possible beneficial influence of diet, and because other environmental toxicants may well interfere. The acceptable daily intake limits are mostly based on animal data and may be too high, due to the differences in the sensitivity of different animal species. The prevention involves first reduction of mycotoxin levels in foodstuffs and further increasing the intake of diet components such as vitamins, antioxidants and substances known to prevent carcinogenesis.
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            Enzyme-linked immunosorbent assay (ELISA) based on superparamagnetic nanoparticles for aflatoxin M1 detection.

            Five different clones of antibodies developed against the aflatoxin M(1) were investigated by using the classical indirect and direct competitive Enzyme-Linked Immunosorbent Assay (ELISA) formats, and also the direct competitive ELISA based on the use of the superparamagnetic nanoparticles. The purpose of this study was to assess if not so friendly time classical ELISA procedures can be further improved, by reducing the coating, blocking and competition time. Here we showed that a complete dc-ELISA (coating, blocking and competition step) based on the use of superparamagnetic nanoparticles can be performed in basically 40 min, if coating step (20 min) should be taken into account. Moreover, the standard analytical characteristics of the proposed method fulfil the requirements for detecting AFM(1) in milk, in a wide linear working range (4-250 ng/L). The IC(50) value is 15 ng/L. The matrix effect and the recovery rate were assessed, using the European Reference Material (BD282, zero level of AFM(1)), showing an excellent percentage of recovery, close to 100%.
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              Aflatoxin: Toxicity to Dairy Cattle and Occurrence in Milk and Milk Products - A Review

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                Author and article information

                Journal
                Sensors (Basel)
                Sensors (Basel, Switzerland)
                Molecular Diversity Preservation International (MDPI)
                1424-8220
                2010
                20 October 2010
                : 10
                : 10
                : 9439-9448
                Affiliations
                Laboratoire IMAGES EA 4218, Bâtiments S, Université de Perpignan Via Domitia, 52 Avenue Paul Alduy, 66860 Perpignan Cedex, France
                Author notes
                [* ] Author to whom correspondence should be addressed; E-Mail: nathalie.paniel@ 123456univ-perp.fr .
                Article
                sensors-10-09439
                10.3390/s101009439
                3230981
                22163418
                b91f642a-3292-4e26-8604-94961ac1c9c2
                © 2010 by the authors; licensee MDPI, Basel, Switzerland.

                This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license ( http://creativecommons.org/licenses/by/3.0/).

                History
                : 9 September 2010
                : 30 September 2010
                : 10 October 2010
                Categories
                Article

                Biomedical engineering
                milk,aflatoxin m1,electrochemical immunosensor,mycotoxin,superparamagnetic nanoparticles,horseradish peroxidase (hrp)

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