Arduini Fabiana, Neagu Daniela, Pagliarini Valeria, Scognamiglio Viviana, Leonardis Maria Antonietta, Gatto Emanuela, Amine Aziz, Palleschi Giuseppe, Moscone Danila
Dipartimento di Scienze e Tecnologie Chimiche, Università di Roma Tor Vergata, Via della Ricerca Scientifica, 00133 Rome, Italy; Consorzio Interuniversitario Biostrutture e Biosistemi "INBB", Viale Medaglie d'Oro 305, Rome, Italy.
Dipartimento di Scienze e Tecnologie Chimiche, Università di Roma Tor Vergata, Via della Ricerca Scientifica, 00133 Rome, Italy; Consorzio Interuniversitario Biostrutture e Biosistemi "INBB", Viale Medaglie d'Oro 305, Rome, Italy.
Talanta. 2016 Apr 1;150:440-8. doi: 10.1016/j.talanta.2015.12.048. Epub 2015 Dec 18.
In this study, we report a novel assay for the combined on site detection of aflatoxin B1 (AFB1) and ochratoxin A (OTA), through a colorimetric biosensing system for AFB1 and a fluorimetric detection for OTA, exploiting the capability of the portable fibre optic spectrometer to perform both analyses. AFB1 was detected using the acetylcholinesterase (AChE) enzyme that is inhibited by this toxin, and the degree of inhibition was quantified by the Ellman's spectrophotometric method, obtaining a detection limit of 10 µg L(-1). OTA quantification was performed by monitoring its intrinsic fluorescence in methanol, reaching a detection limit of 0.1 µg L(-1). In order to successfully apply the analytical tool in the food analysis, immunoaffinity columns were used. Clean-up and quantification of both AFB1 and OTA in millet samples was obtained by HPLC-dedicated AflaOchra-Test HPLC™ (Vicam™) and Afla-OtaCLEAN™ (LC-Tech) immunoaffinity columns, followed by absorption/fluorescence detection. Millet samples which were fortified with both OTA (50 µg kg(-1)) and AFB1 (20 µg kg(-1)), gave recovery values of 100 ± 6% for OTA, and 110 ± 10% for AFB1, using AflaOchra-Test HPLC™. Single OTA clean-up and quantification in wine samples was obtained, using an OchraTest immunoaffinity column (Vicam™), reaching a detection limit of 0.3 µg L(-1) and recovery values between 80% and 120%. These results demonstrated the possibility of employing a single clean-up and a cost-effective, and easy to use analytical system for both AFB1 and OTA detection at µg kg(-1) (ppb) level. Furthermore, in the case of positive samples, they could be analysed further, using standard chromatographic procedures, without any additional clean-up step, since the same extraction procedure of standard method is proposed in our method.
在本研究中，我们报告了一种用于黄曲霉毒素B1（AFB1）和赭曲霉毒素A（OTA）联合现场检测的新型检测方法，该方法通过AFB1的比色生物传感系统和OTA的荧光检测系统，利用便携式光纤光谱仪同时进行两种分析的能力。使用受该毒素抑制的乙酰胆碱酯酶（AChE）检测AFB1，并通过埃尔曼分光光度法对抑制程度进行定量，检测限为10 μg L(-1)。通过监测OTA在甲醇中的固有荧光进行OTA定量，检测限为0.1 μg L(-1)。为了成功地将该分析工具应用于食品分析，使用了免疫亲和柱。通过HPLC专用的AflaOchra-Test HPLC™（Vicam™）和Afla-OtaCLEAN™（LC-Tech）免疫亲和柱对小米样品中的AFB1和OTA进行净化和定量，随后进行吸收/荧光检测。使用AflaOchra-Test HPLC™对同时添加了OTA（50 μg kg(-1)）和AFB1（20 μg kg(-1)）的小米样品进行检测，OTA的回收率为100 ± 6%，AFB1的回收率为110 ± 10%。使用OchraTest免疫亲和柱（Vicam™）对葡萄酒样品中的单一OTA进行净化和定量，检测限为0.3 μg L(-1)，回收率在80%至120%之间。这些结果表明，有可能采用单一净化方法，建立一种经济高效且易于使用的分析系统，用于在μg kg(-1)（ppb）水平检测AFB1和OTA。此外，对于阳性样品，由于我们的方法采用了与标准方法相同的提取程序，因此无需任何额外的净化步骤，即可使用标准色谱程序进行进一步分析。
