Electrochemical Microfluidic Immunosensor with Graphene-decorated Gold Nanoporous for Mycotoxin Detection

GILBERTO J. SILVA JUNIOR; LAURA N. FERNANDEZ SOLIS; MARIA A. FERRONI MARTINI; SIRLEY PEREIRA; MARTÍN A. FERNÁNDEZ BALDO; MATÍAS REGIART; MAURO BERTOTTI

Lyon

Encuentro; 74th Annual Meeting of the International Society of Electrochemistry; 2023

International Society of Electrochemistry

T-2 toxin is one of major concern mycotoxins, a type A trichothecenes secondary metabolite produced by several fungal genera, including Fusarium, when they infect grains, especially oats, maize, and wheat [1]. This toxin has attracted much attention worldwide, according to the European Union Commission Recommendation on the presence of T-2 and HT-2 toxins in cereals and cereal-based products (2013/165/EU), the Scientific Technical Commission on Contaminants in the Food Chain (CONTAM Commission) of the European Food Safety Authority (EFSA) established a Tolerable Daily Intake (TDI) of 100 ng kg-1 for both T-2 and HT-2. Sampling and analysis for T-2 and HT-2 toxins must be carried out in accordance with the Regulation (EC) No 401/2006. The limit of detection should be not higher than 25 μg kg-1 for both toxins [2]. Furthermore, evidence shows that T-2 is one of the most toxic compounds among the trichothecenes and the most potent cytotoxic and food-borne mycotoxin [3]. These factors make it essential to develop a sensitive, specific, rapid and portable method for T-2 detection during food safety control.In this work, we have developed and characterized an electrochemical microfluidic immunosensor for T-2 toxin quantification in corn and wheat samples. T-2 toxin detection was carried out using a competitive immunoassay method based on the use of monoclonal anti-T-2 antibodies immobilized on the poly(methyl methacrylate) (PMMA) microfluidic central channel. The platinum wire working electrode at the end of the channel was in situ modified by a single-step electrodeposition procedure with reduced graphene oxide (RGO)-nanoporous gold (NPG). The obtained nanostructured electrode was characterized by electrochemical impedance spectroscopy (EIS), cyclic voltammetry (CV), scanning electron microscopy (SEM), energy dispersive spectrometry (EDS), and x-ray diffraction (DRX).T-2 toxin in the sample was allowed to compete with T-2-horseradish peroxidase (HRP) conjugated for the specific recognizing sites of immobilized anti-T-2 monoclonal antibodies. The HRP, in the presence of hydrogen peroxide (H2O2) catalyzes the oxidation of 4-tert-butylcatechol (4-TBC), whose back electrochemical reduction was detected on the nanostructured electrode at -0.15 V. Thus, at low T-2 concentrations in the sample, more enzymatically conjugated T-2 will bind to the capture antibodies and therefore we will have a higher current. The calculated detection limits for electrochemical detection and commercial ELISA procedure were 0.10 μg kg-1 and 10 μg kg-1, and the intra and inter-assay coefficients of variation were below 5.35% and 6.87%, respectively. The total assay time was 15 min. The microfluidic immunosensor showed higher sensitivity and lower detection limits than the standard ELISA method. Also, our microfluidic immunosensor minimizes the waste of expensive reagents, showing outstanding physical and chemical stability over one month. Finally, miniaturized immunosensors to T-2 toxin will significantly contribute to the farm´s faster, direct, and secure in situ analysis.