Abstract
Mycotoxins are the secondary toxic metabolites produced naturally by fungi. Analysis of mycotoxins is essential to minimize the consumption of contaminated food and feed. In this present work, an ultrasensitive electrochemical immunosensor for the detection of aflatoxin B1 (AFB1) was successfully developed based on an indirect competitive enzyme-linked immunosorbent assay (ELISA). Various parameters of ELISA, including antigen–antibody concentration, blocking agents, incubation time, temperature and pH of reagents, were first optimized in a 96-well microtiter plate to study the antigen–antibody interaction and optimize the optimum parameters of the assay. The optimized assay was transferred onto the multi-walled carbon nanotubes/chitosan/screen-printed carbon electrode (MWCNTs/CS/SPCE) by covalent attachment with the aid of 1-Ethyl-3-(3-dimetylaminopropyl)-carbodiimide (EDC) and N-hydroxysuccinimide (NHS). Competition occurred between aflatoxin B1-bovine serum albumin (AFB1–BSA) and free AFB1 (in peanut sample and standard) for the binding site of a fixed amount of anti-AFB1 antibody. Differential pulse voltammetry (DPV) analysis was used for the detection based on the reduction peak of TMB(ox). The developed immunosensor showed a linear range of 0.0001 to 10 ng/mL with detection limit of 0.3 pg/mL. AFB1 analysis in spiked peanut samples resulted in recoveries between 80% and 127%. The precision of the developed immunosensor was evaluated by RSD values (n = 5) as 4.78% and 2.71% for reproducibility and repeatability, respectively.
Highlights
Mycotoxins are toxic fungal metabolites that can contaminate primary food products as a result of mold growth
The optimized enzyme-linked immunosorbent assay (ELISA) was transferred onto the screen-printed carbon electrode (SPCE) that was modified with multi-walled carbon nanotubes (MWCNTs) and chitosan (CS), which was proven in our previous work to significantly increase the sensitivity and conductivity [29]
The concentrations of aflatoxin B1 (AFB1) standards immobilized on the microtiter well were varied from 0.00001 to 1000 ng/mL, just like the concentrations of rabbit anti-AFB1 antibody which were in the dilution range of 1/2500 to 1/640,000 (v/v)
Summary
Mycotoxins are toxic fungal metabolites that can contaminate primary food products as a result of mold growth. Of all the 18 known analogues of aflatoxins, aflatoxin B1 (AFB1 ) is the most toxic, mutagenic, teratogenic and carcinogenic and its toxicity is ten times than that of potassium cyanide, 68 times of arsenic, 416 times of melamine, 70 times of dimethylnitrosamine and 10,000 times of benzene hexachloride [7]. It is classified as a group 1 carcinogen by the International Agency for Research on Cancer (IARC) [8]
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