Rapid Immunoenzyme Assay of Aflatoxin B1 Using Magnetic Nanoparticles

Abstract

The main limitations of microplate-based enzyme immunoassays are the prolonged incubations necessary to facilitate heterogeneous interactions, the complex matrix and poorly soluble antigens, and the significant sample dilutions often required because of the presence of organic extractants. This study presents the use of antibody immobilization on the surface of magnetic particles to overcome these limitations in the detection of the mycotoxin, aflatoxin B1. Features of the proposed system are a high degree of nanoparticle dispersion and methodologically simple immobilization of the antibodies by adsorption. Reactions between the immobilized antibodies with native and labeled antigens are conducted in solution, thereby reducing the interaction period to 5 min without impairing the analytical outcome. Adsorption of immunoglobulins on the surface of magnetic nanoparticles increases their stability in aqueous-organic media, thus minimizing the degree of sample dilution required. Testing barley and maize extracts demonstrated a limit of aflatoxin B1 detection equal to 20 pg/mL and total assay duration of 20 min. Using this method, only the 3-fold dilution of the initial methanol/water (60/40) extraction mixture in the microplate wells is necessary. The proposed pseudo-homogeneous approach could be applied toward immunodetection of a wide range of compounds.

A Magnetic Nanoparticle Based Enzyme-Linked Immunosorbent Assay for Sensitive Quantification of Zearalenone in Cereal and Feed Samples

“…Magnetic nanoparticles are recently used in assays of biomedical and food-safety fields with the advantages of uniform diameters and even distribution in solution [ 24 , 25 , 26 ]. Complexes between the nanoparticles and antibodies are formed by covalent immobilization.…”

Sensitive detection of aflatoxin B1 molecules on gold SPR chip surface using functionalized gold nanoparticles

“…The obtained LOD value was 1.4 nM with the advantage that this simple method may be extended to the analysis of other mycotoxins. Urusov et al (2014b) also used AuNPs but without bCD and determined the limit of detection to 160 pg/mL if detected visually, and to 30 pg/mL via instrumental detection. This is significantly lower than the LOD of 2 ng/mL achieved by conventional lateral flow analysis using the same reagents.…”

“…It is very useful to develop new and more sensitive methods for quantitative determination of hydrophobic molecules (Du et al 2007;Kham et al 2007;Pál et al 2009;Varga et al 2009). Although numerous procedures can detect and determine the aflatoxin derivatives, the most typical one is the time-delayed analysis of aflatoxin derivatives by thin-layer chromatography, high-performance liquid chromatography, overpressuredlayer chromatography, and enzyme-linked immunosorbent assay (Li and Zhang 2009;Manetta et al 2005;Moricz et al 2007;Peiwu et al 2009;Urusov et al 2014b;Var et al 2007). The detection of mycotoxins by SPR from food-stuff has already been reported in several publications and those with low molecular weight (aflatoxin, ochratoxin A) were determined by SPR immunosensors (Daly et al 2000;Hodnik and Anderluh 2009;Homola 2008;Li et al 2012;Yuan et al 2009).…”

Advances in Antibody Preparation Techniques for Immunoassays of Total Aflatoxin in Food

“…The method was established by Wei et al [ 20 ] and further improved by Fuentes et al [ 21 ]. Urusov et al [ 22 ] prepared an AFB1 polyclonal antibody (pAb) with an IC50 of 1.6 ng/mL and a CR of 82.6% of AFB2, but its specificity required further improvement. The route of synthesis of the AFB1-BSA antigen by the MOA method is shown in Figure 3 .…”