Antibody conjugated magnetic nanoparticle based colorimetric assay for the detection and quantification of aflatoxin B1 in wheat grains

Abstract

Aflatoxin B1 (AFB1) is a most potent carcinogenic secondary metabolite produced by Aspergillus flavus. As a food safety concern, development of a rapid, cost effective, sensitive and easy to use method for the detection of aflatoxin is of prime requirement. In this study, AFB1 was conjugated with bovine serum albumin (BSA), and AFB1-BSA conjugate was purified by HPLC. Purification was confirmed by UV-Vis spectroscopy, FTIR and MALDI-TOF mass spectrometry. The polyclonal antibody was raised against AFB1-BSA conjugate in rabbit and purified by protein A sepharose and BSA sepharose affinity columns. Iron oxide nanoparticles (MNPs) were synthesised by co-precipitation method and their surface was functionalised with (3-aminopropyl) triethoxysilane (APTES). Size of APTES conjugated MNPs was determined by electron microscopy, and characterised by several biophysical techniques. The purified anti-AFB1 antibody was conjugated with surface functionalised MNPs and the conjugation was confirmed by determining the sizes of free and antibody conjugated MNPs by field emission scanning electron microscope where increase of particle sizes from 10-20 to 40-50 nm was observed due to antibody conjugation. Anti-AFB1 antibody conjugated MNPs were used for capturing AFB1 from the aflatoxin spiked wheat grains with a recovery percentage of more than 80% and used effectively five times. The captured AFB1 was then quantified by a sensitive colorimetric assay where colourless AFB1 was first converted into coumaric acid by NaOH. Subsequently, coumaric acid reacted with 2,6-dibromoquinone-4-chloroimide (DBQC) to a green-coloured indophenol product which was quantified spectrophotometrically. AFB1 contamination as low as 2 μg/kg in wheat grains was detected by the developed technique suggesting its potential application for both qualitative and quantitative analysis of aflatoxins present in feed and food materials.