ABSTRACT
In the present study, a colorimetric biosensor strategy is devised in combination with apta-magnetic separation assisted with DNAzyme based colorimetric detection of Aflatoxin B1 (AFB1). The optimized analytical procedures consisted of the capture of AFB1 by biotinylated aptamer conjugated to streptavidin magnetic beads and detection by a colorimetric signal from a DNAzyme modified aptamer in presence hemin and H2O2/TMB (3', 3', 5, 5'- tetramethylbenzidine). The DNA concentration, incubation time, hemin, and NaCl concentrations were evaluated and optimized. The visual optical signal thus generated could determine the presence of AFB1 in the given sample. The selectivity of the method with other mycotoxins was evaluated. The linear range of AFB1 from 0 to 200 ppb was assessed and detected as low as 40 ppb visually. The absorbance of blue color generated by the catalytic reaction was in a linear correlation with AFB1 concentrations and was able to detect as low as 22.6 ppb (LOD). The suitability of the assay for AFB1 quantification in sorghum and natural samples was also evaluated. Thus, the developed assay could be a reliable, inexpensive, alternative tool for possible use as a screening method for aflatoxins and other mycotoxins.
Subject(s)
Aflatoxin B1/analysis , Aptamers, Nucleotide/chemistry , Biosensing Techniques/methods , Colorimetry/methods , DNA, Catalytic/chemistry , Food Contamination/analysis , Immunomagnetic Separation/methods , Aflatoxin B1/chemistry , Aflatoxin B1/isolation & purification , Benzidines/chemistry , Biotin/chemistry , Calibration , Hemin/chemistry , Hydrogen Peroxide/chemistry , Limit of Detection , Streptavidin/chemistryABSTRACT
Aflatoxins are naturally occurring mycotoxins that contaminate food and agro commodities, leading to acute and chronic health conditions in human and animals. In the present work, an attempt was made to generate high-affinity single stranded DNA aptamers that specifically bind to Aflatoxin B1 (AFB1) by a modified Systemic Evolution of Ligands by Exponential Enrichment (SELEX) procedure with the aid of Immunoaffinity columns. Ten rounds of SELEX and alternating three counter SELEX rounds with a cocktail of related and other mycotoxins were performed to enhance the specificity. Resultant 105 aptamers were clustered into 12 groups according to their primary sequence homology. Candidates with lowest Gibbs free energy (dG value) and unique stem loop structures were selected for further characterization. Aptamers, AFLA5, AFLA53, and AFLA71 exhibiting lower Kd values (50.45 ± 11.06, 48.29 ± 9.45, and 85.02 ± 25.74 nM) were chosen for development of ELONA and determination of purification ability of toxin. The detection limit (LOD) of AFLA5 and AFLA71 was 20 and 40 ng/ml, respectively. HPLC analysis implied that selected aptamers were able to recover and quantify 82.2 to 96.21% (LOQ - 53.74 ng) and 78.3 to 94.22% (LOQ - 66.75 ng) of AFB1 from spiked corn samples, respectively. These findings indicate, immunoaffinity based SELEX can pave an alternative approach to screen aptamers against mycotoxin detection and purification.
