Development of a rapid dot-blot assay for ochratoxin A (OTA) detection using peptide conjugated gold nanoparticles for bio-recognition and detection

Abstract

Ochratoxin A is a secondary metabolite produced by fungi and a major mycotoxin contaminating cereal grains and cereal-based products. Although, ochratoxin A is hepatotoxic, nephrotoxic and carcinogenic, its detection is limited due to low awareness, time-consuming conventional methods and false positive results. In this study, a rapid, user-friendly dot-blot assay was developed using peptide conjugated gold nanoparticles (AuNPs) as a detection agent. The peptide binding to ochratoxin A was evaluated by the apparent dissociation constant ( K d app ) measurement using indirect enzyme linked immunoabsorbent assay. A low K d value (1.046 μM) suggested high binding of peptide with ochratoxin A. The potential of peptide-based ochratoxin A recognition was translated to a portable platform using streptavidin functionalized AuNPs conjugated to a biotinylated 11-mer peptide. Dot-blot assay was developed for detection of ochratoxin A with a limit of detection of 0.49 μg/kg. Negligible cross-reactivity with low spot intensities were observed for high concentrations of other mycotoxins like aflatoxin B1 and citrinin, in comparison to control. Further, percent recovery of ochratoxin A from spiked wheat samples by dot-blot was comparable to the conventional high performance liquid chromatography (HPLC). Moreover, detection of ochratoxin A by dot-blot was validated by HPLC in 65 whole wheat, crushed wheat coarse, crushed wheat fine and wheat flour samples with a high correlation with R 2 = 0.93. HPLC and dot-blot confirmed the presence of ochratoxin A ˂ 5 μg/kg in the samples. Therefore, a portable, easy-to-use dot-blot assay for ochratoxin A detection with potential of application at each stage of harvesting, storage, production and distribution can contribute to safe and healthy food and feed. • A rapid, on-site, sensitive visual Dot-blot assay was developed for ochratoxin detection. • Peptide conjugated gold nanoparticles were employed as a recognition label to replace expensive antibodies. • The developed Dot-blot assay had a limit of detection of 0.49 μg/kg for ochratoxin. • High sensitivity and specificity for ochratoxin detection.