Gold nanoparticle-assisted, label-free SELEX coupled with high-throughput NGS for generating highly sensitive and specific DNA aptamers targeting Aflatoxin B1

Abstract

Background

Aflatoxin B₁ (AFB₁) is a highly toxic fungal secondary metabolite that poses serious health risks to humans and animals, prompting the development of sensitive and specific detection technologies. Aptamer-based detection strategies offer a potential alternative to traditional detection methods. This study aimed to develop AFB₁-specific aptamers using a gold nanoparticles-assisted SELEX (AuNP-SELEX) approach, eliminating the need for target immobilization. In this method, aptamers bound to AFB₁ detach from AuNPs while unbound or weakly bound sequences remain associated. The process was further strengthened using High-Throughput SELEX (HT-SELEX), enabling the efficient screening of large libraries for aptamers with minimal off-target binding.

Results

Successive rounds of SELEX led to the enrichment of high-affinity aptamers, which was monitored using a colorimetric AuNP-based assay. The top four aptamers- L2, CPMA1, L1, and L3, showed strong binding affinities with dissociation constants (K_d) of 1.24, 1.6, 2.55, and 5.26 nM, respectively. Circular dichroism spectroscopy confirmed conformational changes in the aptamers upon AFB₁ binding. A fluorescence assay using a FAM-labelled L2 aptamer and graphene oxide also achieved a detection limit of 0.53 nM.

Significance

This study demonstrates the effectiveness of the AuNP-assisted HT-SELEX platform as a rapid, visual, and refined method for selecting aptamers against small molecules like AFB₁. The approach enhances detection sensitivity and specificity, offering a valuable tool for food safety monitoring and mycotoxin detection.