Aptamer-Based Trimeric DNA Nanostructure for High-Affinity Recognition of Allergenic Protein

The detection of trace amounts of the peanut allergenic protein Ara h1, a critical food allergen linked to severe allergic reactions, is vital for the effective prevention of allergic disorders. The unique homotrimeric shape of Ara h1 presents the opportunity to create a homotrimeric molecular recognition element that is perfectly matching its structural scaffold, thereby significantly enhancing binding affinity. The tetrahedral DNA nanostructure scaffold exhibits an edge length of approximately 5.8 nm, with three vertex-extended arms specifically conjugated to TCAPT1. Here, an asymmetrical dumbbell-shaped monomeric DNA aptamer that specifically binds to Ara h1 was first obtained from a prestructured DNA library via magnetic graphene oxide (MGO) based SELEX. This aptamer was subsequently tailored and engineered into a trimeric DNA nanostructure to align spatially with the homotrimeric configuration of Ara h1, achieving a significantly improved binding affinity with a dissociation constant (K_d) of 3.8 nM. To demonstrate the practical application of this valence-matched trimeric DNA nanostructure in sensitive Ara h1 detection, a fluorescent assay was developed using MGO-based platform, capable of detecting Ara h1 with a detection limit as low as 0.4 nM. The proposed approach further demonstrated high selectivity and recovery rates, underscoring its potential for precise detection of the peanut allergen Ara h1 in complex food matrices.