Orthogonal Aptamer–Lectin Co-Recognition Enables Sandwich-Type Assays for Glycoproteins

Sandwich assays are widely used for protein detection, but aptamer-based formats are constrained by the difficulty of identifying two high-affinity aptamers that bind distinct, nonoverlapping epitopes on the target. Here, we present an orthogonal co-recognition strategy for glycoprotein analysis that combines SELEX-derived aptamers targeting non-glycan epitopes with lectin-mediated glycan recognition. Using the peanut allergen Ara h1 as a model glycoprotein, we selected a high-affinity aptamer against non-glycosylated recombinant Ara h1 and showed that it retained strong binding to native glycosylated Ara h1. Competitive binding studies revealed substantial epitope overlap among the top-performing aptamers, highlighting the practical limitation of conventional aptamer-only sandwich assays. Based on aptamer-lectin co-recognition, we first developed a lectin-based enzyme-linked aptamer binding assay and then advanced this concept into an aptamer-captured protein assembly assay, in which a preassembled lectin-mediated target-enzyme complex is captured by a surface-bound aptamer, enabling multivalent capture, increased enzyme loading, and a simplified workflow. The latter achieved sensitive detection of Ara h1 with a limit of detection of 0.14 ng/mL and was readily extended to multiple analytical readouts, including horseradish peroxidase (HRP)-, urease-, and glucose oxidase (GOx)-based outputs. This work establishes a practical and potentially generalizable framework for constructing high-performance sandwich-type assays for glycoproteins.