Stability matters: evaluating the long-term performance of AuNP–DNA conjugates in lateral flow assays through varied conjugation methods and storage buffers

Abstract

This study evaluates five distinct conjugation techniques for attaching DNA to gold nanoparticles (AuNPs), focusing on their applicability in lateral flow assays (LFA). The selected methods include three salt-aging techniques, one polyT-adsorption method, and a rapid butanol dehydration (INDEBT) approach. These methods were chosen based on factors such as popularity, protocol duration, and ionic strength. Additionally, the study investigates the storage stability of DNA–AuNP conjugates in four different buffers: 100 mM Tris-MgCl₂, 10 mM PBS-MgCl₂, PBS, and deionized water. These buffers were selected to evaluate the effects of ionic strength and their compatibility with LFA materials on conjugate stability and performance. Results indicate that salt-aging methods, particularly with water or PBS, maintained LFA functionality up to 21 days. Adsorbed DNA and all samples stored in PBS-MgCl₂ were not functional from the start and aggregated over time. INDEBT and Tris-MgCl₂ conjugates demonstrated initial functionality but degraded due to butanol residues and aggregation. A complementary analysis using electrophoretic mobility shift assays (EMSAs), UV-Vis spectroscopy, and dynamic light scattering (DLS) provided essential insights into conjugate behavior. These results emphasize the importance of buffer selection and conjugation techniques for optimizing DNA–AuNP conjugates in LFAs, ensuring their reliability and effectiveness in diagnostic applications.