Comprehensive parameter screening for poly(A)DNA functionalization on gold nanoparticles for LFA

While AuNP@DNA bioconjugates have enabled diverse nanobiotechnology applications, poly(A)-modified DNA offers a promising and synthetically simpler alternative to costly thiolated DNA. For express diagnostic applications such as lateral flow assay (LFA), reliable production of stable, high‑performance conjugates is essential. However, for poly(A)-based systems, this remains challenging due to the lack of a link between conjugation parameters and function under LFA conditions. Herein, we disclose the comprehensive evaluation of the preparative methods and conditional factors underpinning the synthesis of poly(А)-conjugated gold nanoparticles. By systematically varying nanoparticle size, functionalization protocol, poly(A)-length and orientation (5'- versus 3'-), poly(A)DNA concentration, we optimized conjugate performance. The resulting AuNP@poly(A)DNA bioconjugates were successfully implemented as signaling components in a sandwich-format LFA for the detection of HIV-DNA, achieving a limit of detection (LOD) of 0.58 nM. We demonstrate that the microwave-assisted functionalization protocol is optimal for the preparation of AuNP@poly(A)DNA for LFA. This insight allows synthesis protocols to be tuned either to maximize signal intensity or to minimize non‑specific binding, establishing a design framework for AuNP@DNA point-of-care diagnostics.