Repurposing a Multi-Lamp UV Photoactivation Platform for DNA Capture-Probe Immobilization in Nucleic Acid Lateral Flow Assays

Preparation of a reproducible and functionally accessible DNA capture zone on nitrocellulose is a critical step in nucleic-acid lateral flow assay (NALFA) fabrication. Here, we repurpose and characterize a four-lamp 254 nm UV photoactivation chamber, originally developed for antibody immobilization, for the preparation of DNA capture zones on nitrocellulose. Lamp configuration, exposure time, and capture-DNA spotting concentration were evaluated through the hybridization-dependent capture of DNA-functionalized gold nanoparticles (AuNPs), prepared by freeze-thaw conjugation and used as colorimetric reporters. Smartphone images acquired under fixed geometry were quantified using a grayscale-calibrated ImageJ workflow. Activating all four lamps produced the highest functional hybridization signal, approximately 2.2-fold above the untreated condition. A 5 min exposure was selected as a practical compromise between response magnitude, observed variability, and processing time, and 45 µM capture DNA was selected as the working spotting concentration. In a proof-of-concept competitive assay, UV-treated membranes showed greater separation between the blank and low-target responses, corresponding to an estimated concentration-based limit of detection of approximately 40 nM, compared with approximately 355 nM for untreated membranes under the investigated conditions. These results establish chamber-specific operating conditions and a measurement-guided workflow for UV-assisted fabrication of functional DNA capture zones, while distinguishing functional assay response from direct claims of quantitative DNA retention or a specific attachment mechanism.