Ultrasensitive Point-of-Care Antigen Test for Detection of Neisseria gonorrhoeae Using Plasmon-Enhanced Lateral Flow Assays

Lateral flow assays (LFAs) are widely used in point-of-care (POC) diagnostics, yet the poor sensitivity limits their utility for detecting low-abundance biomarkers. Here, we report a plasmon-enhanced LFA (p-LFA) for Neisseria gonorrhoeae (NG) antigen detection that harnesses an ultrabright fluorescence nanolabel, plasmonic-fluor, as a reporter. The assay targets the highly repetitive H.8 antigen lipoprotein antigen, with surfactant-mediated bacterial membrane disruption enabling improved antigen accessibility. Analytical evaluation demonstrated an extraordinary limit of detection of 0.1 colony forming unit (CFU)/mL. Validation across 14 World Health Organization (WHO) reference NG strains confirmed broad inclusivity. The assay maintained stability under accelerated shelf life conditions and exhibited minimal batch-to-batch variation. Clinical testing of 161 male urine samples yielded a sensitivity of 94.9% and specificity of 98.1%, exceeding the optimal performance criteria outlined by the WHO and Foundation for Innovative New Diagnostics target product profiles. Performance was consistent in both symptomatic and asymptomatic subgroups, and false negatives correlated with samples exhibiting high cycle threshold (Ct) values in molecular assays. To facilitate POC deployment, we demonstrate the feasibility of using low-cost hand-held ($50) and portable ($250) fluorescence readers as read-out devices. Both compact devices showed performance comparable to the benchtop scanner, with a complete workflow under 30 min and an estimated assay cost of <$2 per test. Collectively, the NG p-LFA represents a rapid, sensitive, and cost-effective diagnostic tool, with strong potential for widespread implementation in low- and middle-income countries and other resource-limited settings.