Ultra-Sensitive 3D Lateral Flow Assay Device for SARS-CoV-2 Detection Based on One-Step Dual-Signal Amplification

A one-step nucleic acid lateral flow assay (NALFA) platform was developed for on-site detection of severe acute respiratory syndrome Coronavirus 2 (SARS-CoV-2), in which no additional operation step is required for signal amplification. Polyadenine (polyA) serves as an effective anchoring block for preferential binding with the gold nanoparticles (AuNPs) surface to form probes with a high density of DNA attachment. Furthermore, bimetallic deposition, which is Au deposition and Ag staining through subsequent reductions of Au³⁺ to Au⁰ and Ag⁺ to Ag⁰ to deposit on gold nanoparticles, was used to achieve dual-signal amplification to improve colorimetric detection and sensitivity. These amplification chemistries predried on three-dimensional flow channels were constructed by stacking the papers with well-defined hydrophobic/hydrophilic areas to eliminate interference between chemistries and enhance the stability of reagents. Upon rehydration and mixing with the sample, the probes initiated a two-step metallic deposition, enabling real-time visual detection with a 2.24 nM limit, achieving 100-fold higher signal amplification than conventional LFA. Finally, the NALFA platform was successfully used to detect viral samples of the SARS-CoV-2 Omicron BA.1 variant, delivering visual results in 25 min. We expect that the developed device holds great potential in resource-limited areas.