A Single-step Platinum Nanoparticle-Enhanced Lateral Flow Immunoassay Platform for Rapid Detection of Influenza A Virus

Lateral flow immunoassays (LFIAs) are widely used point-of-care (POC) diagnostic tools, but their limited sensitivity can hinder reliable diagnoses. To address this limitation, we developed a novel POC diagnostic platform for the highly sensitive detection of influenza A virus (IAV). This developed platform integrates platinum nanoparticle-catalyzed 3,3',5,5'-tetramethylbenzidine (TMB) oxidation for reagent-free, single-step signal amplification with smartphone-based image acquisition and quantitative analysis. This combination of catalytic enhancement and digital interpretation enables rapid, objective, and quantitative diagnostic evaluation, offering improved performance over conventional LFIAs. Upon sample application, the sample flows to complete the immunoreaction at the test line, followed by a chromatographically delayed acidic migration that rehydrates the TMB and delivers it to the captured platinum nanoparticles for signal amplification. This reagent-free, timed enhancement results in approximately 100-fold visual signal amplification compared to unenhanced detection, without the need for additional reagents. Additionally, a custom-developed smartphone application automates image acquisition and quantifies intensity ratios to provide the final diagnosis. The platform achieves a limit of detection of 11.6 pg/mL IAV nucleoprotein within 15 min, offering a 1000-fold increase in sensitivity over traditional LFIAs. In clinical trials, it demonstrated excellent performance, with 96.8% sensitivity and 98.4% specificity compared to RT-PCR. The platform also exhibited semiquantitative capability, with a strong inverse correlation (<i>R</i>² = 0.832) between RT-PCR Ct values and intensity ratios. This integrated system provides a rapid, low-cost, and user-friendly solution for accurate viral diagnostics.