Sensitive dual-channel lateral flow immunoassay tagged with high brightness latex microsphere for simultaneous detection of respiratory viral antigens

Respiratory infectious diseases pose a serious threat to human health worldwide. Timely diagnosis is crucial for effective clinical management and infection control. Herein, we developed a dual-channel lateral flow immunoassay (DC-LFIA) for simultaneous sensitive detection of influenza A virus (IAV) and respiratory syncytial virus (RSV) nucleoprotein (NP) antigens. The high brightness latex microspheres (HB-LMs) exhibiting typical absorption peak at 540 nm with uniform size and excellent optical stability, were prepared by a simple surfactant-free emulsion polymerization by mixing Sudan red, which overcame the complexity and color instability met for traditional synthesis methods. After affinity evaluation, two antibody pairs have good affinity for IAV NP and RSV NP were separately identified. Then, combining antibody pairs as probes and HB-LMs as based signal tag, a DC-LFIA was constructed, by which the visual limit of detection is 25 pg/mL, 100 pg/mL for IAV NP and RSV NP, respectively, 10-fold more sensitive than gold nanoparticles based LFIA. The calculated limit of detection (S/N=3) of IAV NP and RSV NP was improved 22.8- and19.5-fold, respectively, in comparison with gold nanoparticles based LFIA. Additionally, the developed sensor shows excellent specificity and reproducibility. Furthermore, the as-proposed method was validated through the analysis of clinical samples, in good agreement with commercial colloidal gold kits. Thus, the developed DC-LFIA has great potential for clinical application in the multiplex detection of respiratory viral antigens.