Development of a dual-Raman signal lateral flow immunoassay with self-assembling nanogapped SERS tags for the simultaneous monitoring of respiratory bacteria and virus

A facile approach for direct, sensitive, and on-site identification of respiratory bacteria and viruses is still urgently needed. Here, we developed a dual-mode surface-enhanced Raman spectroscopy (SERS)–lateral flow immunoassay (LFA) based on self-assembling nanogapped SERS tags (Si@Au/Au) for the ultrasensitive and simultaneous quantification of a common respiratory bacterium (Streptococcus pneumoniae) and respiratory virus (influenza A virus) in complex specimens. Two layers of 20 nm AuNPs and an ultranarrow polyethyleneimine inter-layer (0.5 nm) were self-assembled on the surfaces of SiO₂ nanoparticles (170 nm) via electrostatic adsorption to form Si@Au/Au nanogapped tags, which provided higher stability, superior sensitivity, and more intra-particle hotspots for SERS-LFA techniques. The colorimetric mode of the proposed method enabled the rapid determination of the presence of target respiratory pathogens, while the SERS mode based on two different Raman molecules allowed the quantitative detection of a specific bacterium and virus on a single test zone with detection limits of 16 cells/mL and 29 pfu/mL, respectively. The clinical utility of the proposed method was further confirmed through the detection of 31 real clinical respiratory tract specimens. Thereby, the proposed SERS biosensor has massive potential for ultrasensitive monitoring of pathogenic microorganisms.