Ultrasensitive and multiplex detection of four pathogenic bacteria on a bi-channel lateral flow immunoassay strip with three-dimensional membrane-like SERS nanostickers

A lateral flow immunoassay (LFA) technique for sensitive and multiplexed on-site detection of bacteria remains a challenge. Here, we develop a bi-channel surface-enhanced Raman scattering (SERS)-based LFA by using three-dimensional membrane-like SERS tags as nanostickers (named GO@Au/Ag) for direct and ultrasensitive analysis of multiple pathogens in a single test. The grafting of numerous Ag satellites onto nanosticker significantly increased the relative surface area for bacteria binding and generated efficient SERS hotspots over large area to improve the sensing sensitivity. Antibody-labeled GO@Au/Ag nanostickers can rapidly stick onto the target bacteria and generate superior SERS signals and fluidity on the paper strip, thus conquering the adverse effect of bacteria size and improving the multiplex analysis ability of LFA. The integration of two different Raman reporter molecules into nanostickers allows simultaneous detection of four pathogens on two test lines, which significantly simplifies the reading process of SERS signals. The proposed biosensor can quantitatively detect four different bacteria in real clinical samples with low detection limit (9 cells mL⁻¹ level), short assay time (20 min), high accuracy and excellent stability, indicating its great application potential for on-site detection of pathogens.