Color- and background-free Raman-encoded lateral flow immunoassay for simultaneous detection of carbendazim and imidacloprid in a single test line

Multiple detection based on lateral flow immunoassay (LFA) holds significant research importance across various fields. Recently, the use of multi-color surface-enhanced Raman scattering (SERS) nanotags has enabled simultaneous detection of multiple targets within a single test line (T-line) in LFA, greatly enhancing detection efficiency, reliability, and application flexibility. However, challenges regarding signal overlap in the Raman-fingerprint region (400–1800 cm−1) and the difficulty in determining specific targets based on typical color analysis have persisted. In this study, we developed two types of nanoprobes that encode both color and Raman information to address these challenges in simultaneous multiple detection on a single T-line in SERS-LFA. The nanoprobes were encoded with distinguishable blue and red colors, respectively, enabling colorimetric study in multiplex LFA for qualitative analysis. Additionally, they were encoded with distinctive and non-overlapping Raman signals in the Raman-silent region (1800–2800 cm−1), ensuring accurate and sensitive detection by effectively avoiding interference from each other as well as background signals in the sample matrix. Using carbendazim (CBZ) and imidacloprid (IMI) as model targets, the color- and Raman-encoded SERS-LFA demonstrated promising potential for the simultaneous detection of CBZ and IMI in both standard and real samples, whether in a colorimetric or a SERS mode.