Plasmonic internal standard-decorated nitrocellulose membranes for duplex detection of circulating tumor biomarkers

Surface-enhanced Raman spectroscopy (SERS) technology has been increasingly integrated with lateral flow immunoassay (SERS-LFA) to enable sensitive and quantitative detection, but SERS-LFA still faces notable challenges. One of the biggest problems is signal variations caused by random distributions of plasmonic SERS immunoprobes. Herein, we develop a sensitive, accurate quantification platform that overcomes this problem with a plasmonic internal standard (PIS), referred to as PIS-LFA. The PIS is achieved by decorating nitrocellulose membranes with Raman reporter molecule-embedded silver-gold alloy nanoparticles . By simultaneously measuring signals of plasmonic immunoprobes and IS and calculating their intensity ratios for signal normalization, we can mitigate signal variations and improve assay sensitivity. Comparing with SERS-LFA in carcinoembryonic antigen (CEA) detection, we show that PIS-LFA achieves a 1.8-fold higher uniformity and 3.2-fold lower limit of detection. We further demonstrate that PIS-LFA exhibits accurate quantification capability in duplex detection of CEA and neuron-specific enolase (NSE) in human sera by performing spike-recovery experiments and validating with electrochemiluminescence immunoassays. As a proof-of-concept, PIS-LFA differentiates levels of serum CEA and NSE between 12 early-stage lung cancer patients and 12 healthy individuals. We believe that PIS-LFA will open up avenues for affordable, rapid, and non-invasive disease screening in point-of-care settings.