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.