Colorimetric enhancement of mixed metal nanoparticle based multicolor and multiplex lateral flow immunoassay for analysis of two epithelial-mesenchymal transition-related proteins

• First multiplex LFIA for key EMT biomarkers of E-cad and N-cad. • Colorimetric signals enhanced by a novel nanoparticle mixing strategy. • Simultaneous, specific detection with low pg/mL sensitivity. • Dual-color display allows for clear visual readout without instrumentation. • A promising point-of-care tool for cancer metastasis monitoring. E-cadherin (E-cad) and N-cadherin (N-cad), core members of the cadherin superfamily, are the two most critical biomarkers in the process of epithelial-mesenchymal transition (EMT). The simultaneous detection of these two proteins will play an indispensable role in predicting and monitoring the occurrence and development of tumors. In this work, a multiplex lateral flow immunoassay (mLFIA) based on multicolor mixing effect was creatively developed and applied to the colorimetric sensing of E-cad and N-cad. To achieve this goal, three nanoparticles of Au, core-shell Au@Ag and hollow Au/Ag with characteristic colors of magenta, yellow and cyan were firstly synthesized and denoted as M-AuNPs, Y-Au@Ag NPs and C-hollow Au/Ag NPs, respectively. Then physical mixing between every two kind NPs was attempted and experimental results demonstrated that M-AuNPs + Y -Au@Ag NPs and M-AuNPs + C -hollow Au/Ag NPs combinations (with final colors of reddish and navy blue) showed stronger plasma coupling effect, thereby obtaining two signal probes for E-cad and N-cad with enhanced colorimetric on the mLFIA strips. Such an approach enabled simultaneous, specific and sensitive quantification of E-cad and N-cad with neglectable cross-interference. Under optimal conditions, the developed mLFIA strip demonstrated limits of detection (LOD) of 24.7 pg mL -1 for E-cad and 34.7 pg mL -1 for N-cad. As a proof of concept, this detection platform can be expected to provide important technical support for the occurrence and tracking of tumor EMT process.