Sensitivity Enhancement of Multiplex Lateral Flow Immunoassays by NIR-II Fluorescence and Thermal Contrast

Lateral flow assays (LFAs) are widely used for point-of-care (POC) diagnostics but often suffer from limited sensitivity and specificity compared with laboratory methods. Here, we present a multimodal LFA platform integrating colorimetric, photothermal, and second near-infrared window (NIR-II) fluorescence readouts for enhanced sensitivity and multiplexed detection. Gold nanorods were coupled with bright NIR-II emissive polymer dots to generate plasmon-enhanced fluorescence and efficient photothermal signals within a single probe. As proof-of-concept, carbohydrate antigen 15-3 (CA15-3) and carcinoembryonic antigen (CEA) were selected as target biomarkers for breast cancer screening and broader cancer indication, respectively. Both thermometric and NIR-II fluorescence modes achieved comparable limits of detection for CA15-3 (0.40 and 0.42 U/mL) with a dynamic range of 0–100 U/mL, while CEA was quantified with a detection limit of 0.096 ng/mL. Multiplexed analysis on a single strip allowed simultaneous detection of CA15-3 and CEA with minimal cross-reactivity, and NIR-II fluorescence from test line 2 was intentionally designed to be invisible to the naked eye to avoid interference with rapid CA15-3 screening. Validation with clinical serum samples demonstrated a strong correlation with standard electrochemiluminescence immunoassays. This portable, low-cost platform demonstrates that NIR-II fluorescence and photothermal readouts can be harmonized for sensitive, selective, and multiplexed POC cancer biomarker detection. This universal and signal-amplifying concept can be further adapted to other targets of interest, offering a promising route toward next-generation LFAs.