ABSTRACT The demand for point‐of‐care (POC) diagnostics is driving portable analytical innovation. Despite advances, precise optical tag control remains a key challenge limiting diagnostic accuracy. Here, we present a vertically stacked microarray paper‐based device (µAPAD) enabling high‐throughput surface‐enhanced Raman scattering (SERS) immunoassays for multiplex biomarker detection. The 16‐layer wax‐patterned architecture integrates sample migration, reaction, and capture within one platform. Optimized microfluidic channels ensure uniform nanotag distribution, reducing signal variation from 36.6% to 6.69%, while an agarose hydrogel layer regulates flow to improve immunocapture efficiency. The µAPAD simultaneously analyzes 14 samples for two biomarkers within 30 min. Using carcinoembryonic antigen (CEA) and alpha‐fetoprotein (AFP) as models, it achieves detection limits of 0.34 and 0.69 ng/mL, outperforming conventional ELISA. Tests with spiked serum confirm high analytical accuracy, showing recoveries of 80.3%–139% and RSDs of 6.30%–10.8%. With its high sensitivity, flow precision, scalability, and true multiplexing capability, this low‐cost platform offers great potential for next‐generation POC diagnostics and large‐scale health monitoring.
Qi et al. (Tue,) studied this question.