Vertically Stacked Paper-Based Microarray Device for High-Throughput SERS Detection of Two Cancer Biomarkers

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.