A Paper-Based Electrochemical Immunosensor for Ultrasensitive Screening of Protein Biomarkers Based on Silver Nanoflower Labeling

The early screening of diseases requires rapid and ultrasensitive detection of trace protein biomarkers where even a qualitative readout can provide critical diagnostic evidence. Electrochemical biosensor technology offers high sensitivity and rapid detection, while paper-based electrochemical sensors provide a promising platform for point-of-care testing (POCT) due to their cost-effectiveness and operational simplicity. However, conventional paper-based electrochemical sensors often suffer from limited sensitivity, primarily due to low analyte capture efficiency and insufficient signal amplification of traditional probes. To overcome these bottlenecks, we developed a vertical-flow paper-based electrochemical immunosensor using strategically engineered silver nanoflowers (AgNFs) as signal probes. The vertical-flow architecture directs the sample perpendicularly to the sensing interface, drastically enhancing analyte capture compared to lateral-flow designs. Furthermore, by precisely controlling the density of cysteine linkers on the AgNF surface, we maximized the electroactive area while maintaining dispersibility, significantly amplifying the electrochemical signal. After systematic optimization of assay parameters, including blocking time, probe volume, and washing conditions, the sensor was evaluated using neuron-specific enolase (NSE) as a model biomarker. The device achieved a linear range from 1 fg/mL to 10 pg/mL within a rapid 10-minute assay, demonstrating its potential as a screening tool for early disease warning. By synergizing microfluidic spatial control with nanoscale surface engineering, this work presents a robust and scalable sensing strategy for ultrasensitive screening of trace proteins in point-of-care applications.