Ultrasensitive electrochemical immunosensing of porcine serum albumin using a nanoporous alumina millirod and a carbon/gold nanohybrid

Pork adulteration is a pervasive global issue with significant implications for public health, economic stability, and cultural sensitivities. The methods currently employed to detect pork adulteration are often intricate, time-consuming, and financially burdensome, necessitating the development of more efficient solutions. This research addresses these challenges by introducing an innovative electrochemical immunosensor platform designed specifically for the detection of porcine serum albumin (PSA), a reliable biomarker for pork adulteration in raw meats. The proposed platform integrates a nanoporous alumina (NPA) millirod as the biorecognition surface and a self-standing nanohybrid composed of multi-walled carbon nanotubes, polyelectrolytes, and gold nanoparticles (MWCNT/PE/AuNP) as an enhanced signaling probe. The NPA millirod fabrication was optimized at 80 V for 6 h to achieve ideal pore structure for antibody immobilization, while surface functionalization with 2% (v/v) APTES for 4 h ensured stable bioconjugation. Optimization of the MWCNT/PE/AuNP nanohybrid focused on maximizing AuNP adsorption by tailoring the polyelectrolyte (PE) layer configuration via a layer-by-layer assembly. The optimal configuration was identified as a triple-layer structure (PAH–PSS–PAH), which significantly enhanced AuNP adsorption through electrostatic and coordination interactions, with no further improvement beyond this configuration due to steric hindrance and binding site saturation