Dual-Signal Electrochemical Aptasensor Based on High-Entropy Alloy Nanosheets for Calprotectin Detection in...

The development of biofunctional electrochemical aptasensors represents an emerging frontier in diagnostic technology, yet achieving precise and ultrasensitive detection of inflammatory bowel disease (IBD) biomarkers for early stage diagnosis remains a critical challenge. To overcome this, we engineered a novel material-based electrochemical aptasensor by using amino acid-functionalized high-entropy alloy nanosheets (HEANSs@AAs) for calprotectin (CP) detection. The fabricated HEANSs@AAs demonstrate remarkable electrochemical performance through their multielemental composition and mesoporous architecture, featuring abundant active sites enriched with several functional groups, which improve the surface chemistry of the composite for efficient immobilization of NH₂-aptamer. The HEANSs@AAs-based platform enables dual signal amplification through accelerated electron transfer kinetics and improved surface reactivity. The developed aptasensor demonstrates an ultrawide dynamic range (5 pg mL^-1 to 100 ng mL^-1) with a limit of detection (LOD) of 2.02 pg mL^-1 (S/N = 3). Significantly, the aptasensor exhibits 5.32% signal retention after 8 days storage at 4 °C and demonstrates an outstanding relative standard deviation (RSD) of 1.92%, revealing remarkable stability and reproducibility. The fabricated sensor revealed acceptable recovery rates in human serum samples between 96.6 and 97.6%, with inter-assay RSD below 2.7%, confirming its efficiency for clinical use. This work establishes a robust platform for early IBD diagnosis through precise CP quantification and introduces a versatile strategy for developing high-performance biosensors based on entropy-stabilized nanomaterials, thereby advancing multiplex biomarker detection in point-of-care diagnostic applications.