Label-Free Bioelectronic Impedimetric Immunosensing Device for Alkaline Phosphatase Detection Using Graphene Oxide and Chitosan–Silk Fibroin–Polycaprolactone–Hydroxyapatite-Based Hybrid

Bone, a dynamic tissue with diverse functions in the human body, plays vital roles, such as providing structural support and protection to organs, serving as the site for blood cell formation, and acting as the primary storage site for calcium. Osteoblasts, bone-forming cells, express alkaline phosphatase (ALP), which binds to the cell surface or matrix vesicles and can be released into the bloodstream. Consequently, ALP can be found in the serum and is commonly used as a biomarker in clinical studies to assess conditions related to bone diseases as well as other disorders. This study presents a label-free approach for detecting ALP using electrochemical impedance spectroscopy (EIS) using a composite probe consisting of graphene oxide (GO) and chitosan (Ch)–silk fibroin (SF)–polycaprolactone (PCL)–hydroxyapatite (HAp) matrix. The GO and composite Ch–SF–PCL–HAp matrix are sequentially deposited on the glassy carbon electrode and further immobilized with anti-ALP antibodies. The biosensor probe is characterized using physical techniques and electrochemical analysis. The biosensor’s analytical performance is assessed using EIS, and it shows a limit of detection of 1.74 (±0.26) U/L with a linear dynamic range of 30–500 U/L, which falls well within the clinical range of ALP detection. The developed biosensor probe exhibits high selectivity for ALP (k_sel < 0.04) when tested against interfering molecules in serum. Furthermore, the system is tested with fetal bovine serum and human serum to detect ALP in biological samples.