Label-Free Bioelectronic\nImpedimetric Immunosensing\nDevice for Alkaline Phosphatase Detection Using Graphene Oxide and\nChitosan–Silk Fibroin–Polycaprolactone–Hydroxyapatite-Based\nHybrid

Bone, a dynamic tissue with diverse functions in the\nhuman body,\nplays vital roles, such as providing structural support and protection\nto organs, serving as the site for blood cell formation, and acting\nas the primary storage site for calcium. Osteoblasts, bone-forming\ncells, express alkaline phosphatase (ALP), which binds to the cell\nsurface or matrix vesicles and can be released into the bloodstream.\nConsequently, ALP can be found in the serum and is commonly used as\na biomarker in clinical studies to assess conditions related to bone\ndiseases as well as other disorders. This study presents a label-free\napproach for detecting ALP using electrochemical impedance spectroscopy\n(EIS) using a composite probe consisting of graphene oxide (GO) and\nchitosan (Ch)–silk fibroin (SF)–polycaprolactone (PCL)–hydroxyapatite\n(HAp) matrix. The GO and composite Ch–SF–PCL–HAp\nmatrix are sequentially deposited on the glassy carbon electrode and\nfurther immobilized with anti-ALP antibodies. The biosensor probe\nis characterized using physical techniques and electrochemical analysis.\nThe biosensor’s analytical performance is assessed using EIS,\nand it shows a limit of detection of 1.74 (±0.26) U/L with a\nlinear dynamic range of 30–500 U/L, which falls well within\nthe clinical range of ALP detection. The developed biosensor probe\nexhibits high selectivity for ALP (<i>k</i><sub>sel</sub> < 0.04) when tested against interfering molecules in serum. Furthermore,\nthe system is tested with fetal bovine serum and human serum to detect\nALP in biological samples.