Rapid Electrochemical Readout of Alkaline Phosphatase Activity and Phosphate Inhibition in Silica Films

This work introduces a bioelectrochemical platform for the rapid electrochemical readout of alkaline phosphatase (ALP) activity based on enzyme entrapment within sol–gel silica films deposited on glassy carbon electrodes. Fluorescence spectroscopy confirms that immobilization preserves the structural integrity of the enzyme while creating a confined microenvironment within the silica matrix. Electrochemical experiments show that the enzymatic hydrolysis of hydroquinone diphosphate generates hydroquinone inside the film, which is detected voltammetrically at the electrode surface. The steady-state oxidation current provides a direct electrochemical readout of the enzymatic reaction rate, allowing the system response to be described using a modified Michaelis–Menten framework in the presence of phosphate as a competitive inhibitor. The estimated activity should be regarded as an apparent specific activity of the immobilized ALP, reflecting the fraction of enzyme effectively contributing to substrate conversion and electrochemical product detection within the silica-film/electrode. An apparent inhibition constant of 3.64 mM was obtained, in good agreement with reported values for ALP in solution, indicating that the film architecture largely preserves the native enzyme–inhibitor interaction. The use of glassy carbon electrodes enables repeated measurements in different solutions using the same biofunctionalized electrode, providing a practical advantage over disposable screen-printed electrode configurations previously employed for ALP-based inhibition assays.