Electrochemical biosensor on disposable electrodes for the detection of alkaline phosphatase inhibitors in seawater

A disposable electrochemical biosensor for the detection of alkaline phosphatase (ALP) inhibitors in seawater was developed by immobilizing ALP within sol-gel silica and organically modified silica matrices deposited onto screen-printed carbon electrodes. Hydroquinone diphosphate, an electroinactive substrate, enabled amperometric quantification of enzymatic activity through the electrochemical detection of the hydrolysis product, hydroquinone. Fluorescence spectroscopy confirmed that ALP retained its structural integrity after immobilization, although restricted rotational mobility and microenvironmental polarity shifts were observed depending on the silica modifier. All matrices allowed substrate and product diffusion, although reduced apparent activity is detected relative to the free enzyme, because of diffusional constraints. Among the materials tested, isobutyl-modified silica provided the optimal balance between structural stabilization and catalytic accessibility, yielding the highest apparent activity. Inhibition assays using phosphate ion demonstrated a clear dose-dependent suppression of enzymatic activity, with a linear response from 1 to 10 μM and a limit of detection of 0.38 μM. The biosensor exhibits rapid response, portability, and suitability for in situ assessment of phosphate and other ALP-inhibiting contaminants in marine environments.