Phosphorylated Chemiluminescent Probe Based on Spiro-cyclobutyl-phenoxy-dioxetane for the In Situ Light-Activated Sensing of Alkaline Phosphatase

Alkaline phosphatase (ALP) plays important biological roles for many living species, including bacteria. Here, we developed an ALP probe (CL-A1) using the dephosphorylation of an in situ light-activated chemiluminescent species based on a spiro-cyclobutane-substituted dioxetane scaffold. A sequential light-induced oxidation and ALP-mediated dephosphorylation of CL-A1 rapidly generated intense chemiluminescence (CL), which was stronger than that of the commercial CL substrates AMPPD and APS-5 tested in the presence of a signal enhancer. Analysis using Escherichia coli strains confirmed the applicability of CL-A1 for the sensitive detection of endogenous ALP activity using in situ light activation.