Peptide-Driven Assembly of Magnetic Beads for Potentiometric Sensing of Bacterial Enzyme at a Subcellular Level

Bacterial enzymes with different subcellular localizations play a critical ecological role in biogeochemical processing. However, precisely quantifying enzymes localized at certain subcellular levels, such as extracellular enzymes, has not yet been fully realized due to the complexity and dynamism of the bacterial outer membrane. Here we present a magneto-controlled potentiometric sensing platform for the specific detection of extracellular enzymatic activity. Alkaline phosphatase (ALP), which is one of the crucial hydrolytic enzymes in the ocean, was selected as the target enzyme. Magnetic beads functionalized with an ALP-responsive self-assembled peptide (GGGGGFFFpYpYEEE, MBs-peptides) prevent negatively charged peptides from entering the bacterial outer membrane, thereby enabling direct potentiometric sensing of extracellular ALP both attached to the bacterial cell surface and released into the surrounding environment. The dephosphorylation-triggered assembly of peptide-coupled magnetic beads can be directly and sensitively measured by using a magneto-controlled sensor. In this study, extracellular ALP activity of Pseudomonas aeruginosa at concentrations ranging from 10 to 1.0 × 10⁵ CFU mL^-1 was specifically and sensitively monitored. Moreover, this magneto-controlled potentiometric method enabled a simple and accurate assay of ALP activity across different subcellular localizations.