Specializing Carbon Nanozyme Active Sites for Sensitive Alkaline Phosphatase Activity Metal‐Free Detection

As biological enzymes regulate metabolic processes, alkaline phosphatase (ALP) is a critical diagnostic indicator associated with many diseases. To accurately measure the enzyme activity, nanozymactic materials can offer sensitive strategies for ALP detection. However, nanozymes often lack specific target binding sites, and the presence of common co‐components, e. g., metal ions, may cause false‐positive or false‐negative results in enzyme activity determination. Herein, we developed a colorimetric assay for ALP detection using metal‐free nanozymatic carbon dots (CDs). The ALP hydrolysis of pyrophosphate ions (PPi) to phosphate ions (Pi) induces a “turn‐on” response based on the nanozyme activity. This PPi‐induced inhibition mechanism is extensively studied via the Michaelis‐Menten model, revealing that PPi acts as a noncompetitive inhibitor for CDs at a binding site distinct from the common nanozyme active site. With superior responses to ALP substrates, a highly sensitive and selective method is established for sensing ALP activity with a linear range of 0.010–0.200 U/L and a detection limit of 0.009 U/L. This finding explores the recognition and binding behavior of nanozymes, allowing for precise and reliable measurements even in complex samples, and represents a significant breakthrough for nanozyme‐based assays in biological analysis. This study introduces a significant advancement in exploring the target recognition sites of nanozymatic carbon dots for alkaline phosphatase detection. The metal‐free nanozyme‐engineered sensitive and selective approach enables superior colorimetric responses to intrinsic ALP activity with a detection limit of 0.009 U/L.