Fluorimetric detection of leucine aminopeptidase based on copper-initiated cascade amplification

The detection of leucine aminopeptidase (LAP) activity is essential for diagnosing liver cancer and liver injury. This study presents a novel fluorescence-based method designed for the sensitive detection of LAP through cascade catalytic reactions. Leucine-histidine (LeH) was synthesized using histidine and leucine as substrates for LAP. Notably, histidine (His) effectively inhibited the Cu²⁺-catalyzed oxidation of o-phenylenediamine (OPD) to fluorescent 2,3-diaminophenazine (oxOPD), resulting in a diminished fluorescence signal. Given that LAP hydrolyzes LeH to release His, which subsequently inhibits the Cu²⁺-OPD system, LAP activity can be quantitatively assessed by measuring fluorescence changes during the hydrolysis process. The exceptional performance of the Cu²⁺-OPD system in quantifying His allowed for highly sensitive LAP detection, achieving a detection limit as low as 0.05 U/L. Furthermore, the strategy was successfully implemented to evaluate LAP activity in cells.