Colorimetric detection of leucine aminopeptidase based on p-aminophenol-mediated redox cycling between Fe3+-ferrozine and tris(2-carboxyethyl)phosphine

Background Leucine aminopeptidase (LAP) is a promising biomarker for the detection and diagnosis of liver diseases encompassing liver cancer and liver injury. Existing assays for LAP activity have limitations, as most of them rely on substrates that are cumbersome to synthesize and fail to amplify the detection signal. Results Initially, L-pAP was synthesized to serve as a substrate for LAP through the condensation of p -aminophenol (pAP) with leucine. LAP triggered the hydrolyzation of L-pAP into pAP, which then reduced colourless Fe 3+ -ferrozine to purple Fe 2+ -ferrozine. Upon addition of tris(2-carboxyethyl)phosphine, the oxidation product p -quinone imine was quickly converted to pAP, which then continuously reduced Fe 3+ -ferrozine to produce substantial amounts of Fe 2+ -ferrozine that could be readily detected as purple using a commercial microplate reader or visual observation. The findings showed that the redox-cycling process greatly lowered the detection limit of LAP to 0.1 U/L, representing 2 orders of magnitude improvement over conventional detection methods. Moreover, we further expanded the feasibility of the proposed process to the assessment of LAP activity in cancer cell lysate. Significance and novelty This work introduces a novel LAP detection method characterized by simple substrate synthesis and high sensitivity. The detection limit of LAP was reduced through the signal amplification strategy of redox cycling. Our research findings provide a powerful chemical tool for LAP detection, offering enormous potential for future biomedical research and diagnosis of related diseases.