Dual-Readout Analysis of Chymotrypsin Activity Based on Multifunctional Lanthanide Metal-Organic Framework

A ratiometric fluorescence/chromaticity dual-readout method is developed for chymotrypsin (ChT) activity detection, integrating a multifunctional lanthanide metal-organic framework (Ln-MOF) and an enzyme-activated fluorogenic reaction. A novel non-peptide substrate, 3-hydroxyphenyl-4-bromobutanoate (3-HPB), is rationally designed by coupling the catalytic specificity of ChT with a resorcinol-involved azamanidine fluorogenic reaction. Upon ChT-catalyzed hydrolysis, 3-HPB releases resorcinol, which subsequently reacts with dopamine to produce blue fluorescent azamanidine. In this reaction system, Eu-BTEC, a Ln-MOF constructed from Eu³⁺ and pyromellitic acid (H₄BTEC), serves not only as a red fluorescent internal standard, but also facilitates confined catalysis and enhances the fluorogenic reaction. A ratiometric fluorescence method is thus established to detect ChT activity in human saliva. Furthermore, a portable visual device composed of a UV lamp and an illuminance meter enables ratiometric chromaticity (B/R) analysis. Both methods have low detection limits (0.0107 U mL^-1 and 0.0144 U mL^-1) and high accuracy (82.4-107.0% recovery). The ChT activities in real human saliva samples obtained by both methods are in good agreement (p > 0.05). This work not only presents a new strategy for ChT activity detection but also introduces a structure-simple, affinity-high substrate, offering a promising platform for future development of sensitive and reliable enzymatic assays.