Ready-to-Use Colorimetric Platform for Versatile Enzyme Assays through Copper Ion-Mediated Catalysis

Abstract

A low cost and versatile colorimetric platform is developed for selective detections of various enzymes. Similar to peroxidases, free copper ion catalyzes the oxidation of 3,3',5,5'-tetramethylbenzidine (TMB) in the presence of H₂O₂ and turns TMB into a blue product. Bindings from ligands toward copper ions inhibit this catalysis. Enzymes catalyze the reactions of related substrates with generation or consumption the ligands for the binding and thus in turn alter the color changes as responses toward the enzymes. With suitable substrates, exemplary enzymes, including trypsin, acid phosphatase, and tyrosinase, can be sensitively measured, with limits of detection of 0.003 μg/mL, 0.004 U/L, and 0.02 U/mL, respectively. This platform is built with directly available reagents, and the signals can be obtained with inexpensive photometers or visual observations. The low cost and convenience make it suitable for cases where complicated instrumentations are not available, such as point-of-care testing.