Facile colorimetric assay of alkaline phosphatase activity using Fe(II)-phenanthroline reporter

Abstract

We report a versatile approach for the colorimetric assay of alkaline phosphatase (ALP) activity based on the distinctive metal-to-ligand charge-transfer (MLCT) absorption properties of Fe(II)-phenanthroline reporter. In the presence of ALP, the applied substrate ascorbic acid 2-phosphate is enzymatically hydrolyzed to produce ascorbic acid, which then reduces Fe3+ to Fe2+. The complexation of Fe2+ with the bathophenanthroline disulfonate (BPS) ligand generates a blood-red Fe(BPS)34− reporter, which is characterized by an intense MLCT absorption band at 535 nm in the visible range. Under optimal conditions, the spectral output exhibits a good quantitative relationship with ALP activity over the range of 0–220 mU mL−1 with a detection limit of 0.94 mU mL−1. Moreover, the activity of ALP can also be conveniently judged through naked-eye observations. Results indicate that it is highly selective and can be applied to the screening of ALP inhibitors. In addition, it has been successfully employed to detect the endogenous ALP level of undiluted human serum samples, with a detection limit of 1.05 mU mL−1 being achieved. This approach avoids any elaborately designed substrates and holds considerable simplicity and flexibility for reporter design. This study broadens the horizon of the applications of phenanthroline-based transition metal complexes. Furthermore, an efficient and practical method like this has the potential to be widely used in clinical applications and in the point-of-care testing.