Analytical Sciences

Metal-to-Ligand Charge-Transfer-based Visual Detection of Alkaline Phosphatase Activity

Minhui HE,* Qiong HU,* Yaqi MEI,* Baojing ZHOU,** Jinming KONG,* and Xueji ZHANG***

*School of Environmental and Biological Engineering, Nanjing University of Science and Technology, Nanjing 210094, P. R. China
**School of Chemical Engineering, Nanjing University of Science and Technology, Nanjing 210094, P. R. China
***Chemistry Department, College of Arts and Sciences, University of South Florida, Tampa, Florida 33620-4202, USA

The ability to directly detect alkaline phosphatase (ALP) activity in undiluted serum samples is of great importance for clinical diagnosis. In this work, we report the use of the distinctive metal-to-ligand charge-transfer (MLCT) absorption properties of the Cu(BCA)₂⁺ (BCA = bicinchoninic acid) reporter for the visual detection of ALP activity. In the presence of ALP, the substrate ascorbic acid 2-phosphate (AAP) can be enzymatically hydrolyzed to release ascorbic acid (AA), which in turn reduces Cu²⁺ to Cu⁺. Subsequently, the complexation of Cu⁺ with the BCA ligand generates the chromogenic Cu(BCA)₂⁺ reporter, accompanied by a color change of colorless-to-purple of the solution with a sharp absorption band at 562 nm. The underlying MLCT-based mechanism has been demonstrated on the basis of density functional theory (DFT) calculations. Needless of any sequential multistep operations and elaborately designed colorimetric probe, the proposed MLCT-based method allows for a fast and sensitive visual detection of ALP activity within a broad linear range of 20 – 200 mU mL⁻¹ (R² = 0.999), with a detection limit of 1.25 mU mL⁻¹. The results also indicate that it is highly selective and has great potential for the screening of ALP inhibitors in drug discovery. More importantly, it shows a good analytical performance for the direct detection of the endogenous ALP levels of undiluted human serum samples. Owing to the prominent simplicity and practicability, it is reasonable to conclude that the proposed MLCT-based method has a high application prospect in clinical diagnosis.

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