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Talanta. 2018 Jan 1;176:52-58. doi: 10.1016/j.talanta.2017.07.095. Epub 2017 Aug 1.

Fluorescence quenching based alkaline phosphatase activity detection.

Author information

1
School of Environmental and Biological Engineering, Nanjing University of Science and Technology, Nanjing 210094, PR China.
2
School of Chemical Engineering, Nanjing University of Science and Technology, Nanjing 210094, PR China.
3
College of Materials and Chemical Engineering, Zhengzhou University of Light Industry, Zhengzhou 450002, PR China.
4
JiangSu RayMe Biotechnology Co., Ltd, Yixing 214200, PR China.
5
American Advanced Nanotechnology, Houston, TX 77459, United States.
6
School of Environmental and Biological Engineering, Nanjing University of Science and Technology, Nanjing 210094, PR China. Electronic address: j.kong@njust.edu.cn.

Abstract

Simple and fast detection of alkaline phosphatase (ALP) activity is of great importance for diagnostic and analytical applications. In this work, we report a turn-off approach for the real-time detection of ALP activity on the basis of the charge transfer induced fluorescence quenching of the Cu(BCDS)22- (BCDS = bathocuproine disulfonate) probe. Initially, ALP can enzymatically hydrolyze the substrate ascorbic acid 2-phosphate to release ascorbic acid (AA). Subsequently, the AA-mediated reduction of the Cu(BCDS)22- probe, which displays an intense photoluminescence band at the wavelength of 402nm, leads to the static quenching of fluorescence of the probe as a result of charge transfer. The underlying mechanism of the fluorescence quenching was demonstrated by quantum mechanical calculations. The Cu(BCDS)22- probe features a large Stokes shift (86nm) and is highly immune to photo bleaching. In addition, this approach is free of elaborately designed fluorescent probes and allows the detection of ALP activity in a real-time manner. Under optimal conditions, it provides a fast and sensitive detection of ALP activity within the dynamic range of 0-220mUmL-1, with a detection limit down to 0.27mUmL-1. Results demonstrate that it is highly selective, and applicable to 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 and even whole blood samples. Therefore, the proposed charge transfer based approach has great potential in diagnostic and analytical applications.

KEYWORDS:

Alkaline phosphatase (ALP); Bathocuproine disulfonate; Charge transfer; Density functional theory; Fluorescence quenching; Fluorometric assay

PMID:
28917785
DOI:
10.1016/j.talanta.2017.07.095
[Indexed for MEDLINE]

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