Rapid and sensitive detection of alkaline phosphatase based on fluorescent gold nanoclusters and p-nitrophenyl phosphate

So-Hee Kim; Chang Soon Huh; Moon-Moo Kim

doi:10.1016/j.jbiosc.2025.06.004

Rapid and sensitive detection of alkaline phosphatase based on fluorescent gold nanoclusters and p-nitrophenyl phosphate

J Biosci Bioeng. 2025 Jul 4:S1389-1723(25)00138-0. doi: 10.1016/j.jbiosc.2025.06.004. Online ahead of print.

Authors

So-Hee Kim¹, Chang Soon Huh¹, Moon-Moo Kim²

Affiliations

¹ Department of Cosmetics Engineering, and Graduate School of Applied Chemistry and Food Engineering, Dong-Eui University, Busan 47340, Republic of Korea.
² Department of Cosmetics Engineering, and Graduate School of Applied Chemistry and Food Engineering, Dong-Eui University, Busan 47340, Republic of Korea. Electronic address: mmkim@deu.ac.kr.

PMID: 40617799
DOI: 10.1016/j.jbiosc.2025.06.004

Abstract

Alkaline phosphatase (ALP) is an essential enzyme that is involved in various metabolic processes. Abnormal ALP levels are linked to diseases and pathological conditions. Herein, a simple and sensitive assay is reported for ALP detection by using glutathione-conjugated gold nanoclusters (GSH-AuNCs) and p-nitrophenyl phosphate (pNPP), based on the fluorescence quenching mechanism. In the underlying mechanism of this assay, the fluorescence of GSH-AuNCs is initially quenched by pNPP, followed by further quenching caused by p-nitrophenol (pNP), a product of ALP activity. To investigate this mechanism for the diagnostic ALP detection, UV-Vis spectrophotometry, transmission electron microscopy (TEM), and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis were employed, and this method was tested in real samples. The prepared GSH-AuNCs exhibited an absorption peak at 600 nm under excitation at 365 nm. TEM analysis revealed that GSH-AuNCs were spherical in shape and exhibited uniform particle size and distribution. Furthermore, the gradual reduction in fluorescence intensity of GSH-AuNCs was observed with increasing concentration of pNPP increased (0.03 mM-2.7 mM), suggesting the quenching of the fluorescence by pNPP. SDS-PAGE analysis further confirmed the quenching effect of pNPP on GSH-AuNCs. In addition, fluorescence intensity was decreased by the increasing amounts of ALP. The relationship curve revealed a detectable concentration range of 1.95-1000 U/L and the correlation coefficient of 0.976. The developed method was successfully applied to human osteosarcoma MG-63 cell lysates, culture medium, and extracts from root plants for detection of ALP. Therefore, this assay will be beneficial for the diagnosis of ALP activity in clinical medicine.

Keywords: Alkaline phosphatase; Fluorescence quenching; Glutathione; Gold nanoclusters; p-Nitrophenyl phosphate.