A label-free G-quadruplex-based fluorescence assay for sensitive detection of alkaline phosphatase with the assistance of Cu2

Abstract

The level of alkaline phosphate (ALP) is a significant biomarker index in organism. In this work, a label-free and sensitive G-quadruplex fluorescence assay for monitoring ALP activity has been developed with the assistance of Cu²⁺ based on the competitive binding effect between pyrophosphate (PPi) and G-quadruplex-N-methylmesoporphyrin (G4/NMM) complex to Cu²⁺. In the sensing assay, the G4/NMM complex is employed as a signal indicator, while the Cu²⁺ as a quencher and the PPi as recovery agent as well as the hydrolytic substance for ALP. In details, the fluorescence of the G4/NMM complex was efficiently quenched by introducing Cu²⁺ due to the proximal carboxylate groups of NMM coordinating with the Cu²⁺ as well as the unfolding of G-quadruplex by Cu²⁺, while the higher affinity between PPi and Cu²⁺ could lead to the fluorescence recovery. However, in the presence of ALP, the PPi was hydrolyzed to phosphate ions (Pi) which cannot integrate with Cu²⁺, resulting in the fluorescence quenching once again. Thus, a simple and facile way to inspect ALP has been exploited. The proposed assay shows a good linear relationship in the range from 0.5 to 100 U/L with the detection limit of 0.3 U/L. Moreover, the fabricated method is succeeded in detecting ALP in human serum samples, indicating the potential as a profitable candidate in biological and biomedical application.

Keywords: Alkaline phosphatase; Cu(2+); Fluorescence; G-quadruplex; Label-free.