A new substrate for alkaline phosphatase based on quercetin pentaphosphate

Abstract

We describe the characterization and application of quercetin pentaphosphate (QPP), a new fluorimetric substrate for the detection of alkaline phosphatase (ALP) activity. QPP exhibits major absorbance peaks at 260/410 nm and a strong fluorescence at λex/λem = 425/510 nm at alkaline pH. The product of enzymatic reaction between QPP and ALP has a strong absorbance peak at 324 nm with no fluorescence at the investigated wavelengths. The product generated from the enzymatic reaction was found to be proportional to ALP activity, and the ALP activity was monitored by the absorbance difference at 310 nm and 410 nm. The change in absorbance was found to be proportional to the ALP concentration with a linear detection range and a limit of detection of 0.01-16 U L(-1) and 0.766 U L(-1), respectively. The enzyme activity was also monitored by evaluating the change in fluorescence emission at 530 nm with a linear range of 0.01-8 U L(-1) and a detection limit of 0.062 U L(-1). Further, the validity of the new substrate for ALP in conjugated form was tested using Bacillus globigii spores as the model sample. A detection limit of 5998 spores per mL was obtained using QPP as the substrate. Unlike the parent compound, QPP substrate exhibits stability in solution for over three and half months and was stable under storage for over 12 months. The results obtained demonstrate the effectiveness of QPP for ALP and compare well with other fluorescent substrates, such as Fluorescein, Alexa Fluor and Cy5.

Flavonoid-derived anisotropic silver nanoparticles inhibit growth and change the expression of virulence genes in Escherichia coli SM10

“…We have reported the synthesis of phosphorylated quercetin derivatives 26 with signicantly enhanced solubility in water than the parent quercetin. [27][28][29] The anisotropic AgNPs have varying bactericidal effects when prepared in various shapes and sizes. In a wool fabric bacteria study utilizing various AgNPs, it was reported that there were far more colonies of Gram-negative E. coli on the untreated sample compared to the AgNPs treated fabrics.…”

Reactivity, characterization of reaction products and immobilization of lead in water and sediments using quercetin pentaphosphate

“…QPP is very stable in aqueous solution and our previous work reported stability of over 1 year in aqueous solution. 16,31 ICP-OES results showed that the metal ion concentrations could be decreased when QPP binds with toxic Pb(II) in water samples and this could lead to inhibition of the lipid peroxidation reactions. The results obtained from ICP-OES are summarized in Table 1 3.8.2 Environmental application of QPP in the immobilization of lead(II) ions from soil samples.…”

Monitoring of phosphatase and kinase activity using 31P NMR spectroscopy

“…Numerous methods have been developed to address this issue. For phosphatases, methods are mainly based on electrochemistry, 2,3 fluorescence, 4,5 colorimetry, 6,7 high-performance liquid chromatography (HPLC) 8 and surface-enhanced Raman scattering, 9 while for kinases, methods mainly include thin-layer chromatography, 10,11 HPLC, 12 capillary electrophoresis 13 and real-time 1 H NMR analysis, 14,15 assays coupled to the depletion reaction of NADH, 16,17 assay coupled to the luciferase reaction 18 and assay coupled to the phosphatase reaction 19 (Table 1). Each of these commonly used enzyme assays has its own shortcomings, such as a cumbersome sample pretreatment process, complicated operation, less selectivity, and easy interference.…”