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Fluorometric determination of the activity of alkaline phosphatase and its inhibitors based on ascorbic acid-induced aggregation of carbon dots
摘要： The authors describe a fluorometric method for determination of the activity of alkaline phosphatase (ALP) and its inhibitors. Nitrogen and boron co-doped carbon dots (C-dots) with excitation/emission peaks at 490/540 nm act as the fluorescent probe. The C-dots were prepared by hydrothermal carbonization starting from 3-aminophenylboronic acid as the sole precursor. On the basis of the boronic acid-triggered specific reaction with cis-diols, the boronic acid modified C-dots can bind to ascorbic acid that is generated by ALP-catalyzed hydrolysis of ascorbic acid 2-phosphate. This results in particle aggregation and quenching of fluorescence. If the ALP inhibitor Na3VO4 is introduced into the system, the activity of ALP is reduced and the fluorescence of C-dots recovers. This fluorometric method allows for the determination of ALP activity in the range from 0.2 to 6.0 mU mL?1 with a detection limit of 0.16 mU mL?1. The IC50 value for the inhibitor Na3VO4 is 3.6 μM. The method is convenient and cost-effective. It does not require complicated operations and in our perception widens the scope of applications of C-dots in bioanalytical sciences.
关键词： cis-Diols,3-Aminophenylboronic acid,Enzyme inhibition,Fluorometry,Ascorbic acid,Ascorbic acid 2-phosphate,Sodium orthovanadate
更新于2025-11-19 16:46:39
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Binding of Clitoria ternatea L. flower extract with α-amylase simultaneously monitored at two wavelengths using a photon streaming time-resolved fluorescence approach
摘要： The binding of an extract from the flowers of Clitoria ternatea L. to the digestive enzyme α-amylase was investigated. This extract is a mixture of flavonoids, including anthocyanins, and has been previously shown to inhibit the activity this enzyme. This has implications for modulating starch digestion. Since the extract contains a mixture of flavonoids, including anthocyanins, in order to investigate the kinetics, we made use of time-resolved fluorescence to simultaneously monitor two different emission bands emanating from the extract. This measurement was enabled by the use of a “photon streaming” approach and changes in fluorescence lifetime and intensity were used to follow the interaction. A longer wavelength band (655 nm) was ascribed to anthocyanins in the mixture and these were observed to bind at a rate an order of magnitude slower than other flavonoids present in the extract, monitored at a shorter wavelength (485 nm). Changes in the fluorescence emission of the extract upon binding were further assessed by the use of decay associated spectra.
关键词： TCSPC,Butterfly pea,Anthocyanins,Enzyme inhibition,Flavonoids
更新于2025-09-23 15:21:21