Abstract
The authors describe a novel, facile, and sensitive fluorometric strategy based on a Cu2+-thiamine (Cu2+-TH) system for the detection of alkaline phosphatase (ALP) activity and inhibition. The principle of the method is as follows. Under a basic conditions, TH, which does not exhibit a fluorescence signal, is oxidized into fluorescent thiochrome (TC) by Cu2+. Ascorbic acid 2-phosphate (AAP), which is the enzyme substrate, is hydrolyzed to produce ascorbic acid (AA) by ALP. The newly formed AA then reduces Cu2+ to Cu+, which prevents the oxidation of TH by Cu2+; as a result, the fluorescent signal becomes weaker. On the contrary, in the absence of ALP, AAP cannot reduce Cu2+; additions of Cu2+ and TH result in a dramatic increase of the fluorescent signal. The sensing strategy displays brilliant sensitivity with a detection limit of 0.08 U/L, and the detection is linear in the concentration range of 0.1 to 100 U/L. This approach was successfully applied to ALP activity in human serum samples, indicating that it is reliable and may be applied to the clinical diagnosis of ALP-related diseases.
Highlights
Detection of Alkaline PhosphataseAlkaline phosphatase (ALP), an enzyme broadly found in mammalian tissues, can catalyze the hydrolysis and dephosphorylation processes of multifarious substrates, which include nucleic acids, proteins, and some small molecules [1,2,3]
The aberrant level of ALP in serum is tightly associated with numerous serious illnesses, such as hepatitis, breast and prostatic carcinoma, liver dysfunction, osteopathy, and diabetes, and it is generally recognized as a biomarker in early clinical diagnosis [5,6,7]
The illustration for the principle of the proposed fluorescence-based Cu2+ -TH system in the determination of ALP activity is depicted in Scheme 1
Summary
Alkaline phosphatase (ALP), an enzyme broadly found in mammalian tissues, can catalyze the hydrolysis and dephosphorylation processes of multifarious substrates, which include nucleic acids, proteins, and some small molecules [1,2,3]. Research suggests that ALP is involved in cell regulation and signal transduction processes [4]. Attention has been paid to ALP for its potential use in fast and sensitive detection. A variety of analytical techniques, including electrochemistry, fluorometry, colorimetry, chromatography, and surface-enhanced Raman scattering have been used in the determination of ALP activity [8,9,10,11,12,13,14,15,16,17]. Wu et al established a signal amplified electrochemical method which relied on enzyme-induced metallization (EIM)
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