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Biosensor of alkaline phosphatase based on non-fluorescent FRET of Eu3+-doped oxide nanoparticles and phosphorylated peptide labeled with cyanine dye

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Abstract

Dephosphorylation of biomolecules under the catalysis of alkaline phosphatase (ALP) is a critical physiological process. Abnormal levels of ALP activity have been associated with a number of diseases; thus, a simple and sensitive assay of ALP activity is highly demanded. Herein, to simulate biological conditions, we labeled a hydrosoluble phosphorylated heptapeptide Gly-Pro-Gly-Asn-p-Tyr-Gly-Ala (pGA) with aminated heptamethine cyanine dye (Cy) to give a low fluorescent labeled peptide Cy-pGA. The synthesized Cy-pGA and Eu3+-doped oxide Y0.6Eu0.4VO4 nanoparticles (NPs) were employed respectively as acceptor and donor to in situ form a non-fluorescent Fluorescence Resonance Energy Transfer (FRET) Cy-pGA-NP system, with the help of the strong interaction between Eu3+ ions in the NPs and phosphate group in Cy-pGA. The breaking of the FRET system of Cy-pGA-NP was triggered by the removal of phosphate group in Cy-pGA catalyzed by ALP and resulting in the release of fluorescent Y0.6Eu0.4VO4 NPs. Thus, the formed Cy-pGA-NP as a sensitive sensor can very well respond to the activity of ALP by measuring the time-resolved fluorescent intensity at near-infrared 617 nm (λ ex = 320 nm, delay time 400 μs). This sensor can not only accurately measure the activity of ALP (1–5 mU/mL) in the designed solutions, but it can also be applied to detect the activity of ALP in biological samples, such as cell lysate and human serum, without the interference of autofluorescent background of biosamples and screen ALP inhibitor by a simple mix-and-measure manner.

A biosensor of alkaline phosphatase (ALP) based on non-fluorescent FRET of Eu3+-doped oxide Y0.6Eu0.4VO4 nanoparticles and the phosphorylated heptapeptide labeled with cyanine dye (Cy-pGA)

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Acknowledgements

We are grateful to the financial support from the National Natural Science Foundation of China (No. 21272144) and the Fundamental Research Funds for the Central Universities of Shaanxi Normal University (No. X2015YB06).

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Authors and Affiliations

  1. Chemical Research Institute, School of Chemical Engineering, Northwest University, Xi’an, 710069, China

    Fan Shi Li & Yi Feng Liu

  2. Key Laboratory of the Ministry of Education for Medicinal Resources and Natural Pharmaceutical Chemistry, National Engineering Laboratory for Resource Development of Endangered Crude Drugs in Northwest of China, School of Chemistry & Chemical Engineering, Shaanxi Normal University, Xi’an, 710062, China

    Fan Shi Li, Ya Ling Zhang, Xia Bing Li & Bao Lin Li

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  1. Fan Shi Li
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Correspondence to Bao Lin Li or Yi Feng Liu.

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Li, F.S., Zhang, Y.L., Li, X.B. et al. Biosensor of alkaline phosphatase based on non-fluorescent FRET of Eu3+-doped oxide nanoparticles and phosphorylated peptide labeled with cyanine dye. Anal Bioanal Chem 409, 5491–5500 (2017). https://doi.org/10.1007/s00216-017-0485-5

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