Gold nanocluster-based fluorescent assay for label-free detection of protein kinase and its inhibitors

Gold nanocluster-based fluorescent assay for label-free detection of protein kinase and its... A sensitive fluorometric assay is reported for label-free detection of the activity of protein kinase (PKA) and of its inhibitors. It is based on the europium(III)-modulated fluorescence of peptide-stabilized gold nanoclusters (peptide-AuNCs). Both adenosine-5′-triphosphate (ATP) and adenosine-5′-diphosphate (ADP) (formed from ATP by PKA-catalyzed hydrolysis) enhance the fluorescence of peptide-AuNCs with its excitation/emission peaks of 330/405 nm. The addition of Eu(III) quenches the fluorescence of the ATP/peptide-AuNC system to the original fluorescence level of peptide-AuNCs, while addition of Eu(III) has no effect on the fluorescence of the ATP/PKA/peptide-AuNC system. Based on this “turn-on” method, the activity of PKA can be detected with high sensitivity in 0.05–1.6 U⋅mL−1 activity range, with a detection limit of 0.02 U⋅mL−1 (3σ). The feasibility of this method for screening of inhibitors was also studied. The IC50 value for the commercially available inhibitor H-89 was found to be 43 nM. Moreover, the detection scheme was also applied to monitoring the drug-stimulated activation of PKA in HepG-2 cell lysates. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Microchimica Acta Springer Journals

Gold nanocluster-based fluorescent assay for label-free detection of protein kinase and its inhibitors

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Publisher
Springer Vienna
Copyright
Copyright © 2017 by Springer-Verlag Wien
Subject
Chemistry; Nanochemistry; Nanotechnology; Characterization and Evaluation of Materials; Analytical Chemistry; Microengineering
ISSN
0026-3672
eISSN
1436-5073
D.O.I.
10.1007/s00604-017-2349-2
Publisher site
See Article on Publisher Site

Abstract

A sensitive fluorometric assay is reported for label-free detection of the activity of protein kinase (PKA) and of its inhibitors. It is based on the europium(III)-modulated fluorescence of peptide-stabilized gold nanoclusters (peptide-AuNCs). Both adenosine-5′-triphosphate (ATP) and adenosine-5′-diphosphate (ADP) (formed from ATP by PKA-catalyzed hydrolysis) enhance the fluorescence of peptide-AuNCs with its excitation/emission peaks of 330/405 nm. The addition of Eu(III) quenches the fluorescence of the ATP/peptide-AuNC system to the original fluorescence level of peptide-AuNCs, while addition of Eu(III) has no effect on the fluorescence of the ATP/PKA/peptide-AuNC system. Based on this “turn-on” method, the activity of PKA can be detected with high sensitivity in 0.05–1.6 U⋅mL−1 activity range, with a detection limit of 0.02 U⋅mL−1 (3σ). The feasibility of this method for screening of inhibitors was also studied. The IC50 value for the commercially available inhibitor H-89 was found to be 43 nM. Moreover, the detection scheme was also applied to monitoring the drug-stimulated activation of PKA in HepG-2 cell lysates.

Journal

Microchimica ActaSpringer Journals

Published: Jun 14, 2017

References

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