Graphene oxide-peptide nanocomplex as a versatile fluorescence probe of protein kinase activity based on phosphorylation protection against carboxypeptidase digestion.
Anal Chem
; 85(12): 5746-54, 2013 Jun 18.
Article
em En
| MEDLINE
| ID: mdl-23734972
ABSTRACT
The research on complicated kinomics and kinase-target drug discovery requires the development of simple, cost-effective, and multiplex kinase assays. Herein, we propose a novel and versatile biosensing platform for the detection of protein kinase activity based on graphene oxide (GO)-peptide nanocomplex and phosphorylation-induced suppression of carboxypeptidase Y (CPY) cleavage. Kinase-catalyzed phosphorylation protects the fluorophore-labeled peptide probe against CPY digestion and induces the formation of a GO/peptide nanocomplex resulting in fluorescence quenching, while the nonphosphopeptide is degraded by CPY to release free fluorophore as well as restore fluorescence. This GO-based nanosensor has been successfully applied to sensitively detect two model kinases, casein kinase (CKII) and cAMP-dependent protein kinase (PKA) with low detection limits of 0.0833 mU/µL and 0.134 mU/µL, respectively. The feasibility of this GO-based sensor was further demonstrated by the assessment of kinase inhibition by staurosporine and H-89, in vitro kinase assay in cell lysates, and simultaneous detection of CKII and PKA activity. Moreover, the GO-based fluorescence anisotropy (FA) kinase assay has been also developed using GO as a FA signal amplifier. The proposed sensor is homogeneous, facile, universal, label-free, and applicable for multiplexed kinase assay, presenting a promising method for kinase-related biochemical fundamental research and inhibitor screening.
Texto completo:
1
Base de dados:
MEDLINE
Assunto principal:
Óxidos
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Carboxipeptidases
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Técnicas Biossensoriais
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Proteínas Quinases Dependentes de AMP Cíclico
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Nanopartículas
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Corantes Fluorescentes
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Grafite
Limite:
Humans
Idioma:
En
Ano de publicação:
2013
Tipo de documento:
Article