1 research outputs found
Graphene Oxide–Peptide Nanocomplex as a Versatile Fluorescence Probe of Protein Kinase Activity Based on Phosphorylation Protection against Carboxypeptidase Digestion
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