Developing Novel Protein Kinase Biosensor Based On Nanomaterials

More than2000kinds of kinase genes in humans are found in the HumanGenome Project, among which includes over500kinds of protein kinase genes. Theprotein phosphorylation catalyzed by protein kinases plays a vital role in regulatingthe signal transduction pathways involved in majority of crucial cellular processessuch as cell growth, metabolism, differentiation, proliferation, a nd apoptosis. In manyinstances, the over-expression and aberrant activity of protein kinase inducedabnormal cellular signaling and were associated with a range of diseases includingcancer, diabetes, inflammation, cardiac diseases, and Alzheimer’s disease. Therefore,detection of protein kinase not only is valuable to understand the disease relatedfundamental biological processes but also has the important effect on the applicationsof clinical diagnosis and drug discovery.The developed methods to detect kinase activity relied on the radiolabeledγ-32/33P-ATP, fluorescence, electrochemistry, surface plasmon resonance, and massspectroscopy et al. These methods are effective but have own shortcomings to someextent. For example, the radiolabeled method might pose great threats to humanhealth and environment due to hazardous radioactive reagents. However, developingconvenient, rapid, and cost-effective assays for detecting protein kinase is still in agreat demand but challenging.Based on above-mentioned statements, and researching the references reportedpreviously, we exploited the unique intrinsic characteristics of some emergingnanomaterials （Au nanoparticles, grapheme oxide, etc.） and Carboxypeptidasedigestion; three kinds of assays for rapidly detecting protein kinase were developed.The details are summarized as follow:（1） We present a novel colorimetric gold nanoparticles （AuNPs）/peptide platformfor probing the activity and inhibition of protein kinases based onphosphorylation-induced suppression of carboxypeptidase Y （CPY） cleavage. ThisAuNPs/peptide platform can easily monitor the kinase activity by UV-Visspectrometer or even by the naked eye. The feasibility of the method has beendemonstrated by sensitive measurement of the cAMP-dependent protein kinase （PKA）activity with a low detection limit of0.232mU/μL and assessment of kinaseinhibition by H-89with an IC50value of18.13nM. The assay was also successfully put into practice for the detection of kinase activity in cell lysate. Because of itslabel-free, homogenous and colorimetric merits, the proposed assay presents greatpotential in high-throughput screening for kinase-targeted drug discovery.（2） We developed a novel and rapid fluorescence sensing platform for probingactive protein kinase based on super-quenching capacity of graphene oxid （GO） andspecific recognition of aptameric peptide (FITC-IP₂₀). In this GO/peptide platform,the fluorescence quenching of FITC-IP₂₀adsorbed on GO can be restored by seletivebinding of the aptameric peptide with active protein kinase （PKA）, resulting in thequick turn-on kinase detection （about10min）. The feasibility of the method has beendemonstrated by sensitive measurement of PKA with a low detection limit of0.053mU/μL. The assay was also successfully put into practice for the detection of activekinase in cell lysate. Furthermore, this assay based on GO could serve as a universalplatform for detecting different protein kinases by simply changing to their cognateaptameric peptides. Therefore, the proposed method presents not only a promisingtechnique for protein kinase biosensing, but also a intriguing example to expand theapplication of GO into the research of post-translation modification-related enzyme.（3） We proposed a novel and versatile biosensing platform for detection ofprotein kinase activity based on graphene oxide （GO）-peptide nanocomplex andphosphorylation-induced suppression of carboxypeptidase Y （CPY） cleavage.Kinase-catalyzed phosphorylation protects the fluorophore-labeled peptide probeagainst CPY digestion and induces the formation of GO/peptide nanocomplexresulting in the fluorescence quenching, while nonphospho-peptide is degraded byCPY to release free fluorophore as well as restore fluorescence. This GO-basednanosensor has been successfully applied to sensitively detect the model kinase PKAwith low detection limits of0.134mU/μL, respectively. The feasibility of thisGO-based sensor was further demonstrated by the assessment of kinase inhibition bystaurosporine and H-89, in vitro kinase assay in cell lysates, and simultaneousdetection of CKII and PKA activity. Moreover, the GO-based fluorescence anisotropy（FA） kinase assay has been also developed using GO as FA signal amplifier. Theproposed sensor is homogenous, facile, universal, label-free and applicable formultiplexed kinase assay, presenting a promising method for kinase-relatedbiochemical fundamental research and inhibitor screening.