Analysis Of Protein Kinase Activity Using Gold And CeO 2 Nanoparticles As Sensing Elements

Protein phosphorylation catalyzed by protein kinases is one of the most important post-translation modifications mechanisms in the process of cellular signal transduction. It plays an important role in regulation of fundamental biological processes, including cell proliferation, differentiation and metabolism. Aberrations of kinase activities and protein phosphorylation level will lead to many diseases, so protein kinase has been regarded as an important target, for new drugs research and development. Therefore, both kinase activity analysis and its relate inhibitor screening require simple, low cost, fast and high throughput assaysIn recent years, with the preparation and functional nanomaterials technology trends to be mature, novel nanomaterials with unique optical, electrical, physical and chemical, catalytic properties have been widely applied in the bioanalytical chemistry and biosensors. In this paper, based on the excellent optical properties of gold nanoparticles and the intrinsic oxidase activity as well as fluorescence quenching ability of CeO2NPs, we constructed two kinds of visual inspection methods for detecting of protein kinase activity.1. In the second chapter, a new method was constructed for the visual detection of protein kinase activities based on the peptide phosphorylation-regulated crosslinking of functionalized AuNPs based on the significant regulatory role to the crosslinking aggregation state of gold nanoparticles before and after peptide substrates phosphorylation. In this method, biotin labeled peptide substrates firstly immobilized on the surface of streptavidin functional AuNPs through the interaction between biotin and streptavidin. For the biotinylated kemptide is positively charged, it will connect with another gold nanoparticles through electrostatic interactions, then color changes resulted from aggregation of gold nanoparticles. However, when the protein kinase is present, the negatively charged phosphate group of the biotin-phosphopeptide will no longer combine with the AuNPs through electrostatic force and efficiently prevent the AuNPs from crosslinking and aggregation efficiently, so the red color of AuNPs will be maintain. As a result, we can analysis kinase activities through the changes of solution color or the absorption intensity at 520 nm. This new colorimetric strategy for the detection of protein kinase activity shows many advantages including high sensitivity, simple operation, low-costs. This method can easily detect the PKA activity over a wide range from 0.0005 U/?L to 0.02 U/?L, and the detection limit is 0.0005 U/?L.2. The third chapter in this paper, we find that CeO2 nanoparticles can recognize and adsorb the products of phosphorylated peptide substrates specificity, which is catalyzed by protein kinase. Based on the fact that CeO2 can efficiently quench the fluorescence signal of the molecules adsorbed on its surface and the oxidase activity of CeO2can also be blocked by the adsorbed biomolecules, we have constructed CeO2-based dual-signal readout pathways for the sensitive detection of PKA activities. and it has successfully used in protein kinase activity of sensitivity analysis. On the one hand, by using fluorescence (TAMRA) labeled peptide as substrates, when the protein kinases are absent, peptides cannot be phosphorylated peptides and thus can not be adsorbed on the CeO2 surface. So the reaction liquid remains the original strong fluorescence intensity. when the protein kinases are present, fluorescence labeled substrate peptides were phosphorylated. Because CeO2 with an equilibrium state of Ce3+/Ce4+ on its surface, the phosphorylated peptide will be captured on the CeO2 through the interaction between Ce3+ and phosphate group, and thus the fluorescence of TAMRA will be efficiently quenched. As such, fluorescent signal gradually reduced with the increasing of the protein kinase activity, and PKs activity can be detected in a simple mix-and-read manner. On the other hand, CeO2 has the oxidase mimetic activity owing to the Ce4+on its surface, and the oxidation of organic substrates 3.3',5,5'-tetramethylbenzidine (TMB) by CeO2 can generate blue colored product when CeO2NPs combined with phosphorylated peptide, it's oxidation capacity are inhibited. Therefore, Visual analysis of protein kinase activity can be realized through observing the color change. Similarly, using the characteristic of the CeO2 nanoparticles, we have successfully constructed the fluorescence and color signal enhancement sensing system for the detecting of protein phosphatase activity. This method can easily detect the PKA activity with the detection limit is 0.0001 U/?L.