Phosphorylation-Directed Assembly of a Single Quantum Dot Based Nanosensor for Protein Kinase Assay

	Phosphorylation-Directed Assembly of a Single Quantum Dot Based Nanosensor for Protein Kinase Assay
	Lijuan Wang; Yong Yang; Chunyang Zhang
刊名	ANALYTICAL CHEMISTRY
	2015
英文摘要	Protein kinases play crucial roles in intracellular signal transduction and metabolic pathways, and the monitoring of protein kinase activity is essential to the understanding of fundamental biochemical processes and the clinical diagnosis. Here, we demonstrate the phosphorylationdirected assembly of a single quantum dot (QD)-based nanosensor for sensitive detection of cAMP-dependent protein kinase (PKA). This assay involves (1) the PKA-directed simultaneous phosphorylation and biotinylation of cyanine 5 (Cy5)-labeled substrate peptides, (2) the assembly of phosphorylated and biotinylated peptides onto the surface of the QD, and (3) the illumination of Cy5 by means offluorescence resonance energy transfer (FRET) between the QD and Cy5. With an adenosine triphosphate (ATP) analogue,γ-biotin-ATP, as the phosphoryl donor, the PKA-catalyzed phosphorylation reaction incorporates the biotin-conjugated phosphate group into the substrate peptides to form the biotinylated peptides. The biotin entity subsequently drives the assembly of peptides onto the surface of streptavidin-functionalized QD to form the sandwiched Cy5−peptide−QD nanostructure, enabling the occurrence of FRET between the QD and Cy5. The FRET signal can be easily recorded by either the conventionalfluorescence spectrometer or the total internal reflectionfluorescence (TIRF) microscope. In contrast, the absence of PKA cannot lead to the formation of Cy5−peptide−QD complex and no Cy5 signal can be detected. This protein kinase-actuated FRET assay is straightforward, without the involvement of either washing or separation steps, and has a significant advantage of high sensitivity with a detection limit of 9.3 ×10−6 U/μL. Moreover, this method can be used to estimate the half-maximal inhibitory concentration (IC50) value of PKA inhibitor H-89 (N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide dihydrochloride) and to monitor forskolin (Fsk)/3-isobutyl-1-methylxanthine (IBMX)-triggered activation of PKA in cell lysates, thus holding great potential for further applications in protein kinase-related biological researches and drug discovery.
收录类别	SCI
原文出处	http://pubs.acs.org/doi/abs/10.1021/ac504358q
语种	英语
内容类型	期刊论文
源URL	[http://ir.siat.ac.cn:8080/handle/172644/7361]
专题	深圳先进技术研究院_医药所
作者单位	ANALYTICAL CHEMISTRY
推荐引用方式 GB/T 7714	Lijuan Wang,Yong Yang,Chunyang Zhang. Phosphorylation-Directed Assembly of a Single Quantum Dot Based Nanosensor for Protein Kinase Assay[J]. ANALYTICAL CHEMISTRY,2015.
APA	Lijuan Wang,Yong Yang,&Chunyang Zhang.(2015).Phosphorylation-Directed Assembly of a Single Quantum Dot Based Nanosensor for Protein Kinase Assay.ANALYTICAL CHEMISTRY.
MLA	Lijuan Wang,et al."Phosphorylation-Directed Assembly of a Single Quantum Dot Based Nanosensor for Protein Kinase Assay".ANALYTICAL CHEMISTRY (2015).