title: Highly sensitive protein kinase activity assay based on electrochemiluminescence nanoprobes.
abstract: Herein, we describe a novel electrochemiluminescence (ECL) biosensor for protein kinase activities and inhibition monitoring based on the magnetic beads (MB) technology and signal enhancement of gold nanoparticles (GNP). In this design, ECL nanoprobes were prepared by conjugating GNP with phosphorylated DNA capture probes and tris-(2,2'-bipyridyl) ruthenium (TBR)-cysteamine. Zirconium cations, a specific bridging agent, mediate the linkage between biotin modified phosphorylated peptides and ECL nanoprobes. The complexes were then captured and enriched on the electrode surface by streptavidin-coated MB for ECL reaction. To confirm the feasibility of this biosensor, we employed protein kinase A (PKA) as the model kinase to validate the assay and a satisfactory detection limit of 0.005 U/mL was achieved. The combination of ECL and GNP lays a solid foundation for highly sensitive assay, meanwhile, the coupling of MB surfaces used for separation and capture with unmodified ECL electrode detection results in a greatly simplified and reusable protocol. Thus, our biosensor offers great promise for a highly sensitive and simple assay for protein kinase activity. Furthermore, the inhibition of PKA activity was monitored on the basis of the ECL signals change in response to the concentration of PKA inhibitor.
authors.affiliation: MOE Key Laboratory of Laser Life Science, Institute of Laser Life Science, College of Biophotonics, South China Normal University, Guangzhou 510631, China.
authors.forename: Zhen
authors.initials: Z
authors.lastname: Zhao
authors.forename: Xiaoming
authors.initials: X
authors.lastname: Zhou
authors.forename: Da
authors.initials: D
authors.lastname: Xing
mesh_terms: D015374:Biosensing Techniques / Q000295:instrumentation*; D003217:Conductometry / Q000295:instrumentation*; D004247:DNA / Q000737:chemistry*; D004789:Enzyme Activation; D004867:Equipment Design; D019544:Equipment Failure Analysis; D006046:Gold / Q000737:chemistry; D018189:Immunomagnetic Separation / Q000295:instrumentation*; D008163:Luminescent Measurements / Q000295:instrumentation*; D053758:Nanoparticles / Q000737:chemistry*; D036103:Nanotechnology / Q000295:instrumentation; D011494:Protein Kinases / Q000032:analysis / Q000737:chemistry*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
chemical_list: D006046:Gold; D004247:DNA; D011494:Protein Kinases