Protein kinase assay on peptide-conjugated gold nanoparticles.
We demonstrate that protein kinase can be assayed with high sensitivity on peptide-conjugated gold nanoparticles (AuNPs). Phosphorylation of peptides on the AuNP-monolayers was detected by using an anti-phosphotyrosine antibody (alpha-pY) and Cy3-labeled secondary antibody (Cy3-alpha-mIgG) as a probing molecule. When compared to conventional self-assembled monolayers (SAMs), spherical and three-dimensional geometry of AuNPs led to high surface density of peptide substrate and easy accessibility to enzyme, and consequently the resulting AuNP monolayers gave rise to improved detection sensitivity. Blocking of peptide-conjugated AuNPs with a poly(ethylene glycol) (PEG) also contributed to a higher signal-to-background ratio in kinase and its inhibition assays. The use of AuNPs as the platform surface will enable highly sensitive detection of protein kinases in a high-throughput manner.
Kim
Young-Pil
YP
Department of Biological Sciences, Korea Advanced Institute of Science and Technology (KAIST), Daejeon, South Korea.
Oh
Young-Hee
YH
Kim
Hak-Sung
HS
D015374:Biosensing Techniques / Q000295:instrumentation* / Q000379:methods; D020099:Coated Materials, Biocompatible / Q000737:chemistry; D004867:Equipment Design; D019544:Equipment Failure Analysis; D006046:Gold / Q000737:chemistry*; D007118:Immunoassay / Q000295:instrumentation* / Q000379:methods; D053758:Nanoparticles / Q000737:chemistry* / Q000648:ultrastructure; D010455:Peptides / Q000737:chemistry*; D011485:Protein Binding; D011494:Protein Kinases / Q000032:analysis* / Q000737:chemistry*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
D020099:Coated Materials, Biocompatible; D010455:Peptides; D006046:Gold; D011494:Protein Kinases