Microarray-based detection of protein binding and functionality by gold nanoparticle probes.
We report a microarray format for the detection of proteins and protein functionality (kinase activity) based on marking either specific antibody-protein binding or peptide phosphorylation events by attachment of gold nanoparticles followed by silver deposition for signal enhancement. The attachment of the gold nanoparticles is achieved by standard avidin-biotin chemistry. The detection principle is resonance light scattering. Highly selective recognition of standard proteins (proteins A and G) down to 1 pg/mL for proteins in solution and 10 fg for proteins on the microarray spots is demonstrated. Enzyme activity of the kinase (PKA) is detected with high specificity down to a limit of 1 fg for an established peptide substrate (kemptide) on the microarray spots. Kinase inhibition by the inhibitor (H89) is shown, demonstrating the potential for high-throughput screening for inhibitors.
Wang
Zhenxin
Z
Centre for Nanoscale Science, Department of Chemistry and School of Biological Sciences, University of Liverpool, Liverpool, L69 7ZD U.K.
Lee
Jason
J
Cossins
Andrew R
AR
Brust
Mathias
M
D006046:Gold / Q000737:chemistry*; D018625:Microscopy, Atomic Force; D053758:Nanoparticles / Q000032:analysis* / Q000737:chemistry* / Q000648:ultrastructure; D010455:Peptides / Q000378:metabolism; D040081:Protein Array Analysis / Q000379:methods*; D011485:Protein Binding; D011506:Proteins / Q000032:analysis* / Q000378:metabolism*; D012996:Solutions
D010455:Peptides; D011506:Proteins; D012996:Solutions; D006046:Gold