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.