Characterization and applications of the dendrimer monolayers to bio-affinity sensing for development of protein chips

Abstract

A parallel analysis of bio-specific interactions on a surface has required the development of biomolecular interfaces in order to retain the activity and the stability of immobilized biomolecules at high densities. As a novel interface, the unique features of a dendrimer monolayer were characterized for protein-ligand interactions. The kinetic and equilibrium analysis of streptavidin-biotin interactions revealed that the dendrimer monolayer renders the unique structural features in terms of the protein coverage and the binding kinetics for streptavidin-biotin interactions, which seem to stem from the synergistic effect caused by the surface orientation of functionalized biotin ligands and the surface orientation. And the layer of tri(ethylene oxide)-attached, fourth-generation poly(amidoamine) dendrimers, more resistant to nonspecific binding of albumin and serum protein, was synthesized and characterized. The specific binding of avidin to the biotinylated layers approached a surface density of $5.2±0.2 ngㆍmm^{-2}$, showing about 92% of full surface coverage. Next, the dendrimer monolayer was implemented as the interfacing layer for chip-based analyses of protein interactions and post-translational modifications. The monolayer of G4 PAMAM dendrimers was adopted to construct the immuno-affinity surface of an antibody layer with high binding capacity of antigenic proteins and a reliable detection. The affinity-captured protein at the immunosensing surface was subjected to direct on-chip tryptic digestion, and the resulting proteolytic peptides were analyzed by using MALDI-TOF MS. The established detection procedure identified the affinity-captured target protein at the level of around 100 fmol and enabled the identification of target protein in biological samples at the level of 0.1 ng/ml. In addition, a chip-based assay of sumoylation, the process of conjugating small ubiquitin-like modifier (SUMO) to a target protein and mediated by multiple enzymes, was develo...