Optimization of Functional Group Concentration of N, N-Dimethylacrylamide-based Polymeric Coatings and Probe Immobilization for DNA and Protein Microarray Applications

We report here a deep investigation into the effect of the concentration of a polymeric coating's functional groups on probe density immobilization with the aim of establishing the optimal formulation to be implemented in specific microarray applications. It is widely known that the ideal performance of a microarray strictly depends on the way probes are tethered to the surface since it influences the way they interact with the complementary target. The N, N-dimethylacrylamide-based polymeric coating introduced by our research group in 2004 has already proven to offer great flexibility for the customization of surface properties; here, we demonstrate that it also represents the perfect scaffold for the modulation of probe grafting. With this aim in mind, polymers with increasing concentrations of N-acryloyloxysuccinimide (NAS) were synthesized and the coating procedure optimized accordingly. These were then tested not only in DNA microarray assays, but also using protein probes (with different MWs) to establish which formulation improves the assay performance in specific applications. The flexibility of this polymeric platform allowed us also to investigate a different immobilization chemistry-specifically, click chemistry reactions, thanks to the insertion of azide groups into the polymer chains-and to evaluate possible differences generated by this modification.

Automated summary

We conducted a detailed investigation on the effect of a polymeric coating's functional group concentration on probe density immobilization, aiming to find the optimal formulation for specific microarray applications. The performance of a microarray is known to be influenced by the way probes are attached to the surface, and our N, N-dimethylacrylamide-based polymeric coating has shown great flexibility for customizing surface properties. Through synthesizing polymers with increasing concentrations of N-acryloyloxysuccinimide (NAS) and optimizing the coating procedure accordingly, we tested their performance in DNA and protein microarray assays to determine the formulation that improves assay performance in specific applications. The flexibility of this polymeric platform also allowed us to explore different immobilization chemistries, specifically click chemistry reactions, and assess the potential differences generated by this modification.