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

Abstract

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.

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“…Chips were pretreated using a HARRICK Plasma Cleaner, PDC-002 (Ithaca, NY, USA), connected to an oxygen line. Copoly azide 4% copolymer (4% of azide groups) was synthesized as reported elsewhere [14]. MCP-4 is the trade name of a polymer purchased from Lucidant Polymers Inc. (Sunnyvale, CA, USA).…”

“…Stabilizer DNA sequence was used without modification. The polymer was synthesized by free radical polymerization as reported in [14]. Briefly, in a two-neck round-bottom flask, 10 mL of anhydrous THF were degassed for 20 min by insufflating argon.…”

“…The polymer was synthesized by post-polymerization modification of copoly NAS positive 4%, similarly to what was described in [14]. In a two-neck round-bottom flask, copoly NAS positive 4% (0.5 g, 0.0483 mmol) was solubilized with 5 mL of anhydrous THF and degassed for 5 min by insufflating argon.…”

“…The approach involves chemisorption of polymers containing click chemistry functional groups, effectively resolving many of the issues encountered in conventional methods. Copolymers derived from N,N-dimethylacrylamide (copoly-DMA) have emerged as highly versatile coatings suitable for a wide range of applications [14]. Notably, they have found extensive use in the development of microarrays and multispot biosensors on various substrates, as supported by relevant references [15].…”

“…This class of copolymers offers rapid and durable surface adhesion, facilitating the covalent attachment of biomolecules while exhibiting excellent antifouling properties [16]. The key precursor of these copolymers, N-acryloyloxysuccinimide (NAS), features a functional moiety that readily reacts with functional groups conducive to click-chemistry reactions, such as azide/alkyne reactions [14]. Furthermore, click-chemistry reactions facilitate the biorthogonal orientation of immobilized probes.…”