Simple One-Step Process for Immobilization of Biomolecules on Polymer Substrates Based on Surface-Attached Polymer Networks

Abstract

For the miniaturization of biological assays, especially for the fabrication of microarrays, immobilization of biomolecules at the surfaces of the chips is the decisive factor. Accordingly, a variety of binding techniques have been developed over the years to immobilize DNA or proteins onto such substrates. Most of them require rather complex fabrication processes and sophisticated surface chemistry. Here, a comparatively simple immobilization technique is presented, which is based on the local generation of small spots of surface attached polymer networks. Immobilization is achieved in a one-step procedure: probe molecules are mixed with a photoactive copolymer in aqueous buffer, spotted onto a solid support, and cross-linked as well as bound to the substrate during brief flood exposure to UV light. The described procedure permits spatially confined surface functionalization and allows reliable binding of biological species to conventional substrates such as glass microscope slides as well as various types of plastic substrates with comparable performance. The latter also permits immobilization on structured, thermoformed substrates resulting in an all-plastic biochip platform, which is simple and cheap and seems to be promising for a variety of microdiagnostic applications.

The Impact of Diffusion on Biochemical Assay Kinetics in 3d-Hydrogel Microarrays

“…To overcome this limitation and to increase the surface density of immobilized biomolecules, different methods based on polymer hydrogels as a new immobilization strategy were described (Rendl et al, 2011;Moschallski et al, 2010;Zubtsov et al, 2007;Rubina et al, 2003). A simple approach developed by Rendl et al allows for fast production of microarrays.…”

“…This results in the functionalization of the surface in a spatially confined manner. The use of plastic substrates abolishes the need of complex substrates functionalized with reactive groups such as NHS or aldehydes (Rendl et al, 2011).…”

“…The photo reactive MABP moiety undergoes n-π* or π-π* transition 1 forming a biradical triplet state. This state enables the molecule to abstract hydrogen atom from any neighboring aliphatic C-H group, then the two resulting carbon radicals are recombined to form C-C bond (Figure 1.9 B) (Rendl et al, 2011;Moschallski, 2007;Toomey et al, 2004).…”

“…Upon UV-irradiation the MABP undergoes n-π* or π-π* forming a biradical triplet state. Then, a hydrogen abstraction from C-H from neighboring protein and then recombines forming C-C stable bond (Rendl et al, 2011 reaction a) mass transport from bulk compartment ( ) to the reaction compartment ( ) and b) the binding reaction (Kusnezow et al, 2006a Figure 3.4 Schematic representation for the formation of depletion zones with increasing the incubation time (Bönisch, 2008 (Rendl, 2009 .18 The kinetic processes involved in the signal development of 3D-hydrogel microarrays. 1) Represents the initial linear phase reflecting the linear signal development due to mass flux from the solution to the surface of the spot, 2) represents the end of phase one and the pseudo steady state, 3) represents the diffusion in the hydrogel and the linear part reflects the diffusion rate in the hydrogel, and 4) thermodynamic equilibrium due to saturation of the binding sites.…”

Microfluidic barcode assay for antibody-based confirmatory diagnostics

“…32 The reactive, functionalized gels provide fixed reagent capture sites distributed throughout the channel bulk, thus yielding a pseudohomogeneous assay. [33][34][35][36] Enhancing the capture site and reagent interactions dramatically, these pseudo-homogeneous approaches reduce interaction distance and the time required for diffusion-based capture.…”