A hydrogel-based versatile screening platform for specific biomolecular recognition in a well plate format

Abstract

Precise determination of biomolecular interactions in high throughput crucially depends on a surface coating technique that allows immobilization of a variety of interaction partners in a non-interacting environment. We present a one-step hydrogel coating system based on isocyanate functional six-arm poly(ethylene oxide)-based star polymers for commercially available 96-well microtiter plates that combines a straightforward and robust coating application with versatile bio-functionalization. This system generates resistance to unspecific protein adsorption and cell adhesion, as demonstrated with fluorescently labeled bovine serum albumin and primary human dermal fibroblasts (HDF), and high specificity for the assessment of biomolecular recognition processes when ligands are immobilized on this surface. One particular advantage is the wide range of biomolecules that can be immobilized and convert the per se inert coating into a specifically interacting surface. We here demonstrate the immobilization and quantification of a broad range of biochemically important ligands, such as peptide sequences GRGDS and GRGDSK-biotin, the broadly applicable coupler molecule biocytin, the protein fibronectin, and the carbohydrates N-acetylglucosamine and N-acetyllactosamine. A simplified protocol for an enzyme-linked immunosorbent assay was established for the detection and quantification of ligands on the coating surface. Cell adhesion on the peptide and protein-modified surfaces was assessed using HDF. All coatings were applied using a one-step preparation technique, including bioactivation, which makes the system suitable for high-throughput screening in a format that is compatible with the most routinely used testing systems.

Immobilization of glycans on solid surfaces for application in glycomics

“…Later, amine-linked poly LacNAc was coupled to the isocyanate groups of the polymer as the first step in creating a layered biomimetic display of extracellular matrix components. [157,158]…”

The Next Step in Biomimetic Material Design: Poly‐LacNAc‐Mediated Reversible Exposure of Extra Cellular Matrix Components

“…In short, prepolymers were diluted in dry THF (100 mg ml −1 ), water was added (THF/water 1/9), prepolymers left for crosslinking for 5 min and spin coated on aminosilanised glass and silicon (2500 rpm, 40 sec, 5 sec acceleration time). 96‐well plates were coated with the prepolymer as described earlier 28. In short, prepolymers were diluted in THF/water as described above, left for 5 min of crosslinking, 400 μL were filled into each well of a CovaLinkTM NH 96‐well plate (NUNC, Langenselbold, Germany) and left for 10 min.…”

Carbohydrate‐Modified Gold Nanoparticles

“…The reaction between amines and isocyanate or isothiocyanate groups (Table 1.3; entries t and u) has also been used to immobilize carbohydrate derivatives on surfaces. Amine-functionalized carbohydrates were successfully immobilized onto 96-well plates coated with an isocyanate-presenting polymer [89]. Using a similar reaction, a mannose derivative containing an isothiocyanate group was attached to amine-terminated surfaces, and the resulting surfaces were used for bacterial capture [90].…”