Analytical and Bioanalytical Chemistry

, Volume 403, Issue 2, pp 517–526

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

  • Meike V. Beer
  • Claudia Rech
  • Sylvia Diederichs
  • Kathrin Hahn
  • Kristina Bruellhoff
  • Martin Möller
  • Lothar Elling
  • Jürgen Groll
Original Paper

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.

Figure

We present a hydrogel coating system for well-plates that can be covalently modified with peptides, sugars or proteins by dip coating. These coatings then allow specific interaction screening of the immobilized ligands with peptides, proteins or cells.

Keywords

Bioassays Immunoassays/ELISA Interaction screening Biofunctionalization Hydrogel coating 96-Well plate format 

Abbreviations

BSA

Bovine serum albumin

ECM

Extracellular matrix

ELISA

Enzyme-linked immunosorbent assay

ELLA

Enzyme-linked lectin assay

FN

Fibronectin

GlcNAc

N-Acetylglucosamine

GSII

Lectin II from Griffonia simplicifolia

HDF

Primary human dermal fibroblast

His6CGL2

Recombinant lectin produced in Escherichia coli BL21

IPDI

Isophorone diisocyanate

LacNAc

N-acetyllactosamine

NCO-sP(EO-stat-PO)

Isocyanate functionalized six-arm star-shaped prepolymer

POD

Peroxidase

SA

Streptavidin

Copyright information

© Springer-Verlag 2012

Authors and Affiliations

  • Meike V. Beer
    • 1
  • Claudia Rech
    • 2
  • Sylvia Diederichs
    • 3
  • Kathrin Hahn
    • 1
  • Kristina Bruellhoff
    • 3
  • Martin Möller
    • 3
  • Lothar Elling
    • 2
  • Jürgen Groll
    • 1
  1. 1.Department of Functional Materials in Medicine and DentistryUniversity of WürzburgWürzburgGermany
  2. 2.Laboratory for Biomaterials, Institute for Biotechnology and Helmholtz Institute for Biomedical EngineeringRWTH Aachen UniversityAachenGermany
  3. 3.Interactive Materials Research Institute (DWI e.V.) and Institute of Technical and Macromolecular ChemistryRWTH Aachen UniversityAachenGermany

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