Analytical and Bioanalytical Chemistry

, Volume 403, Issue 2, pp 517–526 | Cite as

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

References

  1. 1.
    Geistlinger J, Du W, Groll J, Liu F, Hoegel J, Foehr KJ, Pasquarelli A, Schneider EM (2011) P2RX7 genotype association in severe sepsis identified by a novel multi-individual array for rapid screening and replication of risk SNPs. Clin Chim Acta. doi:10.1016/j.cca.2011.05.023
  2. 2.
    Zhang M, Desai T, Ferrari M (1998) Proteins and cells on PEG immobilized silicon surfaces. Biomaterials 19:953–960CrossRefGoogle Scholar
  3. 3.
    Feldman K, Haehner G, Spencer ND, Harder P, Grunze M (1999) Probing resistance to protein adsorption of oligo(ethylene glycol)-terminated self-assembled monolayers by scanning force microscopy. J Am Chem Soc 121:10134–10141CrossRefGoogle Scholar
  4. 4.
    Edinger K, Golzhauser A, Demota K, Woll C, Grunze M (1993) Formation of self-assembled monolayers of n-alkanethiols on gold—a scanning tunneling microscopy study on the modification of substrate morphology. Langmuir 9:4–8CrossRefGoogle Scholar
  5. 5.
    Harder P, Grunze M, Dahint R, Whitesides GM, Laibinis PE (1998) Molecular conformation in oligo(ethylene glycol)-terminated self-assembled monolayers on gold and silver surfaces determines their ability to resist protein adsorption. J Phys Chem B 102:426–436CrossRefGoogle Scholar
  6. 6.
    Mehne J, Markovic G, Proll F, Schweizer N, Zorn S, Schreiber F, Gauglitz G (2008) Characterisation of morphology of self-assembled PEG monolayers: a comparison of mixed and pure coatings optimised for biosensor applications. Anal Bioanal Chem 391:1783–1791CrossRefGoogle Scholar
  7. 7.
    Mrksich M, Whitesides GM (1996) Using self-assembled monolayers to understand the interactions of man-made surfaces with proteins and cells. Annu Rev Biophys Biomol Struct 25:55–78CrossRefGoogle Scholar
  8. 8.
    Peppas NA, Hilt JZ, Khademhosseini A, Langer R (2006) Hydrogels in biology and medicine: from molecular principles to bionanotechnology. Adv Mater 18:1345–1360CrossRefGoogle Scholar
  9. 9.
    Prime KL, Whitesides GM (1991) Self-assembled organic monolayers—model systems for studying adsorption of proteins at surfaces. Science 252:1164–1167CrossRefGoogle Scholar
  10. 10.
    Wang RLC, Kreuzer HJ, Grunze M (1997) Molecular conformation and solvation of oligo(ethylene glycol)-terminated self-assembled monolayers and their resistance to protein adsorption. J Phys Chem B 101:9767–9773CrossRefGoogle Scholar
  11. 11.
    Anderson AS, Dattelbaum AM, Montano GA, Price DN, Schmidt JG, Martinez JS, Grace WK, Grace KM, Swanson BI (2008) Functional PEG-modified thin films for biological detection. Langmuir 24:2240–2247CrossRefGoogle Scholar
  12. 12.
    Kawaguchi T, Shankaran DR, Kim SJ, Gobi KV, Matsumoto K, Toko K, Miura N (2007) Fabrication of a novel immunosensor using functionalized self-assembled monolayer for trace level detection of TNT by surface plasmon resonance. Talanta 72:554–560CrossRefGoogle Scholar
  13. 13.
    Bradner JE, McPherson OM, Mazitschek R, Barnes-Seeman D, Shen JP, Dhaliwal J, Stevenson KE, Duffner JL, Park SB, Neuberg DS, Nghiem P, Schreiber SL, Koehler AN (2006) A robust small-molecule microarray platform for screening cell lysates. Chem Biol 13:493–504CrossRefGoogle Scholar
  14. 14.
    Cheng F, Shang J, Ratner DM (2011) A versatile method for functionalizing surfaces with bioactive glycans. Bioconjugate Chem 22:50–57CrossRefGoogle Scholar
  15. 15.
    Behravesh E, Sikavitsas VI, Mikos AG (2003) Quantification of ligand surface concentration of bulk-modified biomimetic hydrogels. Biomaterials 24:4365–4374CrossRefGoogle Scholar
  16. 16.
    Sung D, Park S, Jon S (2009) Facile method for selective immobilization of biomolecules on plastic surfaces. Langmuir 25:11289–11294CrossRefGoogle Scholar
  17. 17.
    Groll J, Ameringer T, Spatz JP, Moeller M (2005) Ultrathin coatings from isocyanate-terminated star PEG prepolymers: layer formation and characterization. Langmuir 21:1991–1999CrossRefGoogle Scholar
  18. 18.
    Salber J, Grater S, Harwardt M, Hofmann M, Klee D, Dujic J, Huang JH, Ding JD, Kippenberger S, Bernd A, Groll J, Spatz JP, Moller M (2007) Influence of different ECM mimetic peptide sequences embedded in a nonfouling environment on the specific adhesion of human-skin keratinocytes and fibroblasts on deformable substrates. Small 3:1023–1031CrossRefGoogle Scholar
  19. 19.
    Groll J, Fiedler J, Engelhard E, Ameringer T, Tugulu S, Klok HA, Brenner RE, Moeller M (2005) A novel star PEG-derived surface coating for specific cell adhesion. J Biomed Mat Res Part A 74A:607–617CrossRefGoogle Scholar
  20. 20.
    Gasteier P, Reska A, Schulte P, Salber J, Offenhausser A, Moeller M, Groll J (2007) Surface grafting of PEO-based star-shaped molecules for bioanalytical and biomedical applications. Macromol Biosci 7:1010–1023CrossRefGoogle Scholar
  21. 21.
    Cummings RD, Kornfeld S (1984) The distribution of repeating [Galbeta1,4GlcNAcbeta1,3] sequences in asparagine-linked oligosaccharides of the mouse lymphoma cell-lines BW5147 and PHAR2.1—binding of oligosaccharides containing these sequences to immobilized datura–stramonium agglutinin. J Biol Chem 259:6253–6260Google Scholar
  22. 22.
    Fukuda M, Carlsson SR, Klock JC, Dell A (1986) Structures of O-linked oligosaccharides isolated from normal granulocytes, chronic myelogenous leukemia-cells, and acute myelogenous leukemia-cells. J Biol Chem 261:2796–2806Google Scholar
  23. 23.
    Krusius T, Finne J, Rauvala H (1978) Poly(glycosyl) chains of glycoproteins—characterization of a novel type of glycoprotein saccharides from human erythrocyte-membrane. Eur J Biochem 92:289–300CrossRefGoogle Scholar
  24. 24.
    McEver RP, Moore KL, Cummings RD (1995) Leukocyte trafficking mediated by selectin–carbohydrate interactions. J Biol Chem 270:11025–11028CrossRefGoogle Scholar
  25. 25.
    Ujita M, McAuliffe J, Suzuki M, Hindsgaul O, Clausen H, Fukuda MN, Fukuda M (1999) Regulation of I-branched poly-N-acetyllactosamine synthesis—concerted actions by i-extension enzyme, I-branching enzyme, and β1,4-galactosyltransferase I. J Biol Chem 274:9296–9304CrossRefGoogle Scholar
  26. 26.
    Zhou DP (2003) Why are glycoproteins modified by poly-N-acetyllactosamine glycoconjugates? Curr Prot Pept Sc 4:1–9CrossRefGoogle Scholar
  27. 27.
    Elola MT, Wolfenstein-Todel C, Troncoso MF, Vasta GR, Rabinovich GA (2007) Galectins: matricellular glycan-binding proteins linking cell adhesion, migration, and survival. Cell Mol Life Sci 64:1679–1700CrossRefGoogle Scholar
  28. 28.
    Hughes RC (2001) Galectins as modulators of cell adhesion. Biochimie 83:667–676CrossRefGoogle Scholar
  29. 29.
    Goetz H, Beginn U, Bartelink CF, Grunbauer HJM, Moeller M (2002) Preparation of isophorone diisocyanate terminated star polyethers. Macromol Mater Eng 287:223–230CrossRefGoogle Scholar
  30. 30.
    Rech C, Rosencrantz RR, Křenek K, Pelantová H, Bojarová P, Römer C, Hanisch F-G, Křen V, Elling L (2011) Combinatorial one-pot synthesis of poly-N-acetyllactosamine oligosaccharides with leloir-glycosyltransferases. Adv Synth Catal 353:2492–2500CrossRefGoogle Scholar
  31. 31.
    Sauerzapfe B, Křenek K, Schmiedel J, Wakarchuk WW, Pelantová H, Křen V, Elling L (2009) Chemo-enzymatic synthesis of poly-N-acetyllactosamine (poly-LacNAc) structures and their characterization for CGL2-galectin-mediated binding of ECM glycoproteins to biomaterial surfaces. Glycoconjugate J 26:141–159CrossRefGoogle Scholar
  32. 32.
    Lee SH, Ruckenstein E (1988) Adsorption of proteins onto polymeric surfaces of different hydrophilicities—a case-study with bovine serum-albumin. J Colloid Interface Sci 125:365–379CrossRefGoogle Scholar
  33. 33.
    Walser PJ, Haebel PW, Kunzler M, Sargent D, Kues U, Aebi M, Ban N (2004) Structure and functional analysis of the fungal galectin CGL2. Structure 12:689–702CrossRefGoogle Scholar

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

Personalised recommendations