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Mario Beyer,
Ines Block,
Kai König,
Alexander Nesterov,
Simon Fernandez,
Thomas Felgenhauer,
Christopher Schirwitz,
Klaus Leibe,
Ralf F Bischoff,
Frank Breitling,
Volker Stadler
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ABSTRACT: Combinatorial synthesis of peptides on solid supports (1), either as spots on cellulose membranes (2) or with split-pool-libraries on polymer beads (3), substantially forwarded research in the field of peptide-protein interactions. Admittedly, these concepts have specific limitations, on one hand the number of synthesizable peptide sequences per area, on the other hand elaborate decoding/encoding strategies, false-positive results and sequence limitations. We recently established a method to produce high-density peptide arrays on microelectronic chips (4). Solid amino acid microparticles were charged by friction and transferred to defined pixel electrodes onto the chip's surface, where they couple to a functional polymer coating simply upon melting (Fig. 16.1 A-D,F). By applying standard Fmoc chemistry according to Merrifield, peptide array densities of up to 40,000 spots per square centimetre were achieved (Fig. 16.1G). The term "Merrifield synthesis" describes the consecutive linear coupling and deprotecting of L-amino acids modified with base-labile fluorenylmethoxy (Fmoc) groups at the N-terminus and different acid-sensitive protecting groups at their side chains. Removing side chain protecting groups takes place only once at the very end of each synthesis and generates the natural peptide sequence thereby.
Methods in molecular biology (Clifton, N.J.) 02/2009; 570:309-16.
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ABSTRACT: We synthesized various graft copolymer films of poly(ethylene glycol) methacrylate (PEGMA) and methyl methacrylate (MMA) on silicon to examine the dependency of protein-surface interactions on grafting composition. We optimized atom transfer radical polymerizations to achieve film thicknesses from 25 to 100 nm depending on the monomer mole fractions, and analyzed the resulting surfaces by X-ray photoelectron spectroscopy (XPS), ellipsometry, contact angle measurements, and atomic force microscopy (AFM). As determined by XPS, the stoichiometric ratios of copolymer graftings correlated with the concentrations of provided monomer solutions. However, we found an unexpected and pronounced hydrophobic domain on copolymer films with a molar amount of 10-40% PEGMA, as indicated by advancing contact angles of up to 90 degrees . Nevertheless, a breakdown of the protein-repelling character was only observed for a fraction of 15% PEGMA and lower, far in the hydrophobic domain. Investigation of the structural basis of this exceptional wettability by high-resolution AFM demonstrated the independence of this property from morphological features.
Langmuir 08/2008; 24(15):8151-7. · 4.19 Impact Factor
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Volker Stadler,
Thomas Felgenhauer, Mario Beyer,
Simon Fernandez,
Klaus Leibe,
Stefan Güttler,
Martin Gröning,
Kai König,
Gloria Torralba,
Michael Hausmann,
Volker Lindenstruth,
Alexander Nesterov,
Ines Block,
Rüdiger Pipkorn,
Annemarie Poustka,
F Ralf Bischoff,
Frank Breitling
Angewandte Chemie International Edition 02/2008; 47(37):7132-5. · 13.45 Impact Factor
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Mario Beyer,
Alexander Nesterov,
Ines Block,
Kai König,
Thomas Felgenhauer,
Simon Fernandez,
Klaus Leibe,
Gloria Torralba,
Michael Hausmann,
Ulrich Trunk,
Volker Lindenstruth,
F Ralf Bischoff,
Volker Stadler,
Frank Breitling
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ABSTRACT: Arrays promise to advance biology through parallel screening for binding partners. We show the combinatorial in situ synthesis of 40,000 peptide spots per square centimeter on a microchip. Our variant Merrifield synthesis immobilizes activated amino acids as monomers within particles, which are successively attracted by electric fields generated on each pixel electrode of the chip. With all different amino acids addressed, particles are melted at once to initiate coupling. Repetitive coupling cycles should allow for the translation of whole proteomes into arrays of overlapping peptides that could be used for proteome research and antibody profiling.
Science 01/2008; 318(5858):1888. · 31.20 Impact Factor
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ABSTRACT: Complementary metal oxide semiconductor (CMOS) microelectronic chips fulfill important functions in the field of biomedical research, ranging from the generation of high complexity DNA and protein arrays to the detection of specific interactions thereupon. Nevertheless, the issue of merging pure CMOS technology with a chemically stable surface modification which further resists interfering nonspecific protein adsorption has not been addressed yet. We present a novel surface coating for CMOS microchips based on poly(ethylene glycol)methacrylate graft polymer films, which in addition provides high loadings of functional groups for the linkage of probe molecules. The coated microchips were compatible with the harshest conditions emerging in microarray generating methods, thoroughly retaining structural integrity and microelectronic functionality. Nonspecific adsorption of proteins on the chip's surface was completely obviated even with complex serum protein mixtures. We could demonstrate the background-free antibody staining of immobilized probe molecules without using any blocking agents, encouraging further integration of CMOS technology in proteome research.
Journal of Proteome Research 09/2007; 6(8):3197-202. · 5.11 Impact Factor
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ABSTRACT: Glass slides have been modified with a multifunctional poly(ethylene glycol) (PEG)-based polymer with respect to array applications in the growing field of proteome research. We systematically investigated the stepwise synthesis of the PEG films starting from self-assembled alkyl silane monolayers via monolayer peroxidation and subsequent graft polymerization of PEG methacrylate (PEGMA). Chemical composition was examined by X-ray photoelectron spectroscopy (XPS); infrared spectroscopy provided information about order and composition of the films as well; film thickness was determined by ellipsometry; using fluorescence microscopy and again XPS, the amount of proteins adsorbed on the slides was investigated. The novel support material allows a versatile modification of the amino group surface density up to 40 nmol/cm(2) for the linkage of probe molecules. Further on, we carried out standard peptide synthesis based on the well-established 9-fluorenylmethoxycarbonyl (Fmoc) chemistry, which was monitored by UV/Vis quantification of the Fmoc deblocking and mass spectrometry. The polymer coating is stable with respect to a wide range of chemical and thermal conditions, and prevents the glass surface from unspecific protein adsorption. Finally, we applied our modified glass slides in immunoassays and thus examined specific interactions of monoclonal antibodies with appropriate peptide epitopes.
Biomaterials 07/2006; 27(18):3505-14. · 7.40 Impact Factor
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ABSTRACT: The synthesis and electron paramagnetic resonance (EPR) spectroscopic properties of three novel aromatic nitroxides and potential DNA intercalators, the carbazole-based 3,6-dimethylcarbazole-9-oxyl, as well as the acridane-based 9-acridanylidenemalonitrile-10-oxyl and 9-ethylacridanylidenecyanoacetate-10-oxyl, are described. The two acridane-based nitroxides can be isolated and are stable in solution as well as in the solid state for several days. Continuous wave X-band EPR measurements and density functional theory (DFT) calculations demonstrated that the spin density is delocalized over the whole molecule in all three cases. Furthermore, the DFT calculations provided insight into the molecular and electronic structures of these nitroxides and yielded hyperfine coupling constants which are in very good agreement with the experimental data allowing therefore an unambiguous assignment of the hyperfine couplings.
The Journal of Organic Chemistry 04/2003; 68(6):2209-15. · 4.45 Impact Factor
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ABSTRACT: The novel nitroxide biradical 1,8-bis(3-ethinyl-2,2,5,5-tetramethyl-3-pyrroline-1-oxyl)-naphthalene was synthesized and its structure and magnetic properties were investigated. Using SQUID measurements an antiferromagnetic exchange coupling of 2J=−3.5 K was evaluated. Temperature-dependent measurements of the half-field EPR signal intensity and broken symmetry DFT computations confirm this result. To unravel the mechanisms of the intramolecular exchange interaction calculations on model systems were performed. These revealed a strong ferromagnetic through-bond interaction via the 1,8-substituted naphthalene bridge and a competing strong antiferromagnetic through-space interaction via the acetylene groups. Both interactions are of the same order of magnitude leading to the weak overall coupling observed.
Chemical Physics Letters.
