Automated preparation method for colloidal crystal arrays of monodisperse and binary colloid mixtures by contact printing with a pintool plotter.

Photonic crystals and photonic band gap materials with periodic variation of the dielectric constant in the submicrometer range exhibit unique optical properties such as opalescence, optical stop bands, and photonic band gaps. As such, they represent attractive materials for the active elements in sensor arrays. Colloidal crystals, which are 3D gratings leading to Bragg diffraction, are one potential precursor of such optical materials. They have gained particular interest in many technological areas as a result of their specific properties and ease of fabrication. Although basic techniques for the preparation of regular patterns of colloidal crystals on structured substrates by self-assembly of mesoscopic particles are known, the efficient fabrication of colloidal crystal arrays by simple contact printing has not yet been reported. In this article, we present a spotting technique used to produce a microarray comprising up to 9600 single addressable sensor fields of colloidal crystal structures with dimensions down to 100 mum on a microfabricated substrate in different formats. Both monodisperse colloidal crystals and binary colloidal crystal systems were prepared by contact printing of polystyrene particles in aqueous suspension. The array morphology was characterized by optical light microscopy and scanning electron microscopy, which revealed regularly ordered crystalline structures for both systems. In the case of binary crystals, the influence of the concentration ratio of the large and small particles in the printing suspension on the obtained crystal structure was investigated. The optical properties of the colloidal crystal arrays were characterized by reflection spectroscopy. To examine the stop bands of the colloidal crystal arrays in a high-throughput fashion, an optical setup based on a CCD camera was realized that allowed the simultaneous readout of all of the reflection spectra of several thousand sensor fields per array in parallel. In agreement with Bragg's relation, the investigated arrays exhibited strong opalescence and stop bands in the expected wavelength range, confirming the successful formation of highly ordered colloidal crystals. Furthermore, a narrow distribution of wavelength-dependent stop bands across the sensor array was achieved, demonstrating the capability of producing highly reproducible crystal spots by the contact printing method with a pintool plotter.

Large-scale protein expression for proteome research.

Access to pure and soluble recombinant proteins is essential for numerous applications in proteome research, such as the production of antibodies, structural characterization of proteins, and protein microarrays. Through the German cDNA Consortium we have access to more than 1500 ORFs encoding uncharacterized proteins. Preparing a large number of recombinant proteins calls for the careful refinement and re-evaluation of protein purification tools. The expression and purification strategy should result in mg quantities of protein that can be employed in microarray-based assays. In addition, the experimental set-up should be robust enough to allow both automated protein expression screening and the production of the proteins on a mg scale. These requirements are best fulfilled by a bacterial expression system such as Escherichia coli. To develop an efficient expression strategy, 75 different ORFs were transferred into suitable expression vectors using the Gateway cloning system. Four different fusion tags (E. coli transcription-termination anti-termination factor (NusA), hexahistidine tag (6xHis), maltose binding protein (MBP) and GST) were analyzed for their effect on yield of induced fusion protein and its solubility, as determined at two different induction temperatures. Affinity-purified fusion proteins were confirmed by MALDI-TOF MS.

Custom chemical microarray production and affinity fingerprinting for the S1 pocket of factor VIIa.

The goal of this study was to explore the applicability of surface plasmon resonance (SPR)-based fragment screening to identify compounds that bind to factor VIIa (FVIIa). Based on pharmacophore models virtual screening approaches, we selected fragments anticipated to have a reasonable chance of binding to the S1-binding pocket of FVIIa and immobilized these compounds on microarrays. In affinity fingerprinting experiments, a number of compounds were identified to be specifically interacting with FVIIa and shown to fall into four structural classes. The results demonstrate that the chemical microarray technology platform using SPR detection generates unique chemobiological information that is useful for de novo discovery and lead development and allows the detection of weak interactions with ligands of low molecular weight.