Microarray planar

The manufacture and processing of the protein microarray should be conducted in such a manner that the arrayed proteins remain in their native and active state. For most proteins, this usually means the hydrated state in order to avoid surface denaturation. For antibody arrays which are perhaps more forgiving than other proteins, it has been our experience that while these could be stored cold and dry, it is most important to rehydrate them prior to use. This process is in sharp contrast to the preparation of nucleic acid arrays in which strand melting or denaturahon is necessary to achieve optimal binding to the solid support. While the hybridization process is well understood and can be controlled under thermodynamic principles, the folding and renaturation of proteins on planar (microarray) surfaces is under study. 

Microarrays are solid phase-based assay systems consisting of an array of miniaturized test sites. This allows many tests to be performed in parallel. Planar protein microarray systems consist of capture molecules immobilized in rows and columns in microspots... 

In protein microarrays, capture molecules need to be immobilized in a functional state on a solid support. In principle, the format of the assay system does not limit the choice of appropriate surface chemistry. The same immobilization procedure can be applied for both planar and bead-based systems. Proteins can be immobilized on various surfaces (Fig. 1) (12). Two-dimensional polystyrene, polylysine, aminosilane, or aldehyde, epoxy- or thiol group-coated surfaces can be used to immobilize proteins via noncovalent or covalent attachment (13,14). Three-dimensional supports like nitrocellulose or hydrogel-coated surfaces enable the immobilization of the proteins in a network structure. Larger quantities of proteins can be immobilized and kept in a functional state. Affinity binding reagents such as protein A, G, and L can be used to immobilize antibodies (15), streptavidin is used for biotinylated proteins (16), chelate for His-tagged proteins (17, 18), anti-GST antibodies for GST fusion proteins (19), and oligonucleotides for cDNA or mRNA-protein hybrids (20). 

ISFET based systems have been developed at different laboratories [91 -93] using a planar or three dimensional set up. Such systems which are equipped with micropumps and valves are able to detect ions and other interesting chemical parameters (Fig. 4). Different approaches have been made in the detection of ions and diluted gases. Microarrays for measuring pressure, temperature, pH, 02 and C02 were realized using silicon technology and a hybrid set up of the different components [94]. Blood gas measurements with integrated pH, 02 and C02-electrodes have been tested in real samples [95]. 

Applied substrates require homogeneous and planar surfaces. Planar supports allow accurate scanning and imaging, which rely on a uniform detection distance between the microarray surface and the optical device. Planar solid support materials tend to be impermeable to liquids, allowing for a small feature size and keeping the hybridization volume to a minimum. Flat substrates are amenable to automated manufacture, providing an accurate distance from photo masks, pins, ink-jet nozzles and other manufacturing implements. The flatness affords automation, an increased precision in manufacture, and detection and impermeability. Table 1 shows frequently used support materials... 

For specific detection, realized by a base-pair interaction with suitable labeled targets, the probes must be immobilized on planar substrates in an addressable structure. The power of a microarray is defined by its information content and thus by the number of genes that will be represented. To achieve a high number of features or spots, the size of each spot has to be minimized. In principle, two ways exist for obtaining spatially resolved immobilization ... 

This approach is highly efficient, as thousands to hundreds of thousands or even millions of compounds can be generated easily. In this review, we shall briefly discuss the preparation of these chemical microarrays and follow with a more detailed discussion on the various screening methods. Applications of these microarrays to various biological systems will also be described. The approaches that have been employed for preparing microarrays and the methods used to detect activity in different applications are summarized in Table 1. Schematic representations of the generation of planar chemical arrays and bead-arrays are shown in Figure 1. DNA and... 

Table 2 Planar chemical microarrays vs. OBOC combinatorial library bead-arrays...

Planar chemical microarrays OBOC bead-arrays... 

In this method, peptides or small molecules are prepared by the split-mix synthesis method and cleaved from the resin to form an encoded solution-phase library such that each library compound is tethered to a PNA code via a hydrophilic linker (11). The library then is mixed with the target protein and later exposed to planar oligonucleotide microarrays of predetermined sequences. Alternatively, the encoded soluble library can be hybridized to the oligonucleotide microarrays before incubation with the target protein. 

In the original report, it was used for the screening of protein binding. Like the OBOC method, a whole-cell binding assay also can be applied to the encoded planar chemical microarrays, although this has not been reported. 

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