Microarray surface

Schaeferling M., Schiller S., Paul H., Kruschina M., Pavlickova P., Meerkamp M., Giammasi C., Kambhampati D., Application of self-assembly techniques in the design of biocompatible protein microarray surfaces, Electrophoresis 2002 23 3097-3105. 

In the following sections, the major types of substrates currently used for DNA and protein microarrays will be discussed. Much of what is known regarding microarray surface chemistry and the immobilization of biomolecules comes from work with DNA microarrays. Therefore, many of the examples cited here will be from these studies. Zhu and Snyder (2003) in their review provide good insight into the manufacture and utility of protein microarrays. Here are some points to consider when choosing a substrate for protein microarrays ... 

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. 

Fig.26 Pseudo-color time-resolved image of polystyrene nanobeads containing Pt porphyrin and conjugated to streptavidin immobilized to a biotinylated microarray surface (black teflon coated 96-well glass slide, spot diameter 1 mm, Erie Scientific) in different concentrations (25, 15, 10, 5, Ong streptavidin per well) [167]...

Fig. 5 Reversible oxidation and reduction of electroactive SAMs to generate renewable microarray surfaces for bioanalysis left). Corresponding fluorescent micrographs (right) depicting three cycles of carbohydrate and protein immobilization and release on the same substrate...

Figure 2 Minisequencing reaction on with extension primers immobilized on the microarray surface. The multiplex PCR products of the regions containing the SNPs are allowed to hybridize to the oligonucleotides immobilized on the microarray (A). The primers are extended with fluorescently labelled ddNTPs complementary to the nucleotide at the SNP position. Four different fluorophores, one for each nucleotide is used allowing for a simultaneous detection of the four nucleotides in one single reaction (B). After washing the slide with sodium hydroxide and salt buffers, only the extended primers covalently attached to the surface remains and the fluorescence is measured (C) and the genotypes assigned.

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... 

The performance of protein or antibody microarrays is dependent on various factors. One of these is the use of an appropriate microarray surface for the immobilization of the protein or antibody samples. Most conventional microarray surfaces have been adapted from DNA chip technology. DNA can easily be immobilized by electrostatic interactions of the phosphate backbone onto a positively charged surface. In contrast to DNA, as already mentioned, proteins are chemically and structurally much more complex and show variable charges, which may influence the efficiency of protein attachment. Additionally, proteins lose their structure and biochemical activity easily. For example, globular proteins consist of a hydrophilic exterior and a hydrophobic interior. When immobilized on a hydrophobic surface, the inside of the protein turns out, which may destabilize the structure and, simultaneously, the activity of the protein. These considerations demonstrate the complex requirements for protein immobilization. 

Schaferling M, Kambhampati D. Protein microarray surface chemistry and coupling schemes. In Protein Microarray Technology. 53. Kambhampati, D, ed. 2004. Wiley-VCH Verlag GmbH KGaA, Weinheim. pp. 11-38. 

Near-field scanning microwave microscopy (NSMM) is another system that has been used for label-free detection of both DNA and RNA molecules (42). NSMM monitors the microwave reflectance, a factor that depends on the dielectric permittivity profile across the microarray surface (42). This parameter is, in turn, dependent on the length and surface coverage of the strands, as well as on the hybridization state of the molecules (e.g., unhybridized single-stranded probe vs. hybridized duplex). NSMM technique demonstrated an acceptable resolution (potentially less than 50 pm) and comparable sensitivity to the fluorescent detection (42). 

TABLE 5.6-2. A Partial List of Commercially Available Microarray Surfaces... 

Insert the first slide, containing the Mix 1 autoantibodies, into the scanner. Eor the ScanArray 5000, the microarray surface is inserted face up. 

The use of underivatized saccharides for microarray construction has the unique advantage of preserving the native structures of the carbohydrate molecules. It requires, however, a ready-to-use microarray surface with appropriate surface chemistry that can be directly used to fabricate comprehensive carbohydrate microarrays with underivatized carbohydrates from a wide range of sources. Methods include noncovalent binding of underivatized carbohydrate probes on a chip by passive adsorption and methods for covalently immobilizing underivatized carbohydrates on a slide surface by appropriate chemical-linking techniques. 

In functional protein microarrays, set of proteins of interest or an entire proteome is over expressed, purified and spotted in an addressable microarray surface. The most obvious target molecules that can effectively be used to investigate protein binding and therefore protein microarray analysis are monoclonal antibodies or IgG. However, unlike DNA microarrays, antibody arrays may pose some practical concerns ... 

Two schemes to affix proteins to the microarray surface are generally employed, chemical linkage and peptide fusion tags. In the chemical linkage format, proteins are... 

Figure 3.8 The most frequently used methods to immobilize capture molecules onto microarray surfaces. (A) Antibody adsorption by poly-L-lysine-, nitrocellulose-, or polyvinylidene fluoride-treated surface. (B) Covalent binding using various silane reagents of APTES (3-aminopropyl)triethoxysilane, GPTS (3-gly-cidoxypropyl)trimethoxysilane, and MPTS (3-mercaptopropyl)trimethoxysilane. (C) Affinity interactions by biotin/streptavidine or histidine-tag/nickel-nitrilotri-acetic acid. (D) Diffusion-based (hydrogel) antibody-immobilization technique.

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