Microarray surface chemistry

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 ... 

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. 

In this chapter, we will survey the kinds of solid supports (substrates) and surface chemistries currently used in the creation of nucleic acid and protein microarrays. Which are the best supports and methods of attachment for nucleic acids or proteins Does it make sense to use the same attachment chemistry or substrate format for these biomolecules In order to begin to understand these kinds of questions, it is important to briefly review how such biomolecules were attached in the past to other solid supports such as affinity chromatography media, membranes, and enzyme-linked immxm-osorbent assay (ELISA) microtiter plates. However, the microarray substrate does not share certain unique properties and metrics with its predecessors. Principal among these are printing, spot morphology, and image analysis they are the subjects of subsequent chapters. 

Adsorptive attachment. PLL surfaces work reasonably well for creating cDNA microarrays, but the suitability of this surface chemistry for immobilization of short oligonucleotides has been questioned. However, as we have learned, covalent attachment chemistries can be problematic as well. In either case, if the oligonucleotide is constrained too close to the surface with multiple points of contact, it may not be able to fully participate in hybridization. 

Seong (2002) compared silylated (aldehyde) and silanated (amine and epoxy) compounds from several commercial sources to the performance of an antigen (IgG) microarray. In addition, the efficiency of phosphate-buffered saline (PBS) (pH 7.4) and carbonate (pH 9.6) printing buffers were compared. While the various slides and surface chemistries showed differences in their binding isotherms, they ultimately reached similar levels of saturation. Silylated (aldehyde) slides showed comparable loading in both buffer systems. Apparently, tethering of antibody to the surface by Schiff s base formation of the surface aldehyde and lysine residues on the protein was applicable over a broad pH. However, carbonate buffer increased binding of proteins on silanated surfaces. 

Slides specifically selected for microarray applications should be used. They are available as ultracleaned (an important consideration) and untreated for those who wish to prepare their own surfaces or they can be purchased with a variety of precoated surface chemistries (e.g., lysine, aldehyde, or epoxide). The densities of reactive groups and surface coating uniformity are difficult to control. Thus, if lot-to-lot slide consistency is most important factor, consider using commercially available slides that are quality controlled. 

Angenendt et al. (2003) evaluated several slide surface chemistries for use as protein and antibody microarrays. They reasoned that because proteins vary greatly in their surface charge and relative hydrophobicity, a careful selection of surface chemistry may be important for obtaining optimal performance for a particular protein. Thus, several commercially available slide surface chemistries were evaluated for performance in model arrays... 

Finally, an understanding of the making of a microarray is fundamentally important to those interested in producing "spotted" arrays and properly using them. While complementary (cDNA) microarray fabricahon on glass slides has been well studied, we have less experience with the attachment of oligonucleotides and the preparation of protein arrays. Moreover, additional substrates and surface chemistries that may be better suited for printing proteins are now available. 

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). 

Surface chemistry is a key technology for protein microarray development. The supports used for protein immobilization have to fulfil some important requirements they must provide good quality spots, low background, simplicity of manipulation and compatibility with detection systems. An ideal surface or immobilization procedure for all proteins and applications does not exist however current methods are more than adequate for many applications. Basic strategies for protein immobilization consider covalent versus non-covalent and oriented versus random attachment, as well as the nature of the surface itself [106]. It has been demonstrated that the specific orientation of immobilized antibody ( capture agents ) consistently increases the analyte-binding capacity of the surfaces, with up to 10-fold improvement over surfaces with randomly oriented capture agents [107]. 

A common drawback of glass slides as supports for chemical microarrays, as well as of cellulose or polypropylene sheets, is the protein compatibility of fheir respective surface chemistries. It is inherently difficult to render polypropylene or silanated glass resistant to unspecific protein adsorption and is even more challenging to control a critical parameter such as ligand density on fhese polymers. 

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. 

Adhesion forces recorded by a fibronectin-coated AFM probe correlated with fluorescence measurements of protein adhesion on the polymer array, and both methods proved useful in characterizing protein-polymer interactions on a polymer array (Figure 27). ° While only a demonstration, the ability to discover relationships between surface chemistry and surface properties using this combined multiprong analysis with polymeric microarrays is notably powerful. 

Avoid damaging of microarrayer, sample, and surface chemistry of substrate... 

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