ADVANTAGES OF NITROCELLULOSE FILM-SLIDES AND FILM-PLATES FOR PROTEIN/GLYCOPROTEIN MICROARRAYS
Although many surface binding chemistries are available for microarrays, the two that predominate are nitrocellulose- and silane-based. Nitrocellulose has been a preferred material to immobilize and capture biomolecules for decades (vis Northern, Western and dot blots). Binding of biomolecules to nitrocellulose is by a combination of weak intermolecular forces, probably dominated by hydrophobic and van der Waals, based on observed drying effects on binding energies (Van Oss et al, 1987; Tang et al, 2003; Kingsmore et al, 2006). Silane-based chemistries are designed to link capture molecules covalently using various linkage chemistries (MacBeath and Schreiber 2000; Stears et al, 2003). This methodology has been successfully used in many microarray applications because it provides permanently linked capture molecules for assay (Cahill 2001; Wu et al, 2008).
Therein lies the main disadvantage of covalent linking, stemming from the fact that the conditions for covalent bonding generally cause changes to biomolecules, which compromise the efficiency of capture. This is especially problematic when using proteins or glycoproteins as capture molecules, where function is tied to tertiary and quaternary structure (Zhu et al 2003; Ajikumar et al, 2006). Moreover, when spotting entire proteomes derived from cells, uniform and equivalent covalent linking of heterogeneous species cannot be practically achieved.
In actual practice, the weak (non-covalent) bonding of proteins to nitrocellulose is not reversible under normal spotting and assay conditions used in microarray applications (Stillman et al, 2000; Oh et al, 2006). Thus, the actual advantages of weak bonding in terms of preserving protein activity is not challenged by the potential disadvantages. The one exception is small peptide binding; to nitrocellulose which is not quantitative. However, that limitation can be overcome simply by UV cross-linking under conditions which does not affect protein function (link to our poster).
Porous nitrocellulose films embrace the stereochemical requirements for effective capture with proteins and for this reason are well-used for protein function studies (Madoz-Gúrpide et al, 2001; Zhu et al, 2003). The 3D character of films also increases surface area of binding and for this reason, Films are used extensively for reverse phase protein/glycoprotein microarrays (Paweletz et al, 2001; Balboni et al, 2006).
Increased surface area for binding translates into increased binding capacity in microarray spots. Difference in binding capacity is related to pore size, pore structure, pore density and film thickness. ONCYTE® products use these differences to generate Films that bind at least an order of magnitude more protein than non-porous materials (Renault et al, 2007).