Amine microarray surfaces

The phosphate backbone of DNA molecules often results in undesirable electrostatic interactions with the substrate. Although the electrostatic interactions of DNA can be utilized for physical adsorption of DNA to the surface, this process can also lead to the nonspecific physical adsorption of target DNA on the surface. Rather than sample DNA hybridizing to the probe, it can adsorb to the surface and lead to interferences with the final detection call. Nonspecific adsorption effects have primarily been examined by the microarray community. Blocking strategies have been developed to prevent these nonspecific interactions. Succinic anhydride (SA) and bovine serum albumin (BSA) are two common methods to prevent nonspecific adsorption on amine modified surfaces. Blocking strategies are desired to react with or pas-... 

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. 

However, most coupling chemistries do not go to completion so that the substrate will contain a mixture of functional groups capped with attached probe (SR) while others remain free (S ). These residual reachve functional groups must be capped or blocked in some manner to reduce nonspecific binding to the microarray. Residual surface amines may be capped by reaction with succinic anhydride. This renders the support neutral (SR). Using this abbreviated nomenclature, we can describe common surface modifications for microarray substrates. 

Most immobilizahon chemistries for microarrays currently rely upon derivatization of the substrate with amine-reactive functional groups such as aldehydes, epoxides, or NHS esters. While we can choose from many available surface-reactive chemistries, it is important to keep in mind that they must be compatible with a printing process. Ideally, the biomolecule should react completely and rapidly with the substrate in order to achieve good spot formation. It is also critical that the probe remain or be recoverable in its active state following printing. If too reactive a chemistry is employed there is the possibility for excessive crosslinking that can hinder performance by reducing the number of rotatable bonds in the probe. 

In a different approach, fluorescence-based DNA microarrays are utilized (88). In a model study, chiral amino acids were used. Mixtures of a racemic amino acid are first subjected to acylation at the amino function with formation of A-Boc protected derivatives. The samples are then covalently attached to amine-functionalized glass slides in a spatially arrayed manner (Fig. 10). In a second step, the uncoupled surface amino functions are acylated exhaustively. The third step involves complete deprotection to afford the free amino function of the amino acid. Finally, in a fourth step, two pseudo-Qn nX. om.Qx c fluorescent probes are attached to the free amino groups on the surface of the array. An appreciable degree of kinetic resolution in the process of amide coupling is a requirement for the success of the ee assay (Horeau s principle). In the present case, the ee values are accessible by measuring the ratio of the relevant fluorescent intensities. About 8000 ee determinations are possible per day, precision amounting to +10% of the actual value ((S(S). Although it was not explicitly demonstrated that this ee assay can be used to evaluate enzymes (e.g., proteases), this should in fact be possible. So far this approach has not been extended to other types of substrates. 

Charles, P.T., et al. Fabrication and surface characterization of DNA microarrays using amine- and thiol-terminated oligonucleotide probes. Langmuir, 2003, 19, p. 1586-1591. 

Soper et al. [211] presented the fabrication of DNA microarrays onto PMMA surfaces using a UV modification protocol as shown in Fig. 11. Briefly, the PMMA surface was first activated via exposure to UV irradiation, which produced carboxylic acid functional groups onto its surface. EDC/NHS coupling chemistry was then used to facilitate the formation of succinimidyl ester intermediates on the surface, which allowed for the covalent attachment of amine-terminated... 

DNA hybridization microarrays are generally fabricated on glass, silicon, or plastic substrates. Oligonucleotide DNA is the most common probe used in DNA microarrays. Amine or thiol linkers are added to these oligonucleotide probes to enable attachment to the glass, plastic, or silicon surface of the chip. The attachment of these probes to the surface can be done mechanically through automated means. In a laboratory setup, manual array spotters could be used. 

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