The generation of chemically activated glass surfaces is of increasing interest for the production of microarrays containing DNA, proteins, and low-molecular-weight components. We here report on a novel surface chemistry for highly efficient activation of glass slides. Our method is based on the initial modification of glass with primary amino groups using a protocol, specifically optimized for high aminosilylation yields, and in particular, for homogeneous surface coverages. In a following step the surface amino groups are activated with a homobifunctional linker, such as disuccinimidylglutarate (DSG) or 1,4-phenylenediisothiocyanate (PDITC), and then allowed to react with a starburst dendrimer that contains 64 primary amino groups in its outer sphere. Subsequently, the dendritic monomers are activated and crosslinked with a homobifunctional spacer, either DSG or PDITC. This leads to the formation of a thin, chemically reactive polymer film, covalently affixed to the glass substrate, which can directly be used for the covalent attachment of amino-modified components, such as oligonucleotides. The resulting DNA microarrays were studied by means of nucleic acid hybridization experiments using fluorophor-labeled complementary oligonucleotide targets. The results indicate that the novel dendrimer-activated surfaces display a surface coverage with capture oligomers about twofold greater than that with conventional microarrays containing linear chemical linkers. In addition, the experiments suggest that the hybridization occurs with decreased steric hindrance, likely a consequence of the long, flexible linker chain between the surface and the DNA oligomer. The surfaces were found to be resistant against repeated alkaline regeneration procedures, which is likely a consequence of the crosslinked polymeric structure of the dendrimer film. The high stability allows multiple hybridization experiments without significant loss of signal intensity. The versatility of the dendrimer surfaces is also demonstrated by the covalent immobilization of streptavidin as a model protein.
The DNA microarray-based analysis of single nucleotide polymorphisms (SNPs) is important for the correlation of genetic variations and individual phenotypes, and for locating disease-causing genes. To facilitate the development of surfaces suitable for immobilization of oligonucleotides, we report here a novel method for the surface immobilization of DNA using pre-fabricated polyamidoamine (PAMAM) starburst dendrimers as mediator moieties. Dendrimers containing 64 primary amino groups in their outer sphere are covalently attached to silylated glass supports and, subsequently, the dendritic macromolecules are modified with glutaric anhydride and activated with N-hydroxysuccinimide. As a result of the dendritic PAMAM linker system the surfaces reveal both a very high immobilization efficiency for amino-modified DNA-oligomers, and also a remarkable high stability during repeated regeneration and re-using cycles. The performance of dendrimer-based DNA microarrays in the discrimination of SNPs is demonstrated.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
customersupport@researchsolutions.com
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
This site is protected by reCAPTCHA and the Google Privacy Policy and Terms of Service apply.
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.