Dendrimer-activated solid supports for nucleic acid and protein microarrays.
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.
Benters
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Institute of Organic and Macromolecular Chemistry, University Bremen, FB2, P.O. Box 330440, 28334 Bremen, Germany.
Niemeyer
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CM
Wöhrle
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D001345:Autoradiography; D003432:Cross-Linking Reagents; D050091:Dendrimers; D005898:Glass; D007202:Indicators and Reagents; D009693:Nucleic Acid Hybridization; D009696:Nucleic Acids / Q000737:chemistry*; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D009841:Oligonucleotides / Q000737:chemistry; D010783:Photometry; D011073:Polyamines; D019809:Streptavidin / Q000737:chemistry; D013499:Surface Properties; D013861:Thiocyanates / Q000737:chemistry
D003432:Cross-Linking Reagents; D050091:Dendrimers; D007202:Indicators and Reagents; D009696:Nucleic Acids; D009841:Oligonucleotides; C104700:PAMAM Starburst; D011073:Polyamines; D013861:Thiocyanates; C032582:bitoscanate; D019809:Streptavidin