Immobilization of single-stranded DNA fragments to solid surfaces and their repeatable specific hybridization: covalent binding or adsorption?

The different steps of the immobilization process of single-stranded DNA (ssDNA) on surfaces by means of chemical grafting have been investigated using systematic measurements of grafting and hybridization densities by means of radioactive labelling. The immobilization by chemical grafting to a dense monomolecular layer of N-hydroxysuccinimidyl ester reactive functions attached to silica plates was performed from a dilute solution of amino-terminated oligonucleotides (10 μmol/l). The slow evaporation of the solvent allowed to increase the DNA grafting density by a factor of 10. A precise control of the rinsing process that followed the immobilization reaction allowed the discrimination between covalently bound and adsorbed oligonucleotides. Repeatable grafting densities, high signal-to-noise ratio and specific hybridization could be obtained if the adsorbed materials have been removed to completion at the rinsing steps. Repeatable hybridisation–denaturation cycles of complementary oligonucleotides could then be obtained at the surface. The most prominent advantages of the covalent binding associated with efficient rinsing are a better reproducibility and repeatability, and the reusability for the hybridization.