Covalent strategy for immobilization of DNA-microspots suitable for microarrays with label-free and time-resolved optical detection of hybridization

Sequence-specific detection and quantification of nucleic acids are central steps in many molecular biology procedures which have also been transferre d to chip-based procedures. Hybridization-based assays can be used to quant ify and discriminate between DNA target sequences down to the level of sing le base mismatches. Arrays of DNA probes immobilized on a support enable si multaneous testing of multiple sequences of a single sample. DNA arrays can be produced either by in-situ synthesis of oligonucleotides or by immobili zation of pre-assembled DNA probes. Covalent and directed immobilization im proves the reproducibility and stability of DNA arrays. This is especially interesting with repeated use of transducers or chips. Procedures are described for effective covalent immobilization of pre-assem bled amino-linked oligonucleotides, by use of ink-jet techniques, on a modi fied and heated glass surface, with addressable surface areas ranging from 0.01 mm(2) to a few mm(2). Almost immediate evaporation of the spotted drop lets on the heated surfaces leads to very high surface hybridization capaci ties. The surfaces are suitable for use with a label-free detection method - reflectometric interference spectroscopy (RUS). It is shown that hybridiz ation capacity and non-specific interaction at these DNA-surfaces can be ch aracterized by use of RUS. With a consumption of less than 80 ng.mm(-2) oli gonucleotide and a specific hybridization capacity of more than 300 fmol mm (-2), the activated aminodextran procedure was usually suitable for setting up a DNA array with label-free detection. Non-specific interactions with r andom oligomers or protein (ovalbumin) were low. Up to 150 repeated regener ations (stripping) of the surfaces by acid treatment and denaturing agents, and 50 days of storage, have been possible without significant loss of hyb ridization capacity.