Immobilization of homooligonucleotide probe layers onto Si/SiO2 substrates:: characterization by electrochemical impedance measurements and radiolabelling

Radiolabelling and electrochemical impedance measurements were used to characterize the immobilization of single stranded homooligonucleotides onto silica surfaces and their subsequent hybridization with complementary strands. The immobilization procedure consists of grafting an epoxysilane onto microelectronic grade Si/SiO2 substrates, and coupling oligonucleotides bearing a hexylamine linker onto the epoxy moiety. Radiolabelling was used as a reference method to quantify the amount of immobilized and hybridized oligonucleotides, These results show that the Si/SiO2 substrates modified with an epoxysilane yield a surface concentration of similar to 10(11) strands/cm(2) for the immobilized oligonucleotides, after vigorous washings, and that similar to 36% of these undergo hybridization with complementary strands. The impedance measurements, which provide a direct means of detecting variations in electrical charge accumulation across the semiconductor/oxide/electrolyte structure when the oxide surface is chemically modified, show that the semiconductor's flat band potential undergoes reproducible shifts of -150 and -100 mV following the immobilization and the hybridization step, respectively. These results demonstrate that electrochemical impedance measurements using chemically modified semiconductor/oxide/electrolyte structures of this type offer a viable alternative for the direct detection of complementary DNA strands upon hybridization. (C) 2002 Elsevier Science B.V. All rights reserved.