Immobilization of oligonucleotides on glass surface using an efficient heterobifunctional reagent through maleimide–thiol combination chemistry

An efficient heterobifunctional reagent, N-(3-triethoxysilylpropyl)-4-(N′-maleimidylmethyl) cyclohexanamide (TPMC), was developed for the immobilization of thiol-modified oligonucleotides on an unmodified glass surface. The heterobifunctionality of the reagent was used for the construction of a DNA microarray in which the triethoxysilyl functionality has specificity toward a glass surface, whereas the maleimide functionality has thiol-modified oligonucleotides via a stable thioether linkage. Immobilization of DNA was achieved by two alternative approaches. In the first approach, the reagent TPMC was treated with oligonucleotides to get triethoxysilyl–oligonucleotide conjugate, which was then covalently attached via specific triethoxysilyl functionality to an unmodified glass surface. In the second approach, the reagent was first covalently linked with an unmodified glass surface to get maleimide functionality on a glass surface, which was then used for the immobilization of oligonucleotides via a stable thioether linkage. The applicability of the reagent was explored by hybridization studies with the fluorescein-labeled complementary DNA strand and in mismatch discrimination.