Two methods for the covalent immobilization of single-stranded DNA onto optical fibers for the development of biosensors are reported. In the first method, fused silica optical fibers were derivatized with γ-amino-propyltriethoxysilane (APTES) followed by extension with 12-nitrododecanoic acid via amide formation. The terminal nitro moiety was then reduced to an amine to provide a hydrophobic spacer molecule suitable for covalent attachment of an oligonucleotide. In the second method, fibers were activated with 3-glycidoxypropyltrimethoxysilane and a hydrophilic spacer arm composed of 1–17-diamino-3, 6,9,12,15-pentaoxaheptadecane was covalently attached. The oligonucleotides (dT20) were then bound to either type of spacer arm through a phosphoramidate bond between the amine terminus of the spacer molecule and the 5′-phosphate of the DNA via a carbodiimide mediated condensation reaction. The ability of the immobilized oligomers to hybridize with complementary oligonucleotides was then investigated. Detection of hybridization on the fiber surface was accomplished by staining the immobilized duplexes with the well characterized fluorescent intercalator, ethidium bromide, in a total internal reflection fluorescence (TIRF) configuration. Oligonucleotides were successfully immobilized to the amine terminus of the hydrophobic linker via the carbodiimide coupling reaction and fluorescence intensity increases were observed during hybridization studies. The immobilization of dT20 strands to the hydrophilic linker arm was determined to be unsuccessful by ellipsometry, UV-VIS spectroscopy and hybridization experiments with fluorescent intercalating dyes.
Henke, Lisa, Paul A. E. Piunno, Andrea C. McClure, Ulrich J. Krull
Analytica Chimica Acta