Planar waveguide biosensors for nucleic acid hybridization reactions

Oligonucleotide probes derived from (1) the T3 RNA polymerase promoter sequence (T3) and (2) prostate-specific antigen messenger RNA (PSA) were prepared and labeled with a red-emitting fluorescent dye (Cy5). The complimentary oligonucleotides (anti T3 and anti PSA) were prepared and labeled with biotin. Initially, a feasibility study was performed in which the hybridization rate of the T3/anti T3 oligonucleotide pair was examined. Specifically, biotinylated anti T3 was immobilized to a neutravidin-coated waveguide and solutions containing increasing concentrations of Cy5-labeled T3 were injected into the biosensor. Fluorescence emission was detected with an evanescent wave imaging fluorometer. The hybridization reaction proceeded rapidly with a significant amount of binding occurring during the first 5 minutes. A Michaelis-Menton kinetics model was used to analyze hybridization rate data and gave values of 78 nanomolar for the apparent affinity of the hybridization reaction and 1.4 picomolar for the analytical sensitivity of the hybridization assay. In subsequent studies the hybridization rate of the PSA/anti PSA oligonucleotide pair was examined. Biotinylated anti PSA was immobilized to the waveguide and solutions containing increasing concentrations of Cy5-labeled PSA were injected into the biosensor. The hybridization rate observed for formation of the PSA/anti PSA pair was comparable to the high rates observed for the T3/anti T3 pair. Lastly, the selectivity of the biosensor was examined using an oligonucleotide probe derived from human glandular kallikrein (hGK), which exhibits a high degree of homology to PSA. The two oligonucleotide probes (PSA and hGK) only differed in 7 out of 20 positions. Interestingly, the hybridization rate observed for Cy5-Iabeled hGK was very low-not statistically different from the non-specific binding rate of the hybridization assay.