Real-time detection and quantification of DNA hybridization by an optical biosensor

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Abstract
The use of an optical biosensor, the resonant mirror, for direct and rapid detection of DNA-DNA hybridization has been demonstrated. Biotinylated oligonucleotide probes were immobilized on the sensor surface, via streptavidin, and the hybridization of a complementary target oligonucleotide (40-mer) was monitored in real time. The interaction at the sensor surface was shown to be sequence specific under conditions of low stringency. Regeneration of the surface-immobilized probe was possible, allowing reuse without a significant loss of hybridization activity. A comparison of probes indicated that the relative position of complementary sequence and the length of probe affected the hybridization response obtained. The potential of the sensor for quantitation of a hybridized DNA target was investigated. From radiolabeling data, the lowest amount of hybridized target sequence which could be determined directly was 19.9 fmol/mm2 (263 pg/mm2) of sensor surface. The dependence of the sensor response on the concentration of probe and target oligonucleotide was established. Utilizing the assay as an end-point determination method, the lowest detectable concentration of target oligonucleotide (40-mer) was 9.2 nM. This compares favorably to other sensor methods described previously without the requirement for labels.

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