Controlling the density of nucleic acid oligomers on fiber optic sensors for enhancement of selectivity and sensitivity

Abstract The immobilization of oligonucleotides to solid surfaces is relevant to the development of biosensor and microarray technologies. The density of oligonucleotide immobilization determines the charge density at the surface by means of ionizable phosphate groups, and may result in an interfacial dielectric constant, pH and ionic strength that are unlike those of bulk solution. The density of immobilization may affect the extent of interactions between neighbouring oligomers, as well as interactions between the immobilized oligomers and the substrate surface. Experiments were done to examine the effects of immobilization density and solution conditions on the sensitivity, selectivity and dynamic range of hybridization assays done using a fiber optic nucleic acid biosensor based on total internal reflection fluorescence (TIRF). Such immobilized nucleic acid films first required activation by thermal denaturation cycling to reach full activity. The effects of non-selective adsorption of oligonucleotides were dependent on ionic strength, and could not be removed independently of hybridization. Increased immobilization density resulted in significantly higher sensitivity but reduced dynamic range in all hybridization assays done. Sensitivity and selectivity were a function of temperature, however, the selectivity of hybridization assays done using these sensors could not be predicted by consideration of thermal denaturation temperatures alone.