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

The immobilization of oligonucleotides to solid surfaces is relevant to the development of biosensor and microarray technologies. The density of oligo nucleotide immobilization determines the charge density at the surface by m eans of ionizable phosphate groups, and any result in an interfacial dielec tric constant, pH and ionic strength that are unlike those of bulk solution . The density of immobilization any affect the extent of interactions betwe en neighbouring oligomers, as well as interactions between the immobilized oligomers and the substrate surface. Experiments were done to examine the e ffects 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 fluorescenc e (TIRF). Such immobilized nucleic acid films first required activation by thermal denaturation cycling to reach full activity. The effects of non-sel ective adsorption of oligonucleotides were dependent on ionic strength, and could not be removed independently of hybridization. Increased immobilizat ion density resulted in significantly higher sensitivity but reduced dynami c range in all hybridization assays done. Sensitivity and selectivity were a function of temperature, however, the selectivity of hybridization assays done using the sensors could not be predicted by consideration of thermal denaturation temperatures alone. (C) 2001 Elsevier Science B.V. All rights reserved.