James H. Watterson

Practical physical aspects of interfacial nucleic acid oligomer hybridisation for biosensor design

Abstract A significant amount of research concerning rapid, selective biomolecular analysis has focused on development of analytical methods that make use of nucleic acid hybridisation as the basis for selective recognition. The development of biosensors based on nucleic acid hybridisation requires consideration of the thermodynamics of hybrid formation at a solid interface, because the relative thermodynamic stability can dictate the selectivity of hybridisation. Careful control of hybridisation conditions such as the density of oligonucleotides, as well as the temperature, pH, and ionic strength, may therefore enhance the selectivity, sensitivity and speed of a nucleic acid hybridisation assay that is located at an interface.

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.