Sensitivity and specificity of metal surface-immobilized "molecular beacon" biosensors.
The separate developments of microarray patterning of DNA oligonucleotides, and of DNA hairpins as sensitive probes for oligonucleotide identification in solution, have had a tremendous impact on basic biological research and clinical applications. We have combined these two approaches to develop arrayable and label-free biological sensors based on fluorescence unquenching of DNA hairpins immobilized on metal surfaces. The thermodynamic and kinetic response of these sensors, and the factors important in hybridization efficiency, were investigated. Hybridization efficiency was found to be sensitive to hairpin secondary structure, as well as to the surface distribution of DNA hairpins on the substrate. The identity of the bases used in the hairpin stem as well as the overall loop length significantly affected sensitivity and selectivity. Surface-immobilized hairpins discriminated between two sequences with a single base-pair mismatch with high sensitivity (over an order of magnitude difference in signal) under identical assay conditions (no change in stringency). This represents a significant improvement over other microarray-based techniques.
Du
Hui
H
Department of Chemistry, and The Center for Future Health, University of Rochester, Rochester, New York, USA.
Strohsahl
Christopher M
CM
Camera
James
J
Miller
Benjamin L
BL
Krauss
Todd D
TD
D001408:Bacillus anthracis / Q000235:genetics; D001426:Bacterial Proteins / Q000235:genetics; D020137:Base Pair Mismatch; D015374:Biosensing Techniques / Q000379:methods*; D004247:DNA / Q000032:analysis* / Q000235:genetics; D015342:DNA Probes / Q000737:chemistry*; D005798:Genes, Bacterial; D009690:Nucleic Acid Conformation; D009693:Nucleic Acid Hybridization; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D012680:Sensitivity and Specificity; D013211:Staphylococcus aureus / Q000235:genetics
D001426:Bacterial Proteins; D015342:DNA Probes; C096686:FemA protein, Bacteria; C101641:mecR1 protein, Staphylococcus aureus; D004247:DNA