Molecular beacons for DNA biosensors with micrometer to submicrometer dimensions

Ultrasensitive molecular beacon (MB) DNA biosensors, with micrometer to sub micrometer sizes, have been developed for DNA/RNA analysis. The fluorescenc e-based biosensors have been applied in DNA/RNA detection without the meed for a dye-labeled target molecule or an intercalation reagent in the testin g solution. Molecular beacons are hairpin-shaped oligonucleotides that repo rt the presence of specific nucleic acids. We have designed a surface-immob ilizable biotinylated ssDNA molecular beacon for DNA hybridization at a liq uid-solid interface. The MBs have been immobilized onto ultrasmall optical fiber probes through avidin-biotin binding. The MB DNA biosensor has been u sed directly to detect, in real time, its target DNA molecules without the need for a competitive assay. The biosensor is stable and reproducible. The MB DNA biosensor has selectivity with single basepair mismatch identificat ion capability. The concentration detection limits and mass detection limit s are 0.3 nM and 15 amol for a 105-mu m biosensor, and 10 nM and 0.27 amol for a submicrometer biosensor, respectively. We have also prepared molecula r beacon DNA biosensor arrays for simultaneous analysis of multiple DNA seq uences in the same solution. The newly developed DNA biosensors have been u sed for the precise quantification of a specific rat gamma-actin mRNA seque nce amplified by the polymerase chain reaction, (C) 2000 Academic Press.