Optical DNA Biosensor Research Articles

A fiber-optic evanescent wave DNA biosensor based on novel molecular beacons.

We have prepared a novel optical fiber evanescent wave DNA biosensor using a newly developed molecular beacon DNA probe. The molecular beacons (MB) are oligonucleotide probes that become fluorescent upon hybridization with target DNA/RNA molecules. Biotinylated MBs have been designed and immobilized on an optical fiber core surface via biotin-avidin or biotin-streptavidin interactions. The DNA sensor based on a MB does not need labeled analyte or intercalation reagents. It can be used to directly detect, in real-time, target DNA/RNA molecules without using competitive assays. The sensor is rapid, stable, highly selective, and reproducible. We have studied the hybridization kinetics of the immobilized MB by changing the ionic strength of the hybridization solution and target DNA concentration. Our result shows divalent cations play a more important role than monovalent cations in stabilizing the MB stem hybrids and in accelerating the hybridization reaction with target DNA/RNA molecules. The concentration detection limit of the MB evanescent wave biosensor is 1.1 nM. The MB DNA biosensor has been applied to the analysis of specific gamma-actin mRNA sequences amplified by polymerase chain reaction.

Fiber-optic DNA sensor for fluorometric nucleic acid determination.

Single-stranded deoxyribonucleic acid (ssDNA) thymidylic acid icosanucleotides (dT20) were synthesized on the surfaces of derivatized quartz optical fibers to create an optical DNA biosensor. The synthesis made use of an automated solid-phase synthesizer and phosphoramidite synthons. The covalently immobilized oligomers were found to hybridize with complementary ssDNA (cDNA) or ssRNA (cRNA) from solution, and the device was regenerable for multiple cycles of application. Hybridization on optical fibers was detected by the use of the fluorescent DNA stain ethidium bromide (EB). The procedure used hybridization assay techniques and provided a detection limit of 86 ng x mL(-1) cDNA and a sensitivity of 200% fluorescence intensity increase per 100 ng x mL(-1) of cDNA, with one cycle of hybridization analysis requiring 45 min. The sensor has been observed to be regenerable (minimum of five cycles) and to sustain full activity after prolonged storage times (1 year), harsh washing conditions (sonication), and sterilization (autoclaving). The extent of hybridization between the immobilized and complementary nucleic acid strands was determined by UV absorbance thermal denaturation studies wherein all 20 bases on each strand of the nucleic acid were found to be involved in duplex formation.