Towards a fibre optic nucleic acid biosensor: Thiazole orange derivatives as sensitive fluorescent probes to detect DNA hybridization

Single-stranded DNA oligonucleotides (dT20) were immobilized onto the functionalized surface of fused silica optical fibres via a hexaethylene glycol linker. 1-hydroxyethyl-4-[(3-methyl-6-methoxybenzothiazole-2-ylidine)methine] quinolium bromide (TOMEHE) was synthesized and characterized by mass spectrometry and uv-visible absorption spectrophotometry. Absorption of TOMEHE in the presence of single-stranded DNA or double-stranded DNA was found to be linear (r 2 = 0.998 and 0.995, respectively) over the concentration range studied (1:60, 1:30, 1:12, 1:3 and 1:1 dye:base or base pair ratios). Hybridization events were detected using a fibre optic nucleic: acid detection scheme whereby delivery of radiation to the surface of the optical fibre was achieved by total internal reflection. Fluorescence from TOMEHE associated with the DNA membrane was coupled into the fibre and measured. The fluorescence intensity maximum of TOMEHE associated with immobilized dT20 on the surface of the optical fibre was observed to lie at 560 +/- 3 nm. The wavelength of the fluorescence maximum for TOMEHE when associated with hybidized DNA on the fibre surface was not observed to be significantly different from that of single-stranded DNA. TOMEHE demonstrated a 10-fold greater fluorescence enhancement over EB when bound to double-stranded DNA, but also displayed a 2.5 fold enhancement over EB when associated with single-stranded DNA. The linearity of fluorescence for TOMEHE when bound to double-stranded DNA was poor (r2 = 0.566) for the range studied (1:60, 1:30, 1:12, 1:3 and 1:1 dye:base pair ratios), suggesting that this dye may not be suitable for incorporation into the nucleic acid detection strategy described herein.