Two-photon fluorescence excitation of macroscopic areas on planar waveguides.

In this paper, we report the first successful demonstration, to our knowledge, of two-photon fluorescence excitation (TPFE) using planar thin-film waveguide structures of macroscopic excitation dimensions (square millimeters to square centimeters in size). The high intensity of excitation light required for TPFE is available not only at a single focus point but along the whole trace of the beam guided in the waveguide structure. Line profiles of the fluorescence excited by TPFE show excellent correlation with the geometry of the launched laser beams. A clear second-order dependence of the fluorescence intensity on the excitation intensity confirms the two-photon character of fluorescence generation. Spectra of the emission generated by one-photon excitation and by two-photon excitation show only minor differences.

Fiber-optic evanescent wave biosensor for the detection of oligonucleotides.

An automated optical biosensor system based on fluorescence excitation and detection in the evanescent field of a quartz fiber was used to detect 16-mer oligonucleotides in DNA hybridization assays. A biotinylated capture probe was immobilized on the fiber surface via avidin or streptavidin. The hybridization with fluorescein-labeled complementary strands was monitored in real time by fluorescence detection. The double strands formed by hybridization could be dissociated by chemical or thermal regeneration, allowing one to perform hundreds of assay cycles with the same fiber. The signal loss during longtime measurements, i.e., consecutive hybridization assays, can be described by a single-exponential function. Over more than 200 cycles, the net signal decreased by 50% with a signal variation of 2.4% after correction for this signal loss. By binding the capture probe with the 5'-end to the optical fiber surface, and by using a 50% (w/w) aqueous urea solution for chemical regeneration, the duration of an assay cycle could be reduced to 3 min. By applying longer assay cycles, the detection limit for the hybridization with a complementary fluorescein-labeled oligonucleotide was 2.0 x 10(-13) M (24 fmol). To detect an unlabeled complementary 16-mer oligonucleotide, competitive hybridization assays were performed, resulting in a detection limit of 1.1 x 10(-9) M (132 pmol). Poly-(acrylic acid) 5100 sodium salt and Tween 20 were used in the hybridization buffer to prevent nonspecific binding caused by ionic or hydrophobic interaction. The amount of nonspecific binding of noncomplementary oligonucleotides was in the range of 1-2%, compared with the specific binding in the different hybridization assays.