The results of preliminary investigations toward the design of an optical biosensor instrument for the selective and direct analysis of low copy numbers of target nucleic acids in native form are reported. A concept development prototype was constructed based on a total internal reflection fluorescence (TIRF) configuration and the use of time correlated single photon counting (TCSPC). Selective detection of interfacial hybrid formation was done by identification of luminescence of characteristic (20ns) lifetime from the intercalant fluorophore ethidium bromide associated with nucleic acid hybrids formed at the interaction surface of optical sensor elements. Results of these investigations suggest that detection limits on the order of 10<sup>7</sup> dye:dsDNA complexes can be achieved when an effective sensor interaction surface of 150 µm diameter is used. The presence of interfacial nucleic acid duplexes at a sensor surface was further verified by thermal denaturation studies. The sensitivity of this concept design prototype was found to be most limited by long lifetime fluorescence intrinsic to the detection optics in conjunction with large amounts of scatter dispersed from the sensor cartridge. Future directions for continued device development are discussed.
We report on development of frequency-doubled diode-pumped ultrashort pulse Yb:KGW laser operating at 520 nm with approximately 200 fs long pulses at a repetition rate of 15 MHz. For ~2 W of absorbed pump power at 980 nm, the laser delivers up to 30 mW of average power at fundamental wavelength of 1040 nm, corresponding to a pulse energy of 2 nJ. The laser radiation was then frequency-doubled in a single pass configuration within a nonlinear BIBO crystal to produce femtosecond green radiation at 520 nm with peak power of ~200 W. The generated second harmonic served as excitation source of optical DNA sensor based on fluorescence lifetime measurements using the time correlated single photon counting (TCSPC) technique.
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Optical Biochips and Biosensors for Nucleic Acids and Proteins