Toward the development of optical nucleic acid biosensors based on TIRF and TCSPC for high sensitivity determinations

Paul A.E. Piunno; Virginijus Barzda; Sarah C. Jantzi; Christopher C. Kotoris; Arkady Major; Sergei Musikhin; Sandeep Raha; Ulrich J. Krull

doi:10.1117/12.628696

13 October 2005 Toward the development of optical nucleic acid biosensors based on TIRF and TCSPC for high sensitivity determinations

Paul A.E. Piunno, Virginijus Barzda, Sarah C. Jantzi, Christopher C. Kotoris, Arkady Major, Sergei Musikhin, Sandeep Raha, Ulrich J. Krull

Author Affiliations +

Proceedings Volume 5969, Photonic Applications in Biosensing and Imaging; 59690R (2005) https://doi.org/10.1117/12.628696
Event: Photonics North, 2005, Toronto, Canada

Abstract

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 10⁷ 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.

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Paul A.E. Piunno, Virginijus Barzda, Sarah C. Jantzi, Christopher C. Kotoris, Arkady Major, Sergei Musikhin, Sandeep Raha, and Ulrich J. Krull "Toward the development of optical nucleic acid biosensors based on TIRF and TCSPC for high sensitivity determinations", Proc. SPIE 5969, Photonic Applications in Biosensing and Imaging, 59690R (13 October 2005); https://doi.org/10.1117/12.628696

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