AVS 46th International Symposium
    Biomaterial Interfaces Group Tuesday Sessions
       Session BI-TuA

Paper BI-TuA3
Surface-plasmon Field-enhanced Fluorescence Spectroscopy and -Microscopy for the Evaluation of the Hybridization Reaction of Oligonucleotides

Tuesday, October 26, 1999, 2:40 pm, Room 613/614

Session: Characterization of Biomaterial Interfaces
Presenter: W. Knoll, Max-Planck-Institut für Polymerforschung, Germany and Stanford Univ.
Authors: W. Knoll, Max-Planck-Institut für Polymerforschung, Germany and Stanford Univ.
D. Kambhampati, Max-Planck-Institut für Polymerforschung, Germany
T. Liebermann, Max-Planck-Institut für Polymerforschung, Germany
T. Neumann, Max-Planck-Institut für Polymerforschung, Germany
Correspondent: Click to Email
Surface plasmon spectroscopy (SPS) is widely used as a surface-sensitive technique to characterize thin film architectures, or to monitor kinetic processes like biorecognition and binding events or photo-reactions in these layers. We describe an extension of the method combining the field-enhancements obtainable at the resonant excitation of surface plasmons with fluorescence detection schemes. Controlling the balance between the evanescent character of the surface mode and the energy (Förster) transfer between the chromophores and (the acceptor states of) the metal substrate sensitivity enhancements of more than 2 orders of magnitude compared to SPS can be achieved (though not label-free). We demonstrate the potential of this mode of operation for the quantitative evaluation of hybridization reactions between surface-immobilized probe oligonucleotides (15-mers) and complements from solution. It is shown that a simple Langmuir adsorption/desorption model describes the experimental results. Single base mismatches can account for a decrease in the equilibrium constant by two orders of magnitude, a second mismatch can give a reduction by another 3 orders. A further extension that will be introduced is the simultaneous observation of several hybridization/dehybridization reactions on a 3 x 3 matrix of 9 different sensor spots by fluorescence microscopy.