Peter Sluka

Complement hybridization from solution to surface-attached probe-oligonucleotides observed by surface-plasmon-field-enhanced fluorescence spectroscopy

Abstract Surface-plasmon field-enhanced fluorescence spectroscopy is applied for the detection of hybridization reactions of fluorophore-labeled 15mer target oligonucleotides from solution to surface-attached probe DNA. The attachment of the catcher strand via a biotin-group at the 5′ end of an additional 15mer of thymines used as spacers, strongly binding to a monolayer of streptavidin at the sensor surface ensures a separation of the chromophores beyond 2 Forster radii, thus preventing any sifnificant loss of fluorescence signal due to energy transfer to the (acceptor states of the) metal (quenching). The high sensitivity thus obtainable is used to quantify the kinetics of binding (hybridization) and dissociation of different oligonucleotides exhibiting full complementarity to the catcher probes or having one or two mismatches in the base sequence, respectively. It is shown that the hybridization process follows a simple Langmuir model with a single mismatch reducing the equilibrium constant by two orders of magnitude, and a second mismatch causing another three orders in reduction.