Detection of PNA:DNA Hybridization by Surface Plasmon Resonance-induced Fluorescence
Fast and sensitive detection with high fidelity of selected sequences in PCR products is a goal of DNA diagnostics. To achieve this, the DNA of interest is generally captured using complementary probe molecules either in solution or at surfaces, and hybridisation can be detected in various ways including monitoring changes in optical and electrochemical properties. We describe here a method for detecting hybridisation at a surface. For this purpose, single-stranded PNA was immobilised on a gold surface through formation of a mixed self-assembled monomer of a thioalkane and a thioalkane-conjugated PNA. The formation of the monolayer could be monitored by surface-plasmon resonance (SPR), was complete within 1 hour and remained stable thereafter. Hybridisation of complementary single-stranded DNA with the immobilised PNA could also be detected using classical SPR. However, greater sensitivity could be achieved by using the surface plasmons to induce fluorescence (SPRF) from a dye that absorbs at the wavelength used to excite the plasmons (in this case, HeNe laser, 633 nm). We have developed a method for detection of hybridisation that eliminates the need for covalent labelling of the DNA with a fluorescent probe prior to hybridisation by employing a dye under conditions where it exhibit increased fluorescence quantum yield with PNA:DNA hybrids compared to single-stranded PNA. Background fluorescence due to interaction with single-stranded DNA is minimized due to the selective amplification of the signal from dyes bound lying closest to the surface. i.e. those interacting with the hybrid. Introduction of mismatches in the DNA strand leads to a loss of the SPRF signal. PNA has several advantages over DNA as a capture probe for the technique we describe here. For instance, single-stranded DNA immobilised at the surface would induce higher fluorescence in the dye than PNA and lower the sensitivity of hybridisation detection. Also from a thermodynamic aspect, the stability of PNA:DNA hybrids is reduced much more by single-base mismatches than DNA:DNA hybrids.
Eimer Tuite, Malin Ardhammar, Torsten Liebermann, 1Thomas Neumann, 1Wolfgang Knoll, 1Peter Nielsen,* and Bengt Norden
Department of Physical Chemistry, Chalmers Tekniska Högskola,