The chemical modification of DNA for analysis by surface enhanced resonance Raman scattering (SERRS) spectroscopy
The detection of specific DNA sequences is of increasing interest due to the sequencing of the human genome. This can be achieved by the use of covalently labelled oligonucleotide probes detected by sensitive analytical techniques. Surface Enhanced Resonance Raman Scattering (SERRS) spectroscopy is one such technique that provides molecularly specific information about probes at very low concentrations. The enhancement results from adsorption of a chromophore within the probe to a roughened metal surface. Specific SERRS probes were synthesised using benzotriazole as the metal complexing agent and azo dyes as the chromophore. They were coupled to the 5' end of DNA using two strategies. Firstly, coupling was achieved via an amino linker, achieved by reaction of the activated carboxylic acid derivative of the SERRS label with DNA containing a free amine at the 5'end. Secondly, the phosphoramidite of the SERRS label was synthesised and incorporated as the final monomer during the solid phase synthesis of the DNA sequence. Preliminary spectroscopic data was obtained for the labelled oligonucleotides. Ultraviolet melting studies of a DNA sequence labelled with an azobenzotriazole dye show an increase in melting temperature (TM) of 5.42 °C over the same sequence without modification, suggesting that the label confers stability to the double helix. Initial SERRS optimisation experiments allowed the optimum sample conditions to be determined for these novel oligonucleotides. SERRS spectra have been obtained for each labelled oligonucleotide with a detection limit determined at 5x 10⁻⁸ M. A potential application of the labelled oligonucleotides was investigated resulting in the first ever preparation of a SERRS labelled nanoparticle probe. This provides the basis for a specific sequence detection technique based on SERRS.