Singles only: DNA sequences can be induced to spontaneously adsorb to the surfaces of Ag colloids through their nucleotide side chains (see picture). The SERS spectra of these nonspecifically bound strands are sufficiently reproducible that they can be used to identify single‐base mismatches in short (25‐mer and 23‐mer) strands. Subtracting the spectra of different DNA sequences results in difference spectra that contain features corresponding to the exchanged nucleotides.

The SERS spectra of adenine recorded under a broad range of pH values and concentrations using both silver and gold colloids provided evidence for the existence of several distinct species. At high concentration (0.5−10 ppm), the spectra recorded between pH 1 and 11 showed only two distinct spectra, rather than the three forms that would be expected for a compound with two pK a values of 4.2 and 9.8. The spectra at neutral and alkaline pH were identical and assigned to the deprotonated form of adenine on the basis of DFT calculations, isotope shifts, and comparison with the normal Raman spectra of neutral and deprotonated adenine. The spectra at acidic pH were different, consistent with adenine protonation. Neutral adenine was not detected at any pH studied. At low adenine concentration (≤0.25 ppm) on silver (but not gold colloids), the spectra showed the presence of two different, highly scattering Ag+−adenine complexes at neutral and at alkaline pH. These data can rationalize many of the features which were previously reported for adenine SERS spectra but were attributed to reorientation on the enhancing surface or to the use of different substrates.

Unlabelled single- and double-stranded DNA (ssDNA and dsDNA, respectively) has been detected at concentrations ≥10(-9) M by surface-enhanced Raman spectroscopy. Under appropriate conditions the sequences spontaneously adsorbed to the surface of both Ag and Au colloids through their nucleobases; this allowed highly reproducible spectra with good signal-to-noise ratios to be recorded on completely unmodified samples. This eliminated the need to promote absorption by introducing external linkers, such as thiols. The spectra of model ssDNA sequences contained bands of all the bases present and showed systematic changes when the overall base composition was altered. Initial tests also showed that small but reproducible changes could be detected between oligonucleotides with the same bases arranged in a different order. The spectra of five ssDNA sequences that correspond to different strains of the Escherichia coli bacterium were found to be sufficiently composition-dependent so that they could be differentiated without the need for any advanced multivariate data analysis techniques.

DNA sequences attached to Au nanoparticles via thiol linkers stand up from the surface, giving preferential enhancement of the adenine ring breathing SERS band. Non-specific binding via the nucleobases reorients the DNA, reducing this effect. This change in intensity on reorientation was utilised for label-free detection of hybridization of a molecular beacon.

We report the formation of highly scattering silver complexes of adenine, deoxyadenosine and 5'-dAMP under alkaline pH conditions in the colloidal silver solutions which are used for surface-enhanced Raman spectroscopy. These complexes, and other pH-dependent phenomena, help to explain the diversity of previously reported adenine SERS spectra. Using conditions which promote complex formation allows nucleotides to be detected at <1 ppm, even in solutions with high salt concentrations.