There is a need for developing methods that don't require chemical modification of the target biomolecule and can detect binding events with high specificity and sensitivity. In response, we have designed and tested ssDNA and protein sensors based on the concept of Surface Enhanced Raman Spectroscopy (SERS).
The DNA assay contains a surface modified with single stranded PNA (Peptide Nucleic Acid) instead of ssDNA. ssPNA binds to complementary ssDNA more tightly and with better selectivity, relative to ssDNA. Moreover, PNA/DNA hybrids are less sensitive to ionic strength and pH. In this context we have taken advantage of the fact that before hybridization the ssPNA modified surface is neutral, whereas after binding to the complementary ssDNA the charge becomes overall negative. This allows to electrostatically bind positively charged nanoparticles (NPs) and to create SERS active hot spots directly on the hybridized structures, circumventing the need for covalent binding. The addition of a dye with absorption maximum in resonance with the laser wavelength (in our case Rhodamine 6G) and subsequent SERS analysis provide the Raman fingerprint of the dye as proof of the hybridization event. The assay involves commercially available materials and instrumentation.
For the development of the heterogeneous assay for protein detection we take advantage of aptamers. Aptamers are single-stranded oligonucleotides generated via reiterative selection cycles that bind their selected targets, i.e., proteins or small molecules, with high affinities and specificities, via folding into specific ligand binding structures. Here I will report "aptatags", which consist of aptamer-modified silver NP dimers, where the NPs are held together by a dithiolated small molecule that acts at the same time as the optical reporter. It is possible to use these materials to design a heterogeneous method for protein identification that takes advantage of the Raman signal enhancement by metallic nanostructures and the recognition capabilities of aptamers. Aptatags are formed by first linking silver NPs with the organic dithiol molecule, followed by surface modification with thiolated single-stranded DNA (ssDNA) corresponding to the sequence of the aptamer probe. The sensing surface involves a silver layer containing the thiolated capturing aptamer and mercaptohexadecanoic acid to minimize nonspecific binding. The overall process provides excellent selectivity and sensitivity and has been extended to multiplexed detection protocols.
Host: Eric Garfunkel