Multiplexed detection of single-point mutations in human serum by integrating Surface Enhanced Raman Scattering with Polymerase Chain Reaction.

Abstract: The polymerase chain reaction (PCR) is a gold standard in molecular diagnosis due to its high sensitivity, capable of detecting down to one single copy of DNA with high specificity. Nevertheless, the multiplexed capacities of PCR are limited by the light sources used for detecting the fluorescence. To overcome these limitations PCR has been integrated with Surface Enhanced Raman Scattering (SERS). However, the integration of these two technologies still faces some challenges. The SERS spectrum of each fluorophore present in the Raman reporters is specific. Still, when combined in a single set of probes, the interaction among them might mask the results due to the presence of peaks at close wavenumbers in the different SERS spectrum. The different wavelengths of emission from the fluorophores can also contribute to this phenomenon. These characteristics of the Raman reporters, limit the number of targets that can be analyzed simultaneously. To address this issue, it is important that the fluorophore selection is based on their characteristic SERS spectra and the intensity of each fluorophore. Another important step to consider for the integration of these techniques is the unspecific interaction between the nanoparticles and the probes that do not match their specific target during PCR, which can lead to false positives and usually requires complicated extraction methods to prevent this matter. New simple methods can be used to prevent the unspecific interaction of unmatched probes with the nanoparticles and the capture-DNA sequences like blocking DNA sequences capable of hybridizing with the unmatched probes. In this study, we present the SERS Hairpin-blocked Integrated PCR (SHIP), a detection method integrating SERS with PCR using a hairpin-shaped blocking DNA that hybridizes with the unmatched Raman probes, preventing the unspecific binding with the capture-DNA functionalized in the nanoparticles. We designed a set of probes that combine four different fluorophores associated with each specific probe. The probes get cleaved by the polymerase in the presence of the targeted mutation. With the hairpin-shaped blocking DNA implementation, the different probes can be specifically detected without any complicated extraction method by preventing the hybridization of the unmatched probes with the capture-DNA present in the nanoparticles. The analysis of the characteristic peaks from the SERS spectrum of each fluorophore, associated with the specific probes enabled the detection of single-point mutations in human serum up to four different targets. The SHIP system holds great potential for the multiplexed detection of mutations and pathogens for diagnostic purposes.