Role of probe design and bioassay configuration in surface enhanced Raman scattering based biosensors for miRNA detection.

The accurate design of labelled oligo probes for the detection of miRNA biomarkers by Surface Enhanced Raman Scattering (SERS) may improve the exploitation of the plasmonic enhancement. This work, thus, critically investigates the role of probe labelling configuration on the performance of SERS-based bioassays for miRNA quantitation. To this aim, highly efficient SERS substrates based on Ag-decorated porous silicon/PDMS membranes are functionalized according to bioassays relying on a one-step or two-step hybridization of the target miRNA with DNA probes. Then, the detection configuration is varied to evaluate the impact of different Raman reporters and their labelling position along the oligo sequence on bioassay sensitivity. At high miRNA concentration (100-10 nM), a significantly increased SERS intensity is detected when the reporters are located closer to the plasmonic surface compared to farther probe labelling positions. Counterintuitively, a levelling-off of the SERS intensity from the different configurations is recorded at low miRNA concentration. Such effect is attributed to the increased relative contribution of Raman hot-spots to the whole SERS signal, in line with the electric near field distribution simulated for a simplified model of the Ag nanostructures. However, the beneficial effect of reducing the reporter-to-surface distance is partially retained for a two-step hybridization assay thanks to the less sterically hindered environment in which the second hybridization occurs. The study thus demonstrates an improvement of the detection limit of the two-step assay by tuning the probe labelling position, but sheds at the same time light on the multiple factors affecting the sensitivity of SERS-based bioassays.

Real-Time Monitoring of the In Situ Microfluidic Synthesis of Ag Nanoparticles on Solid Substrate for Reliable SERS Detection.

A sharpened control over the parameters affecting the synthesis of plasmonic nanostructures is often crucial for their application in biosensing, which, if based on surface-enhanced Raman spectroscopy (SERS), requires well-defined optical properties of the substrate. In this work, a method for the microfluidic synthesis of Ag nanoparticles (NPs) on porous silicon (pSi) was developed, focusing on achieving a fine control over the morphological characteristics and spatial distribution of the produced nanostructures to be used as SERS substrates. To this end, a pSi membrane was integrated in a microfluidic chamber in which the silver precursor solution was injected, allowing for the real-time monitoring of the reaction by UV-Vis spectroscopy. The synthesis parameters, such as the concentration of the silver precursor, the temperature, and the flow rate, were varied in order to study their effects on the final silver NPs' morphology. Variations in the flow rate affected the size distribution of the NPs, whereas both the temperature and the concentration of the silver precursor strongly influenced the rate of the reaction and the particle size. Consistently with the described trends, SERS tests using 4-MBA as a probe showed how the flow rate variation affected the SERS enhancement uniformity, and how the production of larger NPs, as a result of an increase in temperature or of the concentration of the Ag precursor, led to an increased SERS efficiency.