Label-free detection of miRNAs: role of probe design and bioassay configuration in Surface Enhanced Raman Scattering based biosensors

TL;DR

This study investigates how probe design and bioassay configuration influence the sensitivity of SERS-based biosensors for miRNA detection, highlighting the importance of probe positioning and hybridization strategy.

Contribution

It provides new insights into probe labeling effects and bioassay configurations that optimize SERS detection of miRNAs, including the impact of probe position and hybridization steps.

Findings

Probes closer to the plasmonic surface increase SERS signal at high miRNA concentrations.

At low concentrations, SERS intensity levels off due to hot spot contributions.

Two-step hybridization partially preserves the benefits of probe positioning.

Abstract

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 two different bioassays relying on a one-step or two-step hybridization of the target miRNA with oligonucleotide probes. The detection configuration is varied, exploring the impact of the position of the Raman reporter along the oligo sequence and of the reporter identity on bioassay sensitivity. In the high miRNA concentration regime(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 two configuration is instead recorded at low miRNA concentration. Such effect is explained by an increased contribution of Raman hot spot to the whole SERS signal, as confirmed by simulations of the electric near field for a simplified model of the Ag nanostructures. The beneficial effect of reducing the reporter-to-surface distance is however partially retained for the two-step hybridization assay thanks to the less sterically hindered environment in which the second hybridization occurs. The study thus demonstrates that the limit of detection of the assay can be lowered by tuning the probe labelling position, but sheds at the same time light on the complexity of the bionanointerfaces in SERS and on the multiple factors affecting bioassay sensitivity.