Investigating the reproducibility and repeatability of commercial SERS substrates using a new methodological approach

Surface-enhanced Raman spectroscopy (SERS) is a powerful analytical tool for the observation, the detection and the identification of chemical or biological species at low concentrations due to its high sensitivity, specific fingerprinting capability and real-time detection. However, a key challenge lies in establishing a suitable and reliable measurement protocol to ensure both reproducibility and repeatability when SERS is used as a sensing nanoplatform. In this paper, we propose a specific methodology to investigate the performance of SERS substrates, with a particular focus on their reproducibility and repeatability. Furthermore, we validate our approach on one commercial SERS Hamamatsu substrate from Hamamatsu Photonics by using diluted solution of 4-mercaptobenzoic acid (MBA) at an excitation wavelength of 633 nm. This proposed protocol consists in recording 25 SERS maps equally distributed on the whole surface substrate. For each map, 16 spectra have been acquired and averaged to provide a representative SERS signal. In total, 400 spectra have been collected and analyzed by using the integrated intensities of characteristic MBA bands to determine both reproducibility and repeatability. This approach enables us to quantify signal variations, at the micrometer scale, as well as across the entire substrate. We demonstrated that while the SERS response is highly reproducible locally, it becomes less consistent when evaluated across the full surface. However, the SERS signal is not repeatable at the local scale but it can be repeatable at the whole substrate scale as the average SERS intensity is identical for both SERS measurements. Furthermore, we demonstrated that this method can also be applied to DNA strands thereby demonstrating its effectiveness in evaluating biosensors. Finally, the proposed methodology and protocol can then be used as a standard to precisely evaluate the sensing performances of other substrates.