Integration of SERS active nanotags with mass spectrometry complementary techniques: quantitative capacities study for cancer markers measurement on single cell.

Abstract

Immuno-surface enhanced Raman spectroscopy (iSERS) has the potential to supplant current standard tissue imaging techniques due to its multiplexing and quantitative abilities. However, work is still to be furnished for this achievement. This work explores the potential of iSERS using Au@Ag core@shell nanoparticles as quantitative imaging tags. Au@Ag nanoparticles (≈ 30 nm gold core, ≈ 7 nm silver shell) were functionalized with Raman reporters to form SERS tags. Among five reporters tested at 532 nm, RBITC provided the highest intensity, good synthesis reproducibility (≈ 10% variability) and showed excellent linearity between SERS signal and tag concentration in water (R² > 0.99). However, in buffered solutions mimicking biological conditions, isotonicity induced aggregation and signal loss. Stabilizing polymer coating was successfully applied on the tags, preserving linearity, but increasing variability (≈ 43%) and fluorescent background due to partial dye displacement. Hydrated gelatine gels were then used as tissue-mimicking matrices. Because of gelatine fluorescence at 532 nm and instrumental constraint, measurements were performed at 785 nm, where Oxazine-based tags outperformed other reporters. Raman imaging of gels demonstrated that tags aggregation strongly influences SERS signal intensity and spatial variability. Correlative SEM and mass spectrometry imaging confirmed that SERS differences mainly arose from aggregation rather than nanoparticle quantity, highlighting the need for multi-technique validation. Finally, quantitative analysis in gels was evaluated using two data-processing strategies: intensity-based and pixel-based approaches. Both achieved similar sensitivity (LOD ≈ 2 × 10⁻¹² NP/kg), but the pixel-based method seems to shift the dynamic range toward lower concentrations, making it more suitable for quantitative imaging. Overall, the study demonstrates the potential of SERS tags for quantitative imaging while emphasizing the need to apply robust statistical analysis in future tissue studies.