A Permeabilization Workflow To Enable Specific Multiplexed Profiling Using SERS Nanoparticles.

Surface-enhanced Raman scattering nanoparticles (SERS NPs) are powerful tools for cellular-specific targeting and multiplexed biomarker detection. While they have been effective in labeling extracellular receptors, their application to intracellular targets has been limited by poor membrane permeability and endosomal trapping. Here, we present an optimized permeabilization and staining strategy that enables robust intracellular targeting with SERS NPs. Using breast cancer as a model system, we focused on three clinically relevant biomarkers─human epidermal growth factor 2 (HER2), which has both extracellular and intracellular targets, and estrogen receptor (ER) and progesterone receptor (PR), which are exclusively located inside the cells─to demonstrate the ability of our platform to detect both extracellular and intracellular targets. We conjugated SERS NPs with anti-HER2 antibodies to assess specific binding efficiency across breast cancer cell lines with varying HER2 expression. Flow cytometry revealed a strong correlation between HER2 expression and the specific-to-nonspecific binding ratio, demonstrating over 100-fold specificity for HER2-overexpressing cells. Fluorescence and Raman imaging confirmed high specificity and sensitivity. To extend this approach to intracellular targets, we evaluated three permeabilization agents─Tween 20, Triton X-100, and methanol─and identified Triton X-100 as optimal. It enabled ∼160 nm SERS NPs to access the intracellular space while preserving cell viability. SERS NPs conjugated with anti-ER and anti-PR antibodies revealed significant biomarker binding without compromising cell health, revealing the capability to specifically profile intracellular biomarkers with varying expression levels of ER and PR. Furthermore, multiplexed detection was demonstrated using a cocktail of SERS NPs targeting HER2, ER, and PR in mixed cell populations, mimicking clinical scenarios such as liquid biopsies. Even when target-positive cells were present at low abundance, the NPs retained selective binding and detection capability. Overall, our findings advance the potential of SERS NPs for enhancing breast cancer diagnostics through accurate, multiplexed biomarker targeting.