Fig. 1. ERα-AuNPs incubated in MCF-7 cells showed strong SERS signal with no detectable cell toxicity. (A) Average SERS signal from ERα-AuNPs in MCF-7 cells under different nanotag incubation times ranging from 5 min to 120 min. SERS spectra were collected in fixed MCF-7 cells using a Renishaw InVia system combined with edge Streamline HR high confocality mode at 1 μm resolution in the X and Y directions. A 50× magnification NIR APO Nikon water immersion objective with a 0.75 NA was used on the samples at a laser power of 1.2 mW (10% power) at the sample, from a HeNe 633 nm excitation source with a 0.1 s acquisition time per point, and a 1200 L mm⁻¹ grating in high confocality mode. Windows-based Raman Environment (WiRE™ – Renishaw plc) 4.4 software package was used to pre-process the data for cosmic ray removal and baseline subtraction. (B) Cell viability assay of MCF-7 cells treated with 60 pM ERα-AuNPs (left) and 60 pM PEG5000-AuNPs (nanotags without ERα antibody functionalisation) (right) for 48 h using live/dead staining with Calcein AM and EthD Br-1 assay. Viable cells appear as green (Calcein AM), while non-viable cells appear as red (EthD Br-1). Scale bar 100 μm. (C) Cell viability using trypan blue assay for MCF-7 cells treated with 60 pM BPE-AuNPs, 60 pM PEG5000-AuNPs or 60 pM ERα-AuNPs for 48 h. The average of ten samples from three independent biological replicates is shown. Error bars presented as mean ± standard deviation (SD).