## 2 Materials and Methods  
In this study, we enrolled n = 53 female patients with biopsy confirmed breast cancer, which were referred to the Ion Chiricuta Oncologic Institute Cluj-Napoca, Romania, for mastectomy/lumpectomy. Patients were included irrespective of their stage, grade or histologic type. Male patients presenting with breast cancer were excluded from this study. Morning urine samples were collected in plastic containers and stored at \(-80\)\({}^{\circ}\)C until analysis. Before the SERS measurements, the urine samples were centrifuged for 10 min at 5800 g in order to remove crystals and cell debris. All urine samples were collected from treatment-naive patients. Control urine samples were obtained from n = 22 subjects confirmed to be healthy by clinical examinations. The study was approved by the ethics committee of Iuliu Hatieganu University of Medicine and Pharmacy Cluj-Napoca and all subjects provided written informed consent for enrolling in the study. For the SERS analysis, silver nanoparticles synthesized by reduction with hydroxylamine hydrochloride (hya-AgNPs) were used [20]. Briefly, the nanoparticles were synthesized by mixing 17 mg H2 NOH \(\times\) HCl with 1.2 mL of NaOH solution 1% and 8.8 mL of ultrapure water. Separately, 17 mg of AgNO3 was dissolved in 90 mL of ultrapure water. The two solutions were rapidly mixed under vigorous stirring in order to synthesize the nanoparticles. The pH of the colloid was 7.5 after the synthesis. The fresh colloid was left at room temperature overnight before measurements. All chemicals were purchased from Sigma-Aldrich (Steinheim, Germany). UV-Vis absorption spectra of the silver nanoparticles were recorded using a V-630 Spectrometer (Jasco) by diluting the colloidal solution 10-fold. For acquiring the SERS spectra, 10 uL of urine was added to 90 uL of hya-AgNPs. Then, the colloid was activated by adding 1 uL of Ca(NO3)2 10\({}^{-2}\) M (final concentration of Ca(NO3)2 10\({}^{-4}\) M). A 5 uL droplet from this mixture was then placed on an aluminium foil covered microscope slide and analysed by Raman spectroscopy immediately (in liquid form). The SERS spectra were acquired using an InVia Raman Spectrometer (Renishaw), equipped with a Nd:YAG doubled frequency laser emitting at 532 nm (laser power 20 mW on the sample), which was focused for 40 s on the sample through a 5X microscope objective (NA 0.12). Acquiring SERS spectra from samples in liquid form results in an average SERS spectrum due to the continuous thermal motion of the molecules. Spectra pre-processing consisted of background removal using a linear baseline correction, which eliminated the spectral noise due to autofluorescence, followed by mean normalization. In the case of mean normalization, each intensity value is normalized by the mean intensity of the spectrum. In this way, spectra can be compared even when there are significant differences in the absolute intensities of spectra [21]. The statistical analysis consisted of principal component analysis (PCA) and principal component analysis-linear discriminant analysis (PCA-LDA). PCA is an exploratory data analysis technique that reduces the dimensionality of the data down to a desired number of principal component (PC) score values. The information captured by each PC can be visualized by analysing the corresponding loading plot of the PC. Given that PCA is an unsupervised method, it cannot be used for assessing the classification accuracy. Therefore, we employed  
PCA-LDA, which is a supervised multivariate data analysis technique that requires prior knowledge regarding the group to which samples correspond to (in our case the breast cancer and the control group) in order to calculate figures of merit such as sensitivity, specificity and overall accuracy. Sensitivity is defined as the true positive rate (the percentage of samples in the disease group correctly assigned as such), whereas specificity is defined as true negative rate (the percentage of samples in the control group correctly assigned as such).