A multiplexed, ratiometric method is described that can confidently distinguish between cancerous and noncancerous epithelial prostate cells in vitro. The technique is based on bright surfaceenhanced resonance Raman scattering (SERRS) biotags (SBTs) infused with unique Raman reporter molecules, and carrying cellspecific peptides. Two sets of SBTs were used. One targets the neuropilin-1 (NRP-1) receptors of cancer cells through the RPARPAR peptide. The other functions as a positive control (PC) and binds to both noncancerous and cancer cells through the HIV-derived TAT peptide. Point-by-point 2D Raman maps of the spatial distribution of the two tags were constructed with subcellular resolution from cells simultaneously incubated with the two sets of SBTs. Averaging the SERRS signal over a given cell yielded an NRP/PC ratio from which a robust quantitative measure of the overexpression of the NRP-1 by the cancer cell line was extracted. The use of a local, on-cell reference produces quantitative, statistically robust measures of overexpression independent of such sources of uncertainty as variations in the location of the focal plane, the local cell concentration, and turbidity.surface-enhanced Raman spectroscopy biomarker | cancer cell identification | multiplexing | silver nanoparticles E arly and rapid identification of malignant cells [ideally freeflowing in biological fluids such as urine (1) or blood (2-4)] is a central goal in cancer research. Accordingly, seeking improvements in sensitivity and accuracy of detection and quantification through, for example, the development of cell-specific diagnostic tools and biomarkers is an active enterprise. Although it is known that such cells are present in body fluids, concentrating them to levels appropriate for confident identification and quantification is still a serious challenge, despite significant advances that have been made by, for example, using microfluidics (5). Once collected, the cells must be identified as either cancerous or noncancerous within tolerable confidence limits, which may be improved by, for example, using several nonredundant biomarkers. Numerous promising identification methods have been reported using antibodies. In this article, we report a technique based on surface-enhanced Raman spectroscopy (SERS) and biotags that produce bright surface-enhanced resonance Raman scattering (SERRS) signals, comparable to or exceeding the intensities of fluorescent tags. The SERRS biotags (SBTs) used in this study exploit peptides as recognition moieties. We show that using SBTs ratiometrically can provide highly statistically significant, quantitative measures of neuropilin-1 (NRP-1) overexpression insensitive to normal causes of uncertainty in optical measurements such as variations in focal plane, cell concentration, and turbidity.Normal Raman signals from cells and their infrared (FTIR) absorbance patterns have been previously used to differentiate between cancer and normal cells (6-9). SERS has been used as an alternative immunohistochemistry (IH...