The goal of this application is to develop a technology platform for capture, detection, enumeration and molecular profiling of circulating tumor cells (CTCs) in blood based on unique integration of novel nanomaterials, advanced optical spectroscopy and robust microfluidics. Sensitive and reliable CTC assessment can provide a powerful tool in the clinic for early diagnosis of cancer and metastasis, prognosis of cancer patients, assessment of tumor stage, monitoring of therapeutic response and ultimately aiding in optimization of treatment regimens. Although significant progress has been made during the last decade, CTC detection remains difficult because CTCs are rare events and a heterogeneous population. An ideal technique should capture CTCs of subpopulations with high efficiency and detect them with high sensitivity and specificity without complicated operations and extensive human intervention. We recently demonstrated that rare breast cancer cells in whole blood (1-2 cells/mL blood) can be dually captured with magnetic separation and detected on-line with surface enhanced Raman scattering (SERS) spectroscopy using antibody-conjugated Raman-coded oval shape iron oxide-gold (IO-Au) core-shell nanoparticles (NPs), raising exciting possibilities of new avenues for CTC detection and analysis. In this project, we propose to make multicolor oval shape IO- Au SERS NPs to recognize CTCs with different markers directly in whole blood, to capture them simultaneously via magnetic separation and to detect them subsequently on a microfluidic device at single cell resolution with the capability of molecular profiling of surface makers using SERS spectroscopy. Combining immunomagnetic enrichment with high specificity, multiplexed targeting for capture of CTC subpopulations, multicolor SERS detection with high sensitivity and specificity, microfluidics for handling rare tumor cells and magnetic-metal nanoparticles for dual enrichment and detection, the integrated yet simple and efficient platform will allow for reliable and more sensitive CTC detection and analysis in routine blood draws, helping physicians to make rational decisions for best personalized cancer treatment. Using breast cancer as a model, the project will be accomplished through the following specific aims: (1) Develop multicolor IO-Au SERS NPs targeting different CTC surface markers namely epithelial cell adhesion molecule (EpCAM), epidermal growth factor receptor (EGFR), human epidermal growth factor receptor 2 (HER2) and insulin-like growth factor 1 receptor (IGF1R); (2) Develop a microchip-based system for on-line magnetic enrichment, SERS detection and molecular profiling of CTCs with multiplexed multicolor IO-Au SERS NPs and (3) Assess the capability of our system for detection and molecular profiling of CTCs in blood samples from metastatic breast cancer patients via comparison with conventional flow cytometry. The proposed research will make a high impact in improving the management of patients with breast cancer and other malignancies.