A Covalent Click Chemistry Chip for Highly Efficient Capture and Brightfield Identification of Circulating Tumor Cells

Circulating tumor cells (CTCs) are critical biomarkers for cancer metastasis and recurrence; however, their extremely low abundance in peripheral blood and marked heterogeneity pose substantial challenges for accurate detection. Here, we present a CTC detection platform that combines covalent conjugation via click chemistry, dual-marker targeting (EpCAM and TROP2), and enzyme-mediated brightfield imaging for label-free CTC identification. The platform is based on vertically aligned silicon nanowire arrays modified with an amyloid-like BSA antifouling coating and tetrazine groups. This configuration enables stable immobilization of TCO-modified antibodies. Systematic optimization of key parameters, such as antibody ratio, dosage, nanowire architecture, and incubation time, enabled high-efficiency CTC capture. Compared to conventional antibody-functionalized chips, this platform achieved approximately 2-fold higher capture efficiency in tumor cell models. Under low-abundance conditions (3-10 cells/mL), the chip maintained reliable capture performance across multiple tumor cell lines, and in 1 mL peripheral blood samples from breast cancer patients, 1-5 CTCs were successfully identified with no false positives in healthy donors. Furthermore, benchmarking against representative CTC platforms (CellSearch, NanoVelcro, Parsortix, etc.) demonstrated a distinctive balance between capture performance, operational simplicity, and clinical feasibility. Notably, the brightfield imaging strategy eliminates the need for fluorescence microscopy, enabling cost-effective and rapid CTC detection. Together, these results validate the platform's clinical applicability for low-abundance CTC detection and highlight its potential for real-world liquid biopsy-based cancer monitoring.