Integrated microfluidic single-cell immunoblotting chip enables high-throughput isolation,enrichment and direct protein analysis of circulating tumor cells

Effective capture and analysis of a single circulating tumor cell(CTC)is instrumental for early diagnosis and personalized therapy of tumors.However,due to their extremely low abundance and susceptibility to interference from other cells,high-throughput isolation,enrichment,and single-cell-level functional protein analysis of CTCs within one integrated system remains a major challenge.Herein,we present an integrated multifunctional microfluidic system for highly efficient and label-free CTC isolation,CTC enrichment,and single-cell immunoblotting(ieSCI).The ieSCI-chip is a multilayer microfluidic system that combines an inertia force-based cell sorter with a membrane filter for label-free CTC separation and enrichment and a thin layer of a photoactive polyacrylamide gel with microwell arrays at the bottom of the chamber for single-cell immunoblotting.The ieSCI-chip successfully identified a subgroup of apoptosis-negative(Bax-negative)cells,which traditional bulk analysis did not detect,from cisplatin-treated cells.Furthermore,we demonstrated the clinical application of the ieSCI-chip with blood samples from breast cancer patients for personalized CTC epithelial-to-mesenchymal transition(EMT)analysis.The expression level of a tumor cell marker(EpCAM)can be directly determined in isolated CTCs at the single-cell level,and the therapeutic response to anticancer drugs can be simultaneously monitored.Therefore,the ieSCI-chip provides a promising clinical translational tool for clinical drug response monitoring and personalized regimen development.