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

Abstract

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.

Sickle-like Inertial Microfluidic System for Online Rare Cell Separation and Tandem Label-Free Quantitative Proteomics (Orcs-Proteomics)

“…R e is the Reynolds number of the channel, U m is the maximum fluid velocity, R is the radius of the channel, and C L is the lift coefficient. As mentioned in our previous work and the above equations, 34,35 both the forces are related to the size of the cells and the flow rate in the chip. By adjusting the sample injection flow rate, different sized cells separate slightly from each other.…”

“…As mentioned in our previous work and the above equations, , both the forces are related to the size of the cells and the flow rate in the chip. By adjusting the sample injection flow rate, different sized cells separate slightly from each other.…”

“…The separation channel used in this work was an inertial force-based cell separation chip, which was adjusted from our previous work. , The chip consists of one inlet, zigzag channels for cell focusing, a semicircle channel for cell separation, and two outlets for collecting the separated cells. The width of the zigzag section was 200 μm, and the radius of the inner semicircle curve channel was 6 mm (Figure A).…”

3D-Stacked Multistage Inertial Microfluidic Chip for High-Throughput Enrichment of Circulating Tumor Cells

“…For example, Tang et al used an asymmetric serpentine channel for impedance detection to discriminate CTCs from blood cells [21]. And Abdulla et al combined a serpentine channel and membrane filter to sort cells for downstream single-cell analysis [22].…”

Precise Design Strategies of Nanotechnologies for Controlled Drug Delivery

“…After the creation of the Precision Medicine Initiative (PMI) in 2015 [ 14 ], precision medicine put emphasis on tailoring specific treatment plan to individual by accounting for multiple genetic and epigenetic characteristics [ 15 ]. Nanotechnologies for drug delivery are designed to overcome heterogeneity among patients because of their engineering functional and structural properties, which significantly improves drug specificity, optimizes dosing, and combinatorial strategies [ 16 , 17 , 18 ].…”