Microfluidic-based Fluorescence Detection for Biomarker Analysis

Abstract

In this dissertation, 2 platforms are described with the combination of microfluidics and fluorescence detection for biomarker analysis. The first platform described here is to isolate CTCs with high purity by using 2-stage sorting technique and herringbone features for aliquot stretching, including axial and lateral mixing to spread cells. We achieved a roughly 70-fold improvement in target cell purity to 70%, compared to our previous 1-stage eDAR platform with 1% purity. With the 8.8cm channel and herringbone features, Single CTCs can be collected with less than one contaminating WBC, making sequential eDAR (S-eDAR) amenable to downstream analyses such as single-cell sequencing and single-cell fluorescence in situ hybridization (FISH). S-eDAR was further applied to recover rare cells from concentrated PBMCs at a recovery rate of over 85%, similar to the performance of S-eDAR when using whole blood samples. However, by using concentrated PBMCs, the required sample volume was reduced, and the antibody cost was reduced by 20-fold. We applied S-eDAR to sort rare fetal nucleated red blood cells (fNRBCs) from cord blood and adult female PBMCs into which cord blood cells were added, demonstrating the potential of S-eDAR for isolating fNRBCs from maternal blood for noninvasive prenatal diagnosis. At the end, fNRBCs were successfully enriched from 2nd trimester blood and confirmed with immunostaining. To complement CTCs analysis, the single-molecule sensitive flow platform was applied to enable surface protein profiling on exosomes. The platform was validated with single molecules first, including alexa647 anti-IgG and PE anti-IgG. The recovery rate for these 2 molecules were higher than 90%, indicating the high sensitivity of detecting single molecules with this platform. After validation, DiFi EVs (from DiFi human colorectal cancer cell line) were labeled with a membrane dye (Anepps) and Phycoerythrin (PE)-tagged antibodies (3 common tetraspanin markers, including CD9, CD63 and CD81 and epidermal growth factor receptor (EGFR)). Based the colocalization of fluorescence signals from protein markers and membrane dye, antibody-labeled exosomes can be clearly differentiated from free antibodies. This platform can characterize surface proteins on exosomes and analyze their heterogeneity as a potential complement analysis for CTCs. Furthermore, this platform can characterize EV subpopulations with multiplex protein labeling, which is useful for early detection of cancers and characterize the dynamic change of tumors at different stages and their heterogeneity.