Extracellular Vesicle Profiling Towards Disease Detection by Using Micro/Nano-fluidic Devices

Abstract

Based on the American Cancer Society, in 2020 there will be an estimated 1.8 million new cancer cases diagnosed and about 0.6 million cancer patients will die because of cancer. Meanwhile, millions of people in the United State do not have proper treatment regimens, early diagnosis opportunities, and continuously monitoring recurrence. Point-of-care testing (POCT) is one possible solution to reduce the cost while maintaining disease management capabilities. To achieve the potential of POCT, extracellular vesicles (EVs) have garnered much attention because of the ability to secure these biomarkers in a minimially invasive manner and also, the wealth of information they contain to realize full management of disease for cancer patients. To facilitiate the realization of POCT for cancer diseases, microfluidic and nanofluidic technologies have been recognized as possessing high efficiency, throughput, accuracy, and low-cost to replace conventional benchtop experiments and realized POCT for oncology.We successfully developed a microfluidic system, ExoSearch chip, for cancer diagnosis with on-chip EV isolation using immune-magnetic beads. The ExoSearch chip also included features of continuous flow and customizable capture antibodies, which makes the ExoSearch chip able to target different types of cancer by targeting the appropriate antigen. Three ovarian cancer-related biomarkers, CA-125, EpCAM, and CD24, which reside on the surface of EVs, were analyzed to provide accurate results (p = 0.0001, 0.0009, 0.003, respectively). Furthermore, 3-dimensional (3D) printing technology was used for microfluidic fabrication to boost prototyping capabilities. The EVs were also able to be collected, engineered, and released for immunotherapy. The EVs were modified by cancer-related peptides and were able to trigger an immune response and activate the cytotoxic T cell (CTL) to target tumor cells. Both in vitro and ex-vivo experiments were performed to evaluate the engineered EVs for immunotherapy. Our lab also developed an EV-MAP chip made from thermoplastic materials, which lifted the possibility of chip mass production for clinical applications that require one-time use devices, more binding sites, and faster sample processing rate, which increased the binding capacity and also the sampling efficiency. The EV-MAP chip was used for ovarian cancer plasma sample characterization and for radiation injury diagnosis. The EV related miRNA, miR-92a-3p, and miR-204-5p were also targeted as biomarkers for exposure to ionizing radiation. The combination of total protein expression and miRNA expression indicated that the CD8 expressing EV subpopulation showed upregulated numbers of CD8 expressing EVs without significant changes in protein expression and the CD8 subpopulation did not show major expression of miR-92a-3p or miR-204-5p. Current enumeration platforms for EVs consist of nanoparticle tracking analysis (NTA), electron microscopy (EM), high-resolution flow cytometry (hFC) and are used for both EV size distribution and concentration analysis. However, disadvantages of these technologies include large sample volume requirements, vibration-free operation, temperature consistency, and extensive software analysis, which have reduced the EV translation capacity. We have developed an in-plane nano-Coulter counter (nCC) device for enumerating EVs rapidly. With the concept of resistive pulse sensing (RPS), an electrical signal is generated for each EV when the EV travels through the nanopore. By understanding the electrical signals' frequency and amplitude, both the concentration and size distribution profiles can be collected for each EV sample quickly and efficiently. The nCC chip can also be used for EV enumeration for SARS-CoV-2 viral particle counting to determine viral load of SARS-CoV-2 viral particles enriched from biological samples to screen the infectious status of patients suspected of possessing COVID-19.