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A Rapid and Label-free Method for Isolation and Characterization of Exosomes

Shi, Leilei
http://rave.ohiolink.edu/etdc/view?acc_num=ucin1637063241656663

Abstract Details

2021, PhD, University of Cincinnati, Engineering and Applied Science: Electrical Engineering.
Exosomes are nanoscale membrane vesicles (40-150 nm) produced by almost all kinds of cells, and they carry a wide variety of functional proteins and nucleic acids (particularly messenger RNAs and micro RNAs), which can reflect the status of their originating cells. It has been demonstrated that exosomes act as vehicles for molecular cargos in intercellular communication and thus have been considered as circulating biomarkers for early diagnosis and therapeutics in liquid biopsy. Beyond biomarker applications, exosomes can be used as drug-delivery vehicles with minimal immune response for targeted therapy in personalized medicine. Despite these remarkable attributes, the rapid isolation and non-invasive detection of exosomes have been a challenging task due to their small size and complex nature. The dissertation focuses on the development of a rapid, cost-effective, simple, and label-free method for exosome isolation and characterization. First, we studied the physical principle of particle trapping with a nano/micro-pipette dielectrophoretic (DEP) device. As a model system, carboxylic acid polystyrene (COOH-PS) beads have been used to comprehensively study the micro-/nano-pipette system’s electrokinetic (EK) forces. The correlation between the induced EK forces and the number of trapped particles has been systematically investigated by numerical modeling and experimental observations. Next, we demonstrated the capability of the glass pipette iDEP device to isolate exosomes from conditioned cell culture media and undiluted biofluids, such as plasma, serum, and saliva from healthy donors with minimal sample preparation and high yield. The iDEP device has been demonstrated to be able to extract exosomes from 200 µL sample volumes within 20 minutes. In addition, we developed a proof-of-concept EIS microchip comprised of triangular posts on a substrate for entrapment of nanoparticles using the DEP force. The entrapped particles could be further analyzed based on their impedance measured under an AC field and a wide frequency spectrum (1 kHz to 10 MHz). The device is capable of discriminating between different particles (COOH-PS beads, liposomes, and exosomes of similar size) with different dielectric properties. It has provided a means for characterization and detection of pathogenic nanovesicles based on their unique dielectric properties. Furthermore, the impedance measurement microchip has been further advanced for exosomes' differentiation and characterization. In this novel device, glass micropipettes are assembled with micro-electrodes to realize non-invasive and on-chip characterization of exosomes. The device operates by initially trapping a cluster of exosomes at close proximity of micropipette tips based on the force balance of three electrokinetic forces, and then measuring their impedance at a broad frequency spectrum. This microchip is capable of distinguishing exosomes harvested from different cellular origins at the intermediate and high frequency range (10-50 MHz) based on their unique dielectric properties. Therefore, this device could be further evolved as a simple yet powerful tool for early diagnosis in clinical settings.
Leyla Esfandiari, Ph.D. (Committee Chair)
Chong Ahn, Ph.D. (Committee Member)
Scott Langevin (Committee Member)
Rashmi Jha, Ph.D. (Committee Member)
Greg Harris, Ph.D. (Committee Member)
149 p.
Electrical Engineering
Exosome isolation; Exosome characterization; Nanopipette; Impedance measurement; Label-free; Liquid biopsy

Recommended Citations

Citations

  • Shi, L. (2021). A Rapid and Label-free Method for Isolation and Characterization of Exosomes [Doctoral dissertation, University of Cincinnati]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=ucin1637063241656663

    APA Style (7th edition)

  • Shi, Leilei. A Rapid and Label-free Method for Isolation and Characterization of Exosomes. 2021. University of Cincinnati, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=ucin1637063241656663.

    MLA Style (8th edition)

  • Shi, Leilei. "A Rapid and Label-free Method for Isolation and Characterization of Exosomes." Doctoral dissertation, University of Cincinnati, 2021. http://rave.ohiolink.edu/etdc/view?acc_num=ucin1637063241656663

    Chicago Manual of Style (17th edition)

ucin1637063241656663
198
© 2021, all rights reserved.
This open access ETD is published by University of Cincinnati and OhioLINK.