| Popis: |
Cancer contributes to the most dreaded global disease, and collective types of cancer have been identified as the second largest cause of death in the United States. The high mortality rate of cancer is usually caused by the late stage diagnosis, where cancer cells have metastasized throughout the body. Therefore, the development of accurate and sensitive platforms for cancer detection at an early stage is of great importance. In addition, given the complexity of cancer cell formation, extensive molecular profiles from different classes of cancer-related biomarkers should be evaluated to have a reliable diagnosis, such as mutations in certain gene fragments and over/under-expression of gene activities. Thus, there is an expanding need to investigate powerful tools to detect cancer-related biomarkers that cut across several different classes.This dissertation focuses on the development of electrical impedance sensors to detect different types of cancer-related biomarkers: microRNA, genomic mtDNA and exosomes. First, we report a new nanopore-based detection scheme utilizing a borosilicate micropipette and an assay of complementary r-peptide nucleic acid (r-PNA) probes conjugated to polystyrene beads, to detect miR-204 and miR-210 related to the clear cell Renal Cell Carcinoma (ccRCC). In the detection platform, electroosmotic flow (EOF) is induced as the driving force to transport PNA-beads harboring target miRs to the tip of the pore (sensing zone), which results in pore blockades with unique and easily distinguishable serrated shape electrical signals. The concentration detection limit is investigated to be 1 and 10 fM for miR-204 and miR-210, respectively. The EOF transport mechanism enables highly sensitive detection of molecules with low surface charge density with 97.6% detection accuracy. Next, this PCR-free nanopore-based electrical impedance sensor has been taken a step further and developed as a semi-automated mtDNA sensor. We have omitted the time-consuming sample pretreatment procedure including the DNA digestion and denaturation by designing a new set of bis-PNA and r-PNA assays to detect a segment of the circular double-stranded DNA with high specificity. As a model system for circular DNA, the pTS12 plasmid was constructed with the insertion of a known oligonucleotide DNA sequence into the pUC19 plasmid and the concentration detection limit of 10 pM was established. Furthermore, to investigate the single-base mismatch specificity of our system, skin-derived double-stranded mtDNA with a known point mutation was detected.In addition, a novel frequency-dependent impedance measurement system has been developed to characterize exosomes based on their unique dielectric properties. The system is composed of an insulator-based dielectrophoretic device and an electrical impedance spectroscopy (EIS) to measure the impedance of immobilized vesicles in a wide frequency spectrum. The system showed the ability to discriminate exosomes derived from different cellular origins as well as exosomes from the same cellular origins but different size distributions. The results obtained from the exosomes’ impedance measurements most likely reflect to their differences in membrane and cytosolic compositions at different frequency spectrum and thus, their unique dielectric properties. This proof-of-concept can pave the way for detection and characterization of pathogenic exosomes and other nanovesicles based on their unique dielectric properties. |
