Date of Award

5-2013

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Legacy Department

Bioengineering

Advisor

Vertegel, Alexey

Committee Member

Dean , Delphine

Committee Member

Kornev , Konstantin

Committee Member

Lee , Jeoung Soo

Abstract

Single cell probing has found a number of applications in different areas of research. It can help us to better understand cell-to-cell interactions; it has found numerous applications in immunology, cancer research, detection of pathogenic infections and genetic abnormalities. The single cell analysis is very important in stem cells research and development of cells. The main obstacle in the single cell analysis is the small amount of analyte that a single cell could provide. Another difficulty is connected to the cell-to-cell variability inside the uniform population due to the differences of single cells in size, activity, mitotic stage, and functions.
To overcome these problems several methods of single cell analysis including utilization of fluorescence and microfluidic platforms were developed. However, existing methods for cell isolation and analysis are laborious, costly, taking long time and often lack sensitivity, which is necessary for a single cell level probing. Therefore there is a need for novel high throughput and convenient experimental techniques for analysis of gene expression in individual cells. Fiber-based devices with high surface area that are modified with mRNA capturing element could be an attractive candidate for this purpose. We proposed to develop microfluidic electrospun fiber yarns based biosensors for applications in detection of small amounts of targeted analytes, such as mRNA material of a single cell or specific secondary antibodies molecules.
In order to achieve this goal of improving single cell analysis we produced highly sensitive microfiber probes for PCR analysis of mRNA present in concentrations corresponding to those in a single cell. The bundle of microfibers with high specific surface area was modified with oligo dT25 which specifically binds the poly-A tail of mRNA. Now that RNA is captured by the fiber, relative expression of β-actin by contractile and synthetic vascular smooth muscle cells, as well as by adult and neonatal myocytes, was used as a model to evaluate performance of the fiber-based devices for mRNA extraction. Application of fiber yarns as the mRNA isolation probes for PCR analysis improves usability during the sample collection and processing and allows for focused probing from a desired area.
The sensitivity of an enzyme-linked immunosorbent assay (ELISA) performed directly on the fiber-based devices was also evaluated. Electrospun fiber yarns were covalently coated with a model capture antibody and then used to evaluate presence of the specific secondary antibodies in solutions. The colorimetric ELISA procedure was performed with ABTS substrate to investigate sensitivity of prepared sensors in the ELISA application.
Overall, these fibers provide a new flexible platform for a number of analysis types that can considerably improve its convenience and availability for various applications. This approach is universal and could be utilized to develop fiber-based devices that could be potentially used in point-of-care devices and for forensic analysis.

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