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theses

Immunoassays are one of the most widely performed assays in clinical and research settings due to their sensitivity, specificity, and ability to measure wide ranges of analytes. Recently, immunoassay technology has greatly improved due to the development of multiplex platforms, capable of measuring multiple analytes in a single sample. However, immunoassays are costly, time-consuming, and require relatively large sample volumes that inhibit their use in specific applications. Performing immunoassays using microfluidic devices has been shown to significantly reduce assay time, cost, and sample and reagent consumption. However, previous immunoassay devices possess drawbacks that prevent their broad use, including: low sensitivity, limited dynamic range, inability to change the analyte specificity, specialized reagent requirements, inability to produce quantitative data, and low sample throughput. Therefore, the objective of this dissertation was to develop a microfluidic immunoassay device overcoming the aforementioned limitations. A proof-of-concept device was developed capable of performing 8 parallel immunoassays on commercially available antibody conjugated microbeads. This eliminates the need for specialized reagents while allowing any analyte, for which antibodies are available, to be measured. Furthermore, we developed the first experimentally validated computational fluid dynamic model of antibody antigen binding in microchannels. Design of experiments (DOE) and multi-objective optimization techniques were used in conjunction with the model to optimize an IL-6 immunoassay with a sensitivity of 358 fM using only 1.35 μL of sample volume. The device design was then scaled-up to allow 32 samples to be processed simultaneously. With the expanded device, we demonstrated high-sensitivity, a large dynamic range, and quantification of 6 cytokines (Il-1b, Il-6, IL-10, IL-13, MCP-1, and TNF-a). Finally, we measured in vitro experimental supernatants in parallel using the microdevice and a conventional benchtop assay. The microdevice provided comparable results while reducing sample volume from 50 to 4.2 μL. In summary, we demonstrated a low-volume, highly sensitive assay with a large dynamic range capable of processing large numbers of samples using commercially available reagents. Due to these advantages, the technology in this work has far-reaching in vitro, in vivo, and clinical applications.

ETD_6084

Ph.D.

Includes bibliographical references

by Mehdi Ghodbane

doi:10.7282/T370835S

ETD doctoral

The author owns the copyright to this work.