Publication Date

2017-12-07

Availability

Embargoed

Embargo Period

2019-05-07

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PHD)

Department

Chemistry (Arts and Sciences)

Date of Defense

2017-04-21

First Committee Member

Sylvia Daunert

Second Committee Member

Leonidas G. Bachas

Third Committee Member

Roger M. LeBlanc

Fourth Committee Member

Sapna K. Deo

Abstract

Bioluminescence is the generation of light through a natural oxidation chemical reaction, thus eliminating the need for an external light source which eliminates background fluorescence. This results in higher signal-to-noise ratio when compared to fluorescence, making it suitable for use as a label in ultra-sensitive detection applications. Herein, a multiplexed assay to measure three analytes simultaneously in a single solution by employing the variants of the photoprotein aequorin (AEQ) has been demonstrated. Aequorin, natively found in jellyfish Aequorea victoria, can emit a flash-type light at 469 nm with a half-life around 0.5-1 s after the binding of calcium ions (Ca2+). The emission profiles, decay kinetics, and thermostability of AEQ have been tuned significantly from the native AEQ through site-specific mutations and use of synthetic coelenterazine analogues, expanding the applications of AEQ in versatile and miniaturized analytical systems. Specifically, the AEQ mutants AEQY82F and AEQF113W have been genetically conjugated to three main pro-inflammatory cytokines, namely, interleukin 6, interleukin 8, and tumor necrosis factor alpha (IL-6, IL-8, TNF-α) resulting in cytokines labeled with AEQ mutants. The three fusion proteins in conjunction with different synthetic coelenterazine analogues were applied as bioluminescent labels for the detection of all three cytokines simultaneously through the combination of spatial and temporal windows. In addition, bioluminescence was employed to detect and image cancer cell lines in vitro and in vivo. The bioluminescent protein Gaussia luciferase (Gluc), native from the marine copepod Gaussia princeps, was employed as a sensitive detection label. The surface biomarker epidermal growth factor receptor (EGFR) that is overexpressed in various cancer cells was chosen as the binding target to increase the specificity of the detection reagent in the cancer cells. Gluc was genetically conjugated to the recognition molecule human epidermal growth factor (EGF), the ligand of EGFR, to construct a bioluminescence-based probe that can be directed to the EGFR receptors in the cancer cells, and thus, if such cells are present, then binding between Gluc-EGF and the EGFR occurs and we can visualize the cells by the bioluminescence emission of Gluc. Herein, the EGF-Gluc fusion protein was applied to detect three common cancers including head and neck squamous cell carcinoma (HNSCC), breast cancer, and pancreatic cancer. Furthermore, a different bioluminescent probe for the detection of head and neck cancer, specifically, HNSCC CAL-27 cells, was constructed through chemical conjugation, using aequorin as the reporter. The biomarker CD44 that is overexpressed on the surface of the HNSCC cells was chosen as the binding target. Aequorin mutant S5CF113W was chemically conjugated to the anti-human CD44 antibody to construct a detection probe with specific targeting capability. The anti-CD44 antibody-AEQSC5F113W conjugate was employed in the development of the bioluminescent assay of the HNSCC cells.

Keywords

Bioluminescence; Aequorin; Gaussia luciferase; Cytokines; Cancer biomarker; In vivo imaging

Available for download on Tuesday, May 07, 2019

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