Publication Date
2025
Document Type
Dissertation/Thesis
First Advisor
Nesterova, Irina V.
Degree Name
M.S. (Master of Science)
Legacy Department
Department of Chemistry and Biochemistry
Abstract
This thesis presents the in vitro evaluation of a novel near-infrared (NIR) fluorescent small-molecule probe AB-139 that selectively binds to epidermal growth factor receptor (EGFR) kinase and operates through a turn-on fluorescence mechanism. The sensor addresses several limitations associated with conventional protein-based probes by offering small-molecule specificity, tunable modularity, deep-tissue imaging potential, and reduced background noise. AB-139 comprises four key components: A Zn-phthalocyanine fluorophore that serves as the NIR emission reporter; hydrophilic triethylene glycol chains that enhance aqueous solubility and reduce aggregation; cationic quaternary ammonium groups that facilitate cellular permeability; and a gefitinib-derived recognition anchor that targets the intracellular ATP-binding pocket of EGFR. In aqueous media, AB-139 forms non-fluorescent H-aggregates due to strong π–π stacking of the phthalocyanine cores. Upon binding to EGFR, the anchor engages with the kinase domain and promotes partial deaggregation of the sensor, restoring fluorescence in the 688–694 nm range. Spectroscopic characterization confirmed that the sensor’s fluorescence is minimal in aqueous environments but significantly increases in the presence of EGFR, with a dissociation constant (Kd) of 12 ± 3nM. Specificity studies using structurally unrelated but abundant protein bovine serum albumin (BSA) and a structurally related AKT kinase revealed no significant fluorescence activation, highlighting the selective nature of the sensor for EGFR. To further confirm binding site specificity, two control compounds: AB-138 (lacking the anchor) and ZnPc (lacking both the linker and the anchor) were synthesized and tested under identical conditions. These compounds demonstrated either moderate or negligible fluorescence upon EGFR addition, confirming that EGFR recognition and fluorescence activation critically depend on the presence of the anchoring domain. Competitive displacement assays for FDA-approved EGFR inhibitors, gefitinib and afatinib, were evaluated. These molecules are partially displaced AB-139 from EGFR, causing a measurable decrease in fluorescence, but did not return the signal to baseline, suggesting either incomplete displacement or some kind of interaction of the fluorophore with hydrophobic surfaces on EGFR. In contrast, amodiaquine, a non-EGFR-targeting molecule, failed to disrupt the AB-139–EGFR complex or induce fluorescence in sensor-only experiments, providing further validation of the sensor’s selectivity and mechanism. Taken together, this work demonstrates that the AB-139 probe is capable of selectively detecting EGFR kinase activity in vitro using a NIR turn-on mechanism. Its design enables rapid signal response, high contrast imaging, and strong target specificity.
Recommended Citation
Jadidi, Haniyeh, "Development and In Vitro Evaluation of Novel Near-IR Fluorescent Sensors for EGFR Kinase" (2025). Graduate Research Theses & Dissertations. 8119.
https://huskiecommons.lib.niu.edu/allgraduate-thesesdissertations/8119
Extent
78 pages
Language
en
Publisher
Northern Illinois University
Rights Statement
In Copyright
Rights Statement 2
NIU theses are protected by copyright. They may be viewed from Huskie Commons for any purpose, but reproduction or distribution in any format is prohibited without the written permission of the authors.
Media Type
Text
