Electrochemical Determination of Extracellular Phosphorylation in Neuronal and Neuroblastoma Cells

This thesis describes the investigation, imaging and quantification of the extracellular membrane phosphorylation profiles of human neuron and neuroblastoma cells through the use of surface-based electrochemical sensors. This investigation started by demonstrating electrochemistry as a simple and effective technique to determine the phosphorylation state of neuronal proteins. The nAChR α7 subunit, an abundant and essential surface receptor protein, was used as an example protein and three important ectokinases, PKA, PKC and Src, were utilized to phosphorylate the protein. To confirm these results, gel electrophoresis and XPS studies were performed. Once establishing electrochemistry as a useful methodology, the next phase involves phosphorylating whole fixed neuronal cells and monitoring the phosphorylation profile of each of the three ectokinases described above. The electrochemical techniques were supplemented with fluorescence microscopy and quartz crystal microscopy (QCM). Fluorescence microscopy indicated that each of the kinase phosphorylation reactions had completed within 8 hours and QCM established that the number of phosphorylation sites for neuronal cells by PKA, PKC and Src were 5.7 x106 sites/cell, 5.3 x106 sites/cell, 4.8 x105 sites/cell, respectively. For neuroblastoma cells, the number of sites were determined be 1.1 x106 sites/cell, 1.1 x106 sites/cell and 7.4 x105 sites/cell. It is established that neuroblastoma cells differ significantly from neuronal cells in their extracellular phosphorylation states. Lastly, using the phosphorylation profiles, the percent composition of neuronal and neuroblastoma cells mixtures can be determined. Findings obtained from this thesis establish that mammalian neuronal cells (healthy) and neuroblastoma cells (cancerous/disease state) have different extracellular phosphorylation patterns. Furthermore, it is emphasized that electrochemical techniques provide insights for the design of new histological or diagnostic technology.

Department dc:contributor.department

Chemistry

Year dc:date.issued

2021

Author dc:creator

• Ahmad, Syed Ahmad Ghazali

Advisor dc:contributor.advisor

• Kraatz, Heinz-Bernhard

• Bio-electroanalytical sensors
• Biosensor
• Electrochemistry
• Neuroblastoma
• Neuron
• Phosphorylation

Handle dc:identifier.uri

http://hdl.handle.net/1807/108838

OAI identifier oai:identifier

oai:utoronto.scholaris.ca:1807/108838