Double Pulse Coulometry for Interfacial Biosensing at Platinum Microelectrodes

This dissertation explores the use of a chronocoulometric technique to probe cholesterol efflux from the plasma membrane of cells and tissues. More specifically, a double potential pulse coulometric scheme for cholesterol sensing at platinum microelectrodes was developed and optimized to avoid amperometric measurements needed to reference to a nonzero solution baseline current and baseline shift upon repositioning the electrode in approaching and contacting with the cell plasma membrane. In this double potential pulse scheme, the first pulse (pulse 1) oxidizes hydrogen peroxide at the electrode surface and the second pulse (pulse 2) gauges the background charge at that time. The difference charge (i.e.; pulse 1 - pulse 2) signal arises from gradual accumulation of hydrogen peroxide generated at the interface between the electrode surface and cell plasma membrane. A bare electrode in contact with the plasma membrane serves as the control electrode in these experiments to remove the Faradaic background signal from enzyme-modified electrodes to provide an estimate of the hydrogen peroxide accumulated before pulse 1. The development of the coulometric scheme allows monitoring of background drift in real-time and signal averaging with electrode held in contact with the cell plasma membrane. Chapter 1 describes a general overview of the scope of this research and briefly explains previous work performed by this research group that demonstrates the use of different biosensing strategies to measure cholesterol efflux from the plasma membrane. Chapter 2 describes optimization and characterization studies of the double potential pulse waveform. Previous work with cavity enzyme-modified electrodes led to the optimization of the coulometric method with disk microelectrodes. Importantly, the characterization studies presented in this chapter will show that Faradaic background signal is influenced by platinum electrode surface modification, temperature, contact of cell plasma membrane, and solution chemistries. Chapter 3 describes the analog chronocoulometric detection of plasma membrane cholesterol of excised mouse trachea and frog muscle tissue. Chapter 4 describes the analog chronocoulometric detection of cystic fibrosis (CF) human epithelial cells. Lastly, Chapter 5 outlines future directions with cholesterol cycling from neuron stimulation.

Name thesis:degree_name

Doctor of Philosophy

Level thesis:degree_level

doctoral

Discipline thesis:degree_discipline

Chemistry

Grantor dc:publisher

Case Western Reserve University School of Graduate Studies

Year dc:date

2013

Author dc:creator

• West, Richard H.

Contributors dc:contributor

• Burgess, James
• Dunbar, Robert

• Physical Chemistry
• Cholesterol
• Coulometry
• Microelectrodes

Statement dc:rights

• unrestricted
• This thesis or dissertation is protected by copyright: all rights reserved. It may not be copied or redistributed beyond the terms of applicable copyright laws.

Language dc:language

English

Repository record dc:identifier

http://rave.ohiolink.edu/etdc/view?acc_num=case1344251678

OAI identifier oai:identifier

oai:etd.ohiolink.edu:case1344251678