Date of Graduation

12-2012

Document Type

Dissertation

Degree Name

Doctor of Philosophy in Engineering (PhD)

Degree Level

Graduate

Department

Chemical Engineering

Advisor/Mentor

Hestekin, Christa N.

Committee Member

Beitle, Robert R. Jr.

Second Committee Member

Roper, D. Keith

Third Committee Member

Servoss, Shannon L.

Fourth Committee Member

Koeppe, Roger E. II

Keywords

Pure sciences; Applied sciences; Amyloid beta; Amyloid protiens; Capillary electrophoresis; Disease monitoring; Insulin; Protein aggregation

Abstract

The detection of oligomers and aggregates formed by two amyloid proteins, insulin and amyloid-beta (AB), is of particular importance due to the role which these species play in Diabetes and Alzheimer's disease, respectively. However, existing techniques are limited in the ability to detect insulin and AB; oligomers due to the fact that these early aggregates are transient, present at low concentrations, and difficult to isolate. Improvements must be made to existing techniques or alternative techniques must be explored in order to identify and quantify the size of these oligomeric and aggregate species without disrupting their structure.

Capillary and microchip electrophoresis (CE and ME) are two promising electrophoretic methods for amyloid oligomer and aggregate detection. The present work demonstrated the potential for CE and ME to detect native aggregation of insulin and AB proteins, in particular the formation of oligomers and aggregates. Furthermore, the effect of hydrodynamic size on separation was increased through the use of a highly entangled polymer matrix introduced into the capillary, thus offering the potential to resolve populations of amyloid species. Using this novel separation, we investigated variables such as sample salt concentration, sample preparation method, and fluorescent dye structure. We demonstrated the ability of CE with ultraviolet detection (UV-CE) to detect native insulin aggregates estimated to range in size from 30 - 100 kDa and native AB1-40 aggregates estimated to range in size from 10 - 30 kDa, 10 - 300 kDa and > 300 kDa. These studies demonstrated the successful detection of physiological concentrations (pM) of monomeric fluorescein isothiocyanate labeled insulin (FITC-insulin) and carboxy- fluorescein labeled AB1-42 (FAM-AB1-42) using CE with laser induced fluorescence detection (LIF-CE). However, the detection of oligomeric and aggregate species was altered from unlabeled samples, indicating the need for future work in the study of fluorescent dye attachment.

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