As the move towards the miniaturization of many diagnostic and detection systems continues, the need for increasingly versatile yet sensitive labels for use in these systems also grows. Luminescent reporters provide us with a solution to many of the issues at hand through their unique and favorable characteristics. Bioluminescent proteins offer detection at extremely low concentrations and no interference from physiological fluids leading to excellent detection limits, while the vast number of fluorescent proteins and molecules available allows the opportunity to select a tailored reporter for a specific task. Both provide relatively simply instrumentation requirements and have exhibited great promise with many of the miniaturized systems such as lab-on-a-chip and lab-on-a-CD designs. Herein, we describe the novel employment of luminescent reporters for four distinct purposes. First off, by combining both time and wavelength resolution we have expanded the multiplexing capabilities of the photoprotein aequorin beyond duel-analytes, demonstrating the ability to simultaneously detect three separate analytes. Three semi-synthetic aequorin proteins were genetically conjugated to three pro-inflammatory cytokines (interleukins 1, 6, and 8) resulting in aequorin labeled cytokines with differing emission maxima and half lives to allow for the simultaneous detection of all three in a single solution through the elevated physiological concentration range. Secondly a semi-synthetic aequorin variant has been genetically enhanced to serve as an immunolabel and exhibited the ability to sensitively detect the acute myeloid leukemia marker, CD33, down to the attomole level in addition to improving aequorin imaging capabilities. In the third example, the aequorin complex was rationally, genetically split into two parts and attached to the termini of the cAMP selective cAMP receptor protein (CRP) creating a genetically fused molecular switch. The conformational change experienced by CRP upon the binding of cAMP translates into a loss of bioluminescent signal from aequorin and has shown the ability to respond linearly to cAMP over several orders of magnitude. Lastly, through custom design, a reagentless, portable, fluorescent fiber optic detection system has been developed, capable of being integrated into the body through a heart catheter. The system was able to respond to changes in potassium concentration selectively, reproducibly and reversibly with a fast response time of one minute.
| Identifer | oai:union.ndltd.org:uky.edu/oai:uknowledge.uky.edu:gradschool_diss-1187 |
| Date | 01 January 2011 |
| Creators | Scott, Daniel F. |
| Publisher | UKnowledge |
| Source Sets | University of Kentucky |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | University of Kentucky Doctoral Dissertations |
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