Publication Date

Spring 2011

Document Type

Thesis

Degree Name

Master of Science

Department

Analytical Chemistry

First Advisor

Patty Fu-Giles, Ph.D.

Second Advisor

Phyllis Klingensmith, Ph.D.

Third Advisor

Karen D'Arcy, Ph.D.

Abstract

Non-toxic biosensors are encountering an increase in attention for use in understanding the fate of cells and as a diagnostic tool. Development and incorporation of suitable fluorophores into biological molecules is the key for monitoring proteins in vivo research. This study investigated the enhanced emission of Eu (III) and Tb (III) upon binding to the four DNA bases and their respective nucleotides, found the best ratio for effective energy transfer, and developing nanoparticles to deliver the biosensor into the cells.

It is well known that Eu (III) and Tb (III) exhibit very distinctive photo-characteristics. The luminescence of these two lanthanides is weak due to low absorption cross sections. Conversely, the emission of both trivalent ions, upon irradiation, in aqueous solution, is strong when bound to complex ligand systems. The luminescent enhancement is the result of energy transfer (EnT) and the binding with single-stranded DNA, making these ions perfect candidates for luminescent probes (1). The emission lanthanides theory by G.A. Crosby establishes that the intramolecular energy transfer in a lanthanide complex is when the lowest triplet state energy level of the complex equals or lies above the resonance level of the lanthanide (2)

To overcome the inherently low absorption of lanthanide ions, researchers have developed sensitizing fluorophores that upon excitation, transfer energy to the lanthanide (3) (4). One problem with luminescence in an aqueous solution is that another pathway is available for deactivation of the excited state of the lanthanide, in the form of vibrational energy transfer to water molecules (1). Early research shows that quenching of luminescence is minimized by using ligands which tended to encapsulate the lanthanide ion (1). Longer emission lifetimes and greater quantum yield intensities can be accomplished by either chelation by ligands (5)or encapsulation of the lanthanides. We ascertained the maximum enhancement for the lanthanide ions occurred through the interaction with the base guanine or its nucleotide guanosine 5’-monophosphate disodium salt.

The research initially pursued the encapsulation of the lanthanide ions by single-strand oligonucleotides as a biosensor. However, an alternative delivery method based on inverse micelles and liposomes was developed and it proved to be economical and simple to encapsulate and deliver the biosensor into the cells. The creation of a double emulsion, or water-oil-water system, and the encapsulation (using palmitic acid as surfactant) of the water soluble biosensors were successful. This thesis determined the particle size achieved of 75nm, for both lanthanides had fallen into the nanoemulsions range. Their small size permits the nanoparticles to be injected intravenously(6).

The in vitro toxicity of the nanoparticles, with both luminescence biosensors, was assessed by BCA assay. Results supported both luminescence nanoparticles biosensors were non toxic to human cells. Therefore, these NP’s have a potential to provide a unique detection signature as a contrast agent suitable for medical applications (7).

It has been published that nanoparticles (NPs) can rapidly be transported to the liver (about 90%), then kidneys and other organs (8). After a period of time, the NPs are expelled from the human body through feces and urine, unless the size of the NPs is larger than 200 nm, in which case the NPs are retained / trapped by the liver. The particle size obtained in this research, 75nm, is a good indication that the biosensor will have a safe disposal from the body.

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