Document Type

Thesis - University Access Only

Award Date

2010

Degree Name

Doctor of Philosophy (PhD)

Department / School

Chemistry and Biochemistry

First Advisor

Brian A. Logue

Abstract

In this project, novel methods for the detection of biological markers for cyanide, as well as anthrax, were developed. Although the methods for the detection of these two warfare agents differ, both methods provide significant benefits to current technology. First, a new method for the determination of chronic cyanide exposure was developed, and its applicability was verified with the analysis of biological samples obtained from human smokers and non-smokers. Second, the development of a novel nanoelectrode sensor for DNA fragment detection is described. Exposure to cyanide can occur in a variety of ways, including exposure to smoke from cigarettes or fires,. accidental exposure during industrial processes, and exposure from the use of cyanide as a poison or chemical warfare agent. Confirmation of cyanide exposure is difficult because, in vivo, cyanide quickly breaks down by a number of pathways, including the formation both free and protein-bound thiocyanate. A simple method was developed to confirm cyanide exposure by extraction of protein-bound thiocyanate moieties from cyanide-exposed plasma proteins and subsequent derivatization with pentafluorobenzyl bromide for GC-MS analysis. Thiocyanate levels as low as 2.5 ng mL-1 and cyanide exposure levels as low as 175 μg kg- 1 were detected. This method was then used to analyze plasma from 25 smokers and 25 non-smokers for protein-bound thiocyanate. The amount of protein-bound thiocyanate found in the plasma of smokers, 1.35 ± 0.51 μM, was significantly elevated (p < 0.0001) when compared to non-smokers, 0.65 ± 0.34 μM. These results indicate the effectiveness of analyzing protein-bound thiocyanate in determining instances of chronic cyanide exposure with possible extension to confirmation of acute cyanide exposure. V Biological agents not only pose a danger to society, but can also be difficult to detect in a timely manner, especially during the early stages of infection. One method to successfully determine biological warfare exposure is through the detection of a DNA sequence unique to the pathogen of interest. The objective of this project was to fabricate a novel nanoelectrode device capable of detecting a specific pathogen by DNA sequence. This device utilizes conformational changes of a probe DNA sequence upon hybridization when pathogen DNA is present. The change in DNA conformation causes a change in signal, which allows detection of a specific DNA sequence. The fabrication of the final nanoelectrode was completed using standard lithography and thin film deposition techniques. A ferrocene tag was attached to the probe ssDNA to increase its electrochemical signal and the attachment was confirmed by voltammetry. Results from the device indicate that it is comparable to nanoelectrodes previously reported in literature, with a target DNA detection limit of 10 nM.

Library of Congress Subject Headings

Biochemical markers
Biological weapons
Chemical weapons
Cyanides
Anthrax
Nanotechnology
Biosensors

Publisher

South Dakota State University

Download

Share

COinS
 

Rights Statement

In Copyright