Triacetone triperoxide has been synthesised for a range of experimental studies towards the reactivity, stability and gas phase metal complexation of the explosive. Reaction with transition metals was performed and a series of molten TATP experiments conducted at 120 °C. The effect of a molten state on the degradation, conformation and reactivity of TATP has been studied with the formation of diacetone diperoxide, acetone and acetic acid degradation products observed. The complexation of TATP with a range of metals has been studied by electrospray ionisation-mass spectrometry, yielding [M+Cat]+ ions for all of the alkali metals. The first observations of a [TATP+Ag]+ adduct have been made. The formation of [2TATP+Li+LiX]+ (X = Br, Cl) and [2TATP+Ag]+ sandwich complexes was also observed. Collision cross sections (CCS) for the lithium-containing complexes of TATP were measured by travelling wave ion mobility-mass spectrometry, comparing well to computationally determined structures.

Detection canines represent the fastest and most versatile means of illicit material detection. This research endeavor in its most simplistic form is the improvement of detection canines through training, training aids, and calibration. This study focuses on developing a universal calibration compound for which all detection canines, regardless of detection substance, can be tested daily to ensure that they are working with acceptable parameters. Surrogate continuation aids (SCAs) were developed for peroxide based explosives along with the validation of the SCAs already developed within the International Forensic Research Institute (IFRI) prototype surrogate explosives kit. Storage parameters of the SCAs were evaluated to give recommendations to the detection canine community on the best possible training aid storage solution that minimizes the likelihood of contamination. Two commonly used and accepted detection canine imprinting methods were also evaluated for the speed in which the canine is trained and their reliability. As a result of the completion of this study, SCAs have been developed for explosive detection canine use covering: peroxide based explosives, TNT based explosives, nitroglycerin based explosives, tagged explosives, plasticized explosives, and smokeless powders. Through the use of these surrogate continuation aids a more uniform and reliable system of training can be implemented in the field than is currently used today. By examining the storage parameters of the SCAs, an ideal storage system has been developed using three levels of containment for the reduction of possible contamination. The developed calibration compound will ease the growing concerns over the legality and reliability of detection canine use by detailing the daily working parameters of the canine, allowing for Daubert rules of evidence admissibility to be applied. Through canine field testing, it has been shown that the IFRI SCAs outperform other commercially available training aids on the market. Additionally, of the imprinting methods tested, no difference was found in the speed in which the canines are trained or their reliability to detect illicit materials. Therefore, if the recommendations discovered in this study are followed, the detection canine community will greatly benefit through the use of scientifically validated training techniques and training aids.

Triacetone Triperoxide (TATP) is a primary high explosive that can be synthesized using commercially available starting materials and has grown in use among terrorists over the past several years. Additives present in the precursors were investigated to see if they carry through the TATP synthesis and can be detected in the final product potentially aiding in the identification of the source. Additives identified in the acetones were also identified in pre-blast and in some post-blast samples. However, these additives are present in trace quantities relative to the TATP, which coupled with the volatility and short lifetimes of some of the additives in TATP samples limit their detection in pre-blast and post-blast material. TATP prepared with different acids in the laboratory could generally be discriminated by observing the change in composition of the headspace of the samples upon heating and by IMS analysis of the crystals. The analysis of TATP synthesized on a larger scale was compared to the laboratory results of pre-blast material and post-blast debris. As in the laboratory samples, organic additives were also detected in the large-scale pre-blast samples and the identification of the additives in post-blast debris was consistent with the results obtained in the laboratory detonations.

AN ANALYSIS OF GLOBAL GENE EXPRESSION RESULTING FROM EXPOSURE TO ENERGETIC MATERIALS A Dissertation Presented for the Doctor of Philosophy Degree University of Tennessee, Knoxville VERNON LASHAWN MCINTOSH JR. August 2010 Dedication This dissertation is dedicated to my family. My mother and father Debra and Vernon McIntosh instilled in me the respect for academic excellence and the drive maximize my potential. Early on, my younger brother Kyle started showing signs of a shared interest in biology thus my desire to be a positive role model for him kept me motivated. Last but certainly not least, my loving wife and best friend Nichole has been there to offer love and support throughout my entire undergraduate and graduate degrees. It’s difficult to imagine making it this far without her (and that’s not just because she paid the bills). Abstract Characteristic transcriptional biomarkers have been identified for microbial cultures exposed to 2, 4, 6-trinitrotoluene (TNT), 2, 6-dinitrotoluene (DNT), or triacetone-triperoxide (TATP). This study describes the generation of expression profiles for exposure to each compound, the functional significance of each response, and the identification of the characteristic alterations in gene expression associated with exposure to each compound. Expression profiles were generated from a total of three different candidate organisms: Escherichia coli, Saccharomyces cerevisiae, and Pseudomonas putida. Common to all three organisms, TNT exposure resulted in increased expression of genes involved in toxin resistance and drug efflux systems. The S.cerevisiae and E.coli expression profiles were both characterized by increased expression of genes involved in iron-sulfur cluster assembly, sulfur containing amino acids, sulfate transport and assimilation and the metabolism of nitrogen compounds. Only E.coli and Saccharomyces were used to generate DNT induced expression profiles; both profiles exhibited high degrees of similarity with each organism’s respective TNT profiles. This was especially true of the E.coli profile where 25 of the 30 alterations were also observed after exposure to TNT. A computational discriminant functional analysis was performed to identify characteristic biomarkers for each exposure. For each compound a set of transcriptional biomarkers (10 or less) was developed. An additional set of biomarkers was developed encompassing both TNT and DNT exposure. These sets of genes serve as a transcriptional fingerprint for exposure to each respective compound. The sensitivity and specificity of each transcriptional fingerprint is sufficient to correctly identify exposure to energetic materials against a background of non-energetic compound exposures. This study makes several novel contributions to the greater body of scientific knowledge: • This is the first documented study of the interactions of TATP in any biological system. • This is the first comprehensive gene expression study of the TNT response by P. putida, E.coli or E.coli. • This is the first application of computational class prediction in the development of biomarkers for exposure to energetic materials

Oligoperoxides, H[OO(CH3)2C]nOOH, are formed as side products in the synthesis of the primary high explosive triacetone triperoxide (TATP). Previous tandem mass spectrometry (MSn ) experiments using a quadrupole ion trap reported that the openchained oligoperoxide adducts of ammonium or sodium resulted in different product ions in the mass spectra. Dissociation mechanisms were previously proposed based on MSn experiments, where n > 2; however, a dissociation pathway achieved by an MSn experiment, where n > 2, may not necessarily be the same pathway achieved in an MS2 experiment. For this dissertation research, the collision induced dissociation pathways were investigated for the open-chained oligoperoxides in MS2 experiments utilizing a quadrupole ion trap. Tandem mass spectral experiments were performed for the openchained oligoperoxides ranging from the trimer to the octamer (n=3-8), including both ammonium and sodium adducts. The dissociation pathways were proposed as a result of two-dimensional correlation spectroscopy applied to the mass spectral data, which was referred to as two-dimensional correlation mass spectrometry (2D-CMS). The 2DCMS method was first validated by analysis of simple and more complex kinetic models followed by simple and more complex molecules. To further aid in the elucidation of the dissociation mechanisms, computational chemistry was performed for the optimization of stable precursor and product ion structures and calculations of their relative energies and adduct dissociation energies.

Die vorliegende Arbeit beschreibt die Herstellung und Charakterisierung der ersten Antikörper gegen Triacetontriperoxid (TATP), einem hoch empfindlichen und unkonventionellen (nicht-kommerziellen) Initialsprengstoff. Entscheidend dafür war die Synthese eines TATP-imitierenden Haptens, welches die typische nonagonale Struktur des TATP mit seinen drei Peroxid- und sechs Methylgruppen nahezu perfekt nachbildet, aber den Vorzug einer zusätzlichen Carboxygruppe zur kovalenten Kopplung an Proteine aufweist. Dadurch konnte das TATP-Hapten an Rinderserumalbumin (BSA) gebunden werden, um ein immunogenes Konjugat zu erzeugen, welches die erfolgreiche Immunisierung zweier Säugetierarten, Maus und Kaninchen, ermöglichte. Der Verlauf der In vivo-Immunisierungen wurde durch die Analyse der Tierseren in regelmäßigen Abständen mittels enzymgekoppeltem Immunoassay (ELISA) verfolgt. Die polyklonalen Antikörper beider Spezies waren ungewöhnlich selektiv gegenüber TATP. Jedoch unterschied sich die Affinität der Antikörper der zwei Spezies um das 5000-fache, wobei die Kaninchenseren den Mausseren überlegen waren. Entsprechend war auch die mit Kaninchenserum erreichbare TATP-Nachweisgrenze von 0.01 µg/L deutlich besser im Vergleich zu 50 µg/L, die mit Mausserum erzielt wurden. Der Messbereich des TATP-ELISA mit Kaninchenserum deckte zudem mehr als vier Zehnerpotenzen ab, wie mittels Präzisionsprofil bestimmt wurde. Die erhaltenen TATP-Antikörper aus Kaninchen stehen damit Anwendungen in Nachweissystemen für die sehr empfindliche Detektion von TATP zur Verfügung, die u. a. in sicherheitsrelevanten Bereichen zum Einsatz kommen könnten. Als erste Anwendung wurde ein TATP-ELISA realisiert, der im Rahmen dieser Arbeit ausführlich optimiert wurde. Außerdem wurden erste Schritte zur Entwicklung eines TATP-Schnelltests (LFA) unternommen. Weitere Biosensoren auf Grundlage der neu entwickelten TATP-Antikörper sind denkbar. / The present work decribes the production and characterization of the first antibodies against triacetone triperoxide (TATP), a highly sensitive and improvised (non-commercial) primary explosive. Crucial to this work was the synthesis of a TATP-related hapten that mimics almost perfectly the typical nonagonal structure of TATP with its three peroxide and six methyl groups. Advantageously, it has an additional carboxylic acid group, which provides a conjugation site for covalent attachment to proteins. Thus, the TATP hapten could be linked to bovine serum albumin (BSA) to produce an immunogenic conjugate, allowing the successful immunization of two different mammalian species, mouse and rabbit. The in vivo immunization progress was followed by periodically analyzing the animals’ sera using enzyme-linked immunosorbent assay (ELISA). The polyclonal antibodies of both species were remarkably selective to TATP. The affinity of these TATP-antibodies was, however, different between the two species, with the rabbit sera showing an affinity about 5000-fold superior than the murine one. Consequently, the TATP detection limit of 0.01 µg/L was considerably better using the sera from rabbit in contrast to 50 µg/L when mouse serum was used. The working range of the TATP-ELISA with rabbit sera covers more than four decades, calculated from a precision profile. The obtained TATP antibodies from rabbit are now available for applications in highly sensitive detection systems for TATP, which could be employed, among others, in security-relevant areas. The first application was the realization of a TATP-ELISA, which was extensively optimized within the course of this work. Furthermore, the first steps towards the development of a lateral flow assay (LFA) targeting TATP were taken, making conceivable further biosensor platforms based on the newly developed TATP antibodies.

Die Verbindung von hoch entwickelten Nanomaterialien mit fluoreszenzbasierten Technologien hat sich zu einem aufstrebenden Forschungsbereich entwickelt. Nichtsdestotrotz ist bis heute der Schritt von einem organischen Indikatormolekül zum anwendbaren Sensorsystem ein komplexer Prozess. Diese Arbeit zielte darauf ab, sensorische Materialien verschiedener chemischer Natur für diverse Analyten zu entwickeln, zu charakterisieren und zu etablieren. Hierbei wurden zunächst pH sensitive Fluoreszenzfarbstoffe entwickelt und in dünnen Membranen immobilisiert. Der Teststreifen ermöglicht die Beurteilung von pH-Änderungen mit dem Auge. Darüber hinaus wurde gezeigt, wie diese Farbstoffe auch in eine wasserlösliche Form überführt werden können. Damit konnten lokale pH-Änderungen an der Wachstumsfront von Silikat-Biomorphs detektiert werden. Auch partikuläre Systeme stellten sich als geeignete Materialien heraus. Es konnte gezeigt werden, wie die Silikat-Matrix von Partikeln zu verbesserten Eigenschaften für Farbstoffe führt. Mittels farbstoffbeladener Partikel konnte in einem Lateral-Flow-Assay ein schneller Nachweis von TATP etabliert werden. Ein anderer Ansatz verfolgte das Ziel des sensitiven Nachweises von Quecksilberionen in Wasser. In einem anderen System konnten Silikat-Nanopartikeln so funktionalisiert werden, dass ein sensitiver und selektiver Nachweis von Schwermetallionen und Anionen über ein Quencher-Displacement-Assay gelang. Zusätzlich wurde die einzigartige Oberfläche von Zellulosepartikeln mithilfe eines neu entwickelten Fluoreszenzfarbstoffs untersucht. Die untersuchten Materialien und Strategien zeigen, wie leicht innovative Moleküle für potentielle sensorische Systeme im wässrigen Medium auf Basis von fluoreszierenden Partikeln und dünnen Schichten geschaffen werden können. Das Verhalten der hergestellten Materialien wurde über spektroskopische Methoden evaluiert und dabei, wenn möglich, die Parameter Sensitivität, Selektivität und Ansprechzeit beurteilt. / The combination of fluorescence and nanomaterials has developed into an emerging research area. Nonetheless until now the step from an organic sensory molecule to a final sensor format is a complex endeavor. This thesis aimed at the preparation of particulate and thin-film based platforms for various analytes through combining the features of an appropriate host material with outstanding properties of dyes concomitant with sensitive fluorescence detection techniques. In particular, pH sensitive fluorescent probes were sterically immobilized into a thin membrane. The dip-stick allows the assessment upon change in pH with the eye. Especially a probe working at high basic pH range was converted into a water-soluble analogue and was directly applied at the growth front of silica biomorphs to detect local pH changes. But also particulate structures are suitable host materials. It is shown how the silica matrix of nanoparticles lead to improved optical properties for embedded dyes. The interactions of silica and fluorescent dyes within the pores of mesoporous particles were exploited to develop an actual sensor format based detection of TATP. In another approach it was possible to detect mercury ions in water. Heavy metal ions were also successfully detected in a quencher displacement assay involving receptor-dye functionalized silica nanoparticles. The impact of the unique surface properties of cellulose microparticles was shown by a fluorescent dye which allows an assessment of the surface functional groups and microenvironment through the reactivity and its changes in the optical properties. The performance of the prepared materials were evaluated mostly by spectroscopic methods and if possible assessed in terms of sensitivity, selectivity and response time. The newly developed and investigated materials based on fluorescent particulate and thin-films show the facile application of innovative sensor probes for potentially sensing devices.