Die vorliegende Arbeit behandelt die Oberflächenmodifikation von Nanodiamanten (ND) aus der Detonationssynthese und die anschließende Konjugation von einzel- bzw. doppelsträngiger DNA an die zuvor eingeführten funktionellen Gruppen. Als Ausgangsmaterialien wurden zwei Nanodiamantpulver mit unbekannter Oberflächenbelegung eingesetzt, deren Charakterisierung durch elektronenmikroskopische Methoden erfolgte. Weiterhin wurden kommerziell modifizierte ND mit definierter Oberflächenbelegung (Amino- und Hydroxylgruppen) verwendet. Für potenzielle Anwendungen von ND wird eine monofunktionale Oberfläche angestrebt, die u. a. über Oxidation oder Reduktion der durch den Herstellungsprozess eingeführten primären funktionellen Gruppen realisiert werden kann. Die dadurch erzeugten sekundären Funktionen ermöglichen die kovalente bzw. nichtkovalente Anbindung weiterer Substanzen, z. B. von Biomolekülen, an die Oberflächen der ND-Partikel. Die hier beschriebene Konjugation von DNA, an die mit Carboxyl-, Hydroxyl- oder Aminogruppen modifizierten Partikeloberflächen, erfolgte durch die Generierung von Amid-, Phosphodiester- und Isoharnstoffbindungen. Der Erfolg der Konjugationen wurde mit Hilfe von Infrarotspektroskopie und Fluoreszenzmikroskopie untersucht. Die Fluoreszenz der Konjugate beruhte dabei auf Fluoreszenzfarbstoffen, die an die DNA-Moleküle gebunden waren. Darüber hinaus wird die Herstellung einer kolloidalen ND-Suspension beschrieben, von der die Partikelgrößen und das Zeta-Potenzial bestimmt wurden. Kolloidale Suspensionen ermöglichen aufgrund der geringen Partikelgrößen diverse biologische und medizinische Anwendungen von ND. Mit den hier präsentierten Ergebnissen erweitert sich der Kenntnisstand zur Konjugation von DNA an ND aus der Detonationssynthese. Die angewandte Methodik kann ebenso auf andere Substanzen wie Proteine oder Chemotherapeutika übertragen werden. Derart funktionalisierte Partikel besitzen ein großes Potenzial für die weitere Anwendung in der Biomedizin und Nanotechnologie.:1 Einleitung 1 2 Theoretische Grundlagen 6 2.1 Nanodiamant 7 2.1.1 Historische Betrachtungen zur Detonationssynthese 7 2.1.2 Herstellung von Diamant 8 2.1.3 Aufbereitung von Nanodiamanten aus der Detonationssynthese 11 2.1.4 Struktur und Eigenschaften von Diamant 12 2.1.5 Homogenisierung der Oberflächenbelegung 16 2.1.6 Aggregation und Deaggregation von Nanodiamant-Partikeln 20 2.1.7 Anwendungen von Nanodiamant-Partikeln 21 2.2 Aptamere 26 2.2.1 Strukturbildung und Bindungsmechanismen 26 2.2.2 Zielsubstanzen 28 2.2.3 Vergleich von Aptameren und Antikörpern 29 2.2.4 Herstellung von Aptameren – Der SELEX-Prozess 32 2.2.5 Anwendungsfelder für Aptamere 34 2.3 Konjugation von Nanopartikeln mit Biomolekülen 38 2.4 Herstellung und Charakterisierung von kolloidalen Nanodiamantsuspensionen 46 2.4.1 Herstellung kolloidaler Nanodiamantsuspensionen 46 2.4.2 Bestimmung der Partikelgröße und Partikelgrößenverteilung durch dynamische Lichtstreuung (DLS) 47 2.4.3 Bestimmung des Zeta-Potenzials durch elektrophoretische Licht-streuung (ELS) 48 2.5 Methoden zur Materialcharakterisierung von Nanodiamantpulver 52 2.5.1 Rasterelektronenmikroskopie (REM) 52 2.5.2 Energiedispersive Röntgenspektroskopie (EDX) 53 2.5.3 Transmissionselektronenmikroskopie (TEM) 54 2.6 Nachweismethoden für Modifikation und Konjugatbildung 56 2.6.1 Fourier-Transform-Infrarot- (FT-IR-) Spektroskopie 56 2.6.2 Fluoreszenzmikroskopie 60 3 Material und Methoden 62 3.1 Herstellung und Charakterisierung von kolloidalen Nanodiamantsuspensionen 62 3.1.1 Herstellung kolloidaler Nanodiamantsuspensionen 62 3.1.2 Bestimmung von Partikelgröße, Partikelgrößenverteilung und Zeta-Potenzial 63 3.2 Materialcharakterisierung von Nanodiamantpulver 64 3.2.1 Rasterelektronenmikroskopie (REM) 64 3.2.2 Energiedispersive Röntgenspektroskopie (EDX) 65 3.2.3 Hochauflösende Transmissionselektronenmikroskopie (HRTEM) 65 3.3 Chemische Modifikation von Nanodiamanten 66 3.3.1 Verwendete Materialien und Geräte 67 3.3.2 Einführung von Carboxylgruppen 68 3.3.3 Einführung von Hydroxylgruppen 69 3.3.4 Einführung von Aminogruppen 70 3.4 Herstellung von Nanodiamant-Aptamer-Konjugaten 73 3.4.1 Verwendete Materialien und Geräte 73 3.4.2 Konjugation über Amidbindungen 77 3.4.3 Konjugation über Ester- und Phosphodiesterbindungen 81 3.4.4 Konjugation über Isoharnstoffbindungen 85 3.5 Nachweismethoden für Modifikation und Konjugatbildung 88 3.5.1 Fourier-Transform-Infrarot- (FT-IR-) Spektroskopie 88 3.5.2 Fluoreszenzmikroskopie 89 4 Ergebnisse und Diskussion 92 4.1 Charakterisierung kolloidaler Nanodiamantsuspensionen 92 4.1.1 Bestimmung der Partikelgröße und Partikelgrößenverteilung 92 4.1.2 Bestimmung des Zeta-Potenzials 93 4.2 Materialcharakterisierung von Nanodiamantpulvern 98 4.2.1 Rasterelektronenmikroskopie (REM) 98 4.2.2 Energiedispersive Röntgenspektroskopie (EDX) 101 4.2.3 Hochauflösende Transmissionselektronenmikroskopie (HRTEM) 107 4.3 Fourier-Transform-Infrarot- (FT-IR-) Spektroskopie 117 4.3.1 Nanodiamanten: Originalmaterial und modifizierte Nanodiamanten 118 4.3.1.1 Nanodiamanten – Originalmaterial 118 4.3.1.2 Modifikation mit Carboxylgruppen (ND-COOH) 122 4.3.1.3 Modifikation mit Hydroxylgruppen (ND-OH) 123 4.3.1.4 Modifikation mit Aminogruppen (ND-NH2) 128 4.3.2 Nanodiamant-DNA-Konjugate 138 4.3.2.1 Konjugation über Amidbindungen 140 4.3.2.2 Konjugation über Phosphodiesterbindungen 144 4.3.2.3 Konjugation über Isoharnstoffbindungen 150 4.4 Fluoreszenzmikroskopie an Nanodiamant-DNA-Konjugaten 154 4.4.1 Konjugation über Amidbindungen 154 4.4.2 Konjugation über Phosphodiesterbindungen 157 4.4.3 Konjugation über Isoharnstoffbindungen 161 5 Zusammenfassung und Ausblick 165 6 Literaturverzeichnis 170 Anhang I A-1 Parameter der Partikelgrößen- und Zeta-Potenzial-Messungen I A-2 Nukleotidsequenz von EF1a III A-3 GFP-Filter-Spektrum IV A-4 FT-IR-Spektren von Nanodiamanten V A-5 FT-IR-Spektren von Nanodiamant-DNA-Konjugaten X Verzeichnis der Formelzeichen XIV Abkürzungsverzeichnis XV Eigene wissenschaftliche Beiträge XVIII Danksagung Erklärung / The present study deals with the surface modification of nanodiamonds (ND) from detonation synthesis and the subsequent conjugation of both single and double stranded DNA to previously introduced functional groups. As starting materials two kinds of nanodiamond powders with unknown surface configuration were used. Both types of ND were characterized by electron-microscopic methods. Furthermore, commercially modified ND with defined surface configuration (amino and hydroxyl groups) were applied. Potential applications of ND require a mono-functional surface, that can be realized e. g. via oxidation or reduction of the primary functional groups introduced during the production process. The thereby generated secondary functions permit the covalent or non-covalent linking of further substances onto the surfaces of ND particles. Conjugation of DNA, as described here, onto the carboxyl-, hydroxyl- or aminomodified particle surfaces was accomplished by generating of amino, phosphodiester and isourea bonds. The success of conjugations has been examined by infrared spectroscopy and fluorescence microscopy. The fluorescence of conjugates based on fluorescent dyes bound to the DNA molecules. Furthermore, the fabrication of a colloidal ND suspension is described, of which the particle sizes and the Zeta potential have been determined. Colloidal suspensions facilitate various biological and medical applications of ND on the basis of low particle sizes. The presented results enlarge the state of knowledge about the conjugation of DNA on ND from detonation synthesis. The applied methodology may also be transferred to other substances like proteins or chemotherapeutics. In this way, functionalized particles have a big potential for further application in biomedicine and nanotechnology.:1 Einleitung 1 2 Theoretische Grundlagen 6 2.1 Nanodiamant 7 2.1.1 Historische Betrachtungen zur Detonationssynthese 7 2.1.2 Herstellung von Diamant 8 2.1.3 Aufbereitung von Nanodiamanten aus der Detonationssynthese 11 2.1.4 Struktur und Eigenschaften von Diamant 12 2.1.5 Homogenisierung der Oberflächenbelegung 16 2.1.6 Aggregation und Deaggregation von Nanodiamant-Partikeln 20 2.1.7 Anwendungen von Nanodiamant-Partikeln 21 2.2 Aptamere 26 2.2.1 Strukturbildung und Bindungsmechanismen 26 2.2.2 Zielsubstanzen 28 2.2.3 Vergleich von Aptameren und Antikörpern 29 2.2.4 Herstellung von Aptameren – Der SELEX-Prozess 32 2.2.5 Anwendungsfelder für Aptamere 34 2.3 Konjugation von Nanopartikeln mit Biomolekülen 38 2.4 Herstellung und Charakterisierung von kolloidalen Nanodiamantsuspensionen 46 2.4.1 Herstellung kolloidaler Nanodiamantsuspensionen 46 2.4.2 Bestimmung der Partikelgröße und Partikelgrößenverteilung durch dynamische Lichtstreuung (DLS) 47 2.4.3 Bestimmung des Zeta-Potenzials durch elektrophoretische Licht-streuung (ELS) 48 2.5 Methoden zur Materialcharakterisierung von Nanodiamantpulver 52 2.5.1 Rasterelektronenmikroskopie (REM) 52 2.5.2 Energiedispersive Röntgenspektroskopie (EDX) 53 2.5.3 Transmissionselektronenmikroskopie (TEM) 54 2.6 Nachweismethoden für Modifikation und Konjugatbildung 56 2.6.1 Fourier-Transform-Infrarot- (FT-IR-) Spektroskopie 56 2.6.2 Fluoreszenzmikroskopie 60 3 Material und Methoden 62 3.1 Herstellung und Charakterisierung von kolloidalen Nanodiamantsuspensionen 62 3.1.1 Herstellung kolloidaler Nanodiamantsuspensionen 62 3.1.2 Bestimmung von Partikelgröße, Partikelgrößenverteilung und Zeta-Potenzial 63 3.2 Materialcharakterisierung von Nanodiamantpulver 64 3.2.1 Rasterelektronenmikroskopie (REM) 64 3.2.2 Energiedispersive Röntgenspektroskopie (EDX) 65 3.2.3 Hochauflösende Transmissionselektronenmikroskopie (HRTEM) 65 3.3 Chemische Modifikation von Nanodiamanten 66 3.3.1 Verwendete Materialien und Geräte 67 3.3.2 Einführung von Carboxylgruppen 68 3.3.3 Einführung von Hydroxylgruppen 69 3.3.4 Einführung von Aminogruppen 70 3.4 Herstellung von Nanodiamant-Aptamer-Konjugaten 73 3.4.1 Verwendete Materialien und Geräte 73 3.4.2 Konjugation über Amidbindungen 77 3.4.3 Konjugation über Ester- und Phosphodiesterbindungen 81 3.4.4 Konjugation über Isoharnstoffbindungen 85 3.5 Nachweismethoden für Modifikation und Konjugatbildung 88 3.5.1 Fourier-Transform-Infrarot- (FT-IR-) Spektroskopie 88 3.5.2 Fluoreszenzmikroskopie 89 4 Ergebnisse und Diskussion 92 4.1 Charakterisierung kolloidaler Nanodiamantsuspensionen 92 4.1.1 Bestimmung der Partikelgröße und Partikelgrößenverteilung 92 4.1.2 Bestimmung des Zeta-Potenzials 93 4.2 Materialcharakterisierung von Nanodiamantpulvern 98 4.2.1 Rasterelektronenmikroskopie (REM) 98 4.2.2 Energiedispersive Röntgenspektroskopie (EDX) 101 4.2.3 Hochauflösende Transmissionselektronenmikroskopie (HRTEM) 107 4.3 Fourier-Transform-Infrarot- (FT-IR-) Spektroskopie 117 4.3.1 Nanodiamanten: Originalmaterial und modifizierte Nanodiamanten 118 4.3.1.1 Nanodiamanten – Originalmaterial 118 4.3.1.2 Modifikation mit Carboxylgruppen (ND-COOH) 122 4.3.1.3 Modifikation mit Hydroxylgruppen (ND-OH) 123 4.3.1.4 Modifikation mit Aminogruppen (ND-NH2) 128 4.3.2 Nanodiamant-DNA-Konjugate 138 4.3.2.1 Konjugation über Amidbindungen 140 4.3.2.2 Konjugation über Phosphodiesterbindungen 144 4.3.2.3 Konjugation über Isoharnstoffbindungen 150 4.4 Fluoreszenzmikroskopie an Nanodiamant-DNA-Konjugaten 154 4.4.1 Konjugation über Amidbindungen 154 4.4.2 Konjugation über Phosphodiesterbindungen 157 4.4.3 Konjugation über Isoharnstoffbindungen 161 5 Zusammenfassung und Ausblick 165 6 Literaturverzeichnis 170 Anhang I A-1 Parameter der Partikelgrößen- und Zeta-Potenzial-Messungen I A-2 Nukleotidsequenz von EF1a III A-3 GFP-Filter-Spektrum IV A-4 FT-IR-Spektren von Nanodiamanten V A-5 FT-IR-Spektren von Nanodiamant-DNA-Konjugaten X Verzeichnis der Formelzeichen XIV Abkürzungsverzeichnis XV Eigene wissenschaftliche Beiträge XVIII Danksagung Erklärung

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The primary cell wall is a complex multipolymer system whose composite structure has been mostly determined from chemical and biochemical studies. Although the primary cell wall serves a central role, with regard to the connective properties of fibres, knowledge about the interactions among the polymers, when it comes to the mechanical properties, is very limited. The physical properties of the polymers, i.e. their elastic and viscous deformations, as well as the ultrastructure of the polymers, i.e. the interactions among the polymers in the outer fibre wall layers that lead to this behaviour, are still not fully understood. The aim of this study was to examine how the different wood polymers, viz. lignin, protein, pectin, xyloglucan and cellulose, interact in the outer fibre wall layers of the spruce wood tracheid. The initial objective was to separate an enriched primary cell wall material from a first stage TMP, by means of screening and centri-cleaning. From this material, consisting of the primary cell wall (P) and outer secondary cell wall (S1) materials, thin sheets were prepared and analysed using a number of different analytical methods. The major measuring technique used was dynamic Fourier transform infra-red (FT-IR) spectroscopy in combination with dynamic 2D FT-IR spectroscopy. This technique is based on the detection of small changes in molecular absorption that occur when a sinusoidally stretched sample undergoes low strain. The molecular groups affected by the stretching respond in a specific way, depending on their environment, while the unaffected molecular groups provide no response to the dynamic spectra, by producing no elastic or viscous signals. Moreover, the dynamic 2D FT-IR spectroscopy provides useful information about various intermolecular and intramolecular interactions, which influence the reorientability of functional groups in a polymer material. Measurements of the primary cell wall material, using dynamic FT-IR spectroscopy, indicated that strong interactions exist among lignin, protein and pectin, as well as among cellulose, xyloglucan and pectin in this particular layer. This was in contrast to the secondary cell wall, where interactions of cellulose with glucomannan and of xylan with lignin were dominant. It was also indicated that the most abundant crystalline cellulose in the primary cell wall of spruce wood fibres is the cellulose Iβ allomorph, which was also in contrast to the secondary cell wall, where the cellulose Iα allomorph is more dominant. The presence of strong interactions among the polymers in the primary cell wall and, especially, the relatively high content of pectin and protein, showed that there is a very good possibility of selectively attacking these polymers in the primary cell wall. The first selective reaction chosen was a low degree of sulphonation, applied by an impregnation pretreatment of chips with a very low charge of sodium sulfite (Na2SO3). This selective reaction caused some structural modification of the lignin, a weakening of the interactions between lignin;pectin, lignin;protein and pectin;protein, as well as an increased softening of the sulphonated primary cell wall material, when compared to the unsulphonated primary cell wall material. All this resulted in an increased swelling ability of the material. / QC 20101123

Silicon Oxide/Oxynitride (SiO2/SiON) has been the mainstream material used for gate dielectric for MOS transistors for the past 30 years. The aggressive scaling of the feature size of MOS transistor has limited the ability of SiO2/SiON to work effectively as the gate dielectric to modulate the conduction of current of MOS transistors due to excess leakage current dominated by direct quantum tunneling. Due to this constraint, alternative gate dielectric/high-k is being employed to reduce the leakage current in order to maintain the rate of scaling of MOS transistors. However, the cost involved in the implementation of these new gate dielectric materials are high due to the requirements of a change in the process flow for device fabrication. This work presents the results of a novel processing method implementing the use of rapid thermal processing (RTP) on conventional SiO2/SiON gate dielectric to reduce the gate leakage current by three to five orders of magnitude. Electrical properties of the effect were characterized on fabricated MOS capacitors using semiconductor parameter analyzer and LCR meter. Material characterization was performed using FT-IR to understand the mechanism involved in this novel processing method, named PECE (Phonon-Energy-Coupling-Enhancement). By implementing this novel process, the use of SiO2/SiON as gate dielectric can be scaled further in conventional process flow of device fabrication.

Excipients are used in pharmaceutical formulations as fillers and drug carriers. Their successful function is inextricably linked to their physicochemical properties and, in turn, these properties are directly related to their structure. This thesis is concerned with the structural and spectroscopic characterisation of a selection of excipients by powder and single crystal X-ray diffraction, Raman and IR spectroscopy and MASNMR and an investigation of their stability as a function of temperature, humidity and particle size. As well as being a well-known excipient used in the pharmaceutical industry, lactose is also a common food additive. The diverse usage of lactose has led to a wealth of contradictory information relating to both structure and properties of this material. The first part of experimental work in this thesis identifies the four real lactose polymorphs; the naturally occurring a-lactose monohydrate; the anhydrous stable form of a-lactose; the hygroscopic unstable form of a-lactose; and the anomeric equivalent, p-lactose using powder X-ray diffraction. The work shows that anhydrous lactose formed by solvent dehydration often termed aM is simply the anhydrous stable form of a-lactose formed via a different route. Simple methods for discerning between the polymorphs using standard laboratory equipment are suggested. IlC MASNMR data were collected on all four forms of lactose for the first time and illustrate key differences between the four structures. Single crystal data were successfully collected on the a-lactose monohydrate and refinement carried at low temperature to determine the hydrogen bonded arrangement for the first time. Rietveld refmement of the hygroscopic unstable form of a-lactose using in-situ temperature resolved X-ray diffraction has shown that the hygroscopic form can be produced as a single phase. Refinement of Plactose using the Rietveld method has shown that powder diffraction data were comparable with single crystal data, with respect to structure refinement but attempts at both crystallisation and refinement of the stable anhydrous a-lactose polymorph were unsuccessful due to the complexity of the structure. Powder X-ray diffraction analysis was shown to be an effective tool in the quantification of mixed phase lactose samples with respect to both mixed phase stable anhydrous a-lactose and a-lactose monohydrate; and mixed p-Iactose and a-lactose monohydrate samples. The accuracy of the technique was determined to be at least 5%. Quantification was carried out using relative intensities of a well resolved unique reflection for each phase within the system. Dehydration techniques applied to lactose were applied to other hydrated pharmaceutical sugars; trehalose dihydrate and raffmose pentabydrate. Solid state techniques; powder X-ray diffraction, Raman and IR spectroscopy; showed that discrimination of other sugar hydrates became more complex with increasing levels of hydration.

PVDF, Poly(vinylidene fluoride), is a polymer that has been studied for over four decades due to its good electromechanical properties, stability, and durability in various environments. Currently, PVDF is the only commercially available piezoelectric polymer. PVDF is a polymorph, which indicates the presence of several crystalline phases such as α, β, γ, and δ-phase. Oriented β-phase PVDF exhibits ferroelectric properties and displays the largest piezoelectricity amongst the four phases, which makes it the most desirable phase. Preparing oriented β-phase PVDF is a multi-step process, which is cost intensive, due to the time, labor and energy utilized. The main goal of this work is to prepare oriented β-phase PVDF using the electrospraying technique in a one step process. During the electrospraying process a polymer jet is ejected. This jet disintegrates into droplets due to overwhelming surface tension, resulting in a sprayed coating on the collector substrate. Because of the combination of jet ejection and the high voltage applied between the needle tip and the substrate, the droplets can be stretched and the polymer chains can be oriented. Both the stretching and the high electric field are required for the transformation of α-phase to the oriented β-phase. This study proposes that by using the electrospraying technique it is possible to transform the α-phase to the β-phase in a one step process starting from solution. This research focuses on the processing and characterization of electrosprayed PVDF as well as electrosprayed PVDF/carbon nanotubes (PVDF/CNT) nanocomposites. The specific tasks are to determine the changes to the PVDF phases due to the electrospraying technique, and to determine the changes in the PVDF morphology due to the addition of carbon nanotubes to the polymer matrix.PVDF with two different molecular weights were electrosprayed using different solvents and parameters. Initial observations after electrospraying were that, high boiling point solvents resulted in the spraying of the solution and forming films, whereas a low boiling point volatile solvent such as acetone resulted in the spinning of the solution thus forming non-woven fiber mats. The thermal and electrical properties of the electrosprayed PVDF and PVDF/CNT composites are measured using several characterization techniques, including Modulated Differential Scanning Calorimetry (MDSC), Dielectric spectroscopy, Thermally Stimulated Current (TSC), Fourier Transform Infrared Spectroscopy (FT-IR), and X-Ray Diffraction (XRD). MDSC results show that electrosprayed PVDF has a lower melting point temperature than that of PVDF commercially available pellets. In addition, electrosprayed PVDF/CNT nanocomposites show a linear increase in the percentage of crystallinity with the increase of CNT concentration in the composite. Dielectric spectroscopy results indicate that by increasing the CNT concentration in the composite, the dielectric constant and the polymer conductivity increase.From the four characterizing techniques used, two of them, FT-IR and XRD, show that it is possible to transform α-phase to β-phase PVDF in a one-step process using electrospraying. The other two techniques, TSC and dielectric spectroscopy, show α-phase for the electrosprayed samples without CNT, and some β-phase formation with samples electrosprayed with CNT. These last two techniques; TSC and dielectric spectroscopy have results that differ from the FT-IR and XRD techniques. This contradiction may be a result of the small amounts of β-phase in the sample, which cannot be detected using these techniques. Another reason may be due to the difference in the probing levels between these techniques. XRD and FT-IR probe at the molecular level, whereas TSC and dielectric probe at a much larger scale, which may make it hard to detect small amounts of β-phase.

Several traditional methods have been used to characterise bacteria, such as biochemical, morphological and molecular tests; however, these methods are time-consuming and not always reliable. Recently, modern analytical techniques have emerged as powerful tools offering high-throughput, reliable and rapid analysis in applications, such as clinical and microbiology studies. A variety of modern analytical techniques have been employed for bacterial characterisation, including matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF-MS), liquid chromatography-mass spectrometry (LC-MS), Fourier transform infrared (FT-IR) spectroscopy and Raman spectroscopy. This thesis focused on developing a robust MALDI-TOF-MS methodology to generate mass spectra profiles for the discrimination of clinically-significant bacteria. The data generated from MALDI-TOF-MS analysis are significantly influenced by a number of experimental factors, namely instrument settings, sample preparation, the choice of matrix, matrix additives and matrix preparation as well as sample-matrix deposition methods. The need to optimise experimental variables for bacterial analysis using MALDI-TOF-MS was evident despite the increased application of this analytical tool for clinical microbiology. Experimental optimisation revealed that the choice of matrix is the most important element in MALDI-TOF-MS analysis. Based on this study, a number of different matrices were used to obtain more reproducible mass spectra to classify bacterial samples using a rapid and effective approach. Studies in this thesis indicated that sinapinic acid (SA) is the best matrix for the analysis of proteins from intact bacteria, while 6-aza-2-thiothymine (ATT) and 2,5-dihydroxybenzoic acid (DHB) produced promising results for the analysis of lipid extracts from bacteria. Analytical techniques in combination with multivariate analysis, such as principal components analysis (PCA) and principal component-discriminant function analysis (PC-DFA), were used for bacterial discrimination. Classification was initially undertaken using MALDI-TOF-MS analysis, and subsequently FT-IR spectroscopy, Raman spectroscopy and LC-MS were performed to confirm the classification results. Two main types of bacteria were used for this analysis: 34 strains from seven Bacillus and Brevibacillus species and 35 isolates from 12 Enterococcus faecium strains. The findings showed that the four analytical techniques provide clear discrimination between bacteria at these different levels. Classification of different Bacillus and Brevibacillus bacteria using MALDI-TOF-MS analysis of extracted lipids was confirmed by LC-MS data. In addition, MALDI-TOF-MS data based on extracted lipids and intact bacterial cell proteins were very similar. MALD-TOF-MS analysis of intact enterococci cells produced successful classification with 78% correct classification rate (CCR) at the strain level. FT-IR and Raman spectroscopic data produced very similar bacterial classification with CCR of 89% and 69% at the strain level, respectively. However, classification based on MALDI-TOF-MS data and that based on spectroscopic data were slightly different (Procrustes distance of 0.81, p < 0.001, at the species level). Overall, the findings in this thesis indicate the potential of MALDI-TOF-MS as a rapid, robust and reliable method for the classification of bacteria based on different bacterial preparations.

Chemometrics is a discipline of chemistry which uses mathematical and statistical tools to help in the extraction of chemical information from measured data. With the assistance of chemometric methods, infrared (IR) spectroscopy has become a widely applied quantitative analysis tool. This dissertation explores two challenging applications of IR spectroscopy facilitated by chemometric methods: (1) passive Fourier transform (FT) remote sensing and (2) process monitoring by near-infrared (NIR) spectroscopy. Passive FT-IR remote sensing offers a measurement method to detect gaseous species in the outdoor environment. Two major obstacles limit the application of this method in quantitative analysis: (1) the effect of both temperature and concentration on the measured spectral intensities and (2) the difficulty and cost of collecting reference data for use in calibration. To address these problems, a quantitative analysis protocol was designed based on the use of a radiance model to develop synthetic calibration data. The synthetic data served as the input to partial least-squares (PLS) regression in order to construct models for use in estimating ethanol and methanol concentrations. The methodology was tested with both laboratory and field remote sensing data. Near-infrared spectroscopy has attracted significant interest in process monitoring because of the simplicity in sample preparation and the compatibility with aqueous solutions. For use in process monitoring, the need exists for robust calibrations. A challenge in the NIR region is that weak, broad and highly overlapped spectral bands make it difficult to extract useful chemical information from measured spectra. In this case, signal processing methods can be helpful in removing unwanted signals and thereby uncovering useful information. When applying signal processing as a spectral preprocessing tool and regression analysis for building a quantitative calibration model, optimizing the parameters that specify the details of the methods is crucial. In this research, particle swarm optimization, a population-based optimization method was applied. Digital filtering and wavelet processing methods were evaluated for their utility as spectral preprocessing tools. Both a pump-controlled flowing system and bioreactor runs involving the yeast, Pichia pastoris, were studied in this work. In investigating the bioreactor runs, insufficient reference data resulted in difficulties in employing the PLS calibration method. Instead, the augmented classical least-squares modeling technique was applied since it requires only pure-component or composite spectra of the analyte and background matrix rather than a large set of mixture samples of known analyte concentration.

The application of a hollow waveguide (HW) was investigated as a gas cell for analytical infrared analysis. The analysis was the measurement of carbon monoxide (CO) and nitric oxide (NO) in sidestream cigarette smoke. An FT-IR analysis system was setup with a 3m multi-pass gas cell and a 55cm by 2mm i.d. Ag/AgI coated HW in tandem with individual CO and NO gas analyzers. The HW demonstrated response times an order of magnitude less than the larger volume multi-pass gas cell and slightly faster than the single analyte gas analyzer. Furthermore, it has been demonstrated that the HW provides up to approx. 60% greater sensitivity on a per meter optical path basis than the multi-pass gas cell of the analytes investigated due to increased optical efficiency maximizing the light concentration within the gaseous sample volume. Simulations in 3-D showed the sensitivity could theoretically improve by more than an order of magnitude if the IR beam was coupled more efficiently into the waveguide. Both FT-IR configurations gave statistically equivalent results for CO to the independent analyzers. With the HW increased temporal resolution, inter-puff measurements comparable to the gas analyzer were achieved at a lower spectral resolution. The HW optical configuration was modeled for ray tracing in MATLAB. Simulations in 2-D and 3-D were accomplished. The simulations show a major drawback to HW optimization is the coupling of the infrared beam into the waveguide. As demonstrated in a 3-D simulation, approximately 97% of the rays are rejected when an off-axis parabolic mirror with 25.4mm focal length is used to focus the IR beam into the 2mm i.d. waveguide. Repeating the simulation with longer focal length mirrors showed improved in IR coupling into the waveguide from 3% to 85%. Simulations applying a compound parabolic concentrator show comparable performance to the traditional design of two OAP mirrors to collect rays from the HW and focus onto the detector, but in a much smaller configuration. The simulation routines can be used to further improve the design of this and other optical sensing systems and enhanced by incorporating a spectral component to the simulation.

Bladder cancer is one of the most common urogenital cancers worldwide. Two techniques commonly used for bladder cancer diagnosis are urine cytology and cystoscopy. Cytology is not sensitive for detecting tumors. Cystoscopy is an invasive technique which disturbs patient comfort. In the current study, we used Fourier transform infrared (FT-IR) spectroscopy as a novel method which is rapid and non-invasive to investigate the bladder tumor recurrence using the bladder wash samples collected in the course of control cystoscopy. This study is unique since it is the first one to use the bladder wash sample in the diagnosis of the bladder tumor by using FT-IR spectroscopy. Molecular investigation of the FT-IR spectra revealed many differences between control and tumor samples such as a considerable increase in protein, carbohydrate and nucleic acids content, and changes in protein and carbohydrate structure. On the basis of the spectral differences, cluster analysis was performed to differentiate between the control and tumorous spectra and we reached to an overall sensitivity (including all individuals with tumor) of 91.8%, a PUNLMP sensitivity of 83.3% and a papilloma sensitivity of 77.8% in spectral range 1444-1457 cm-1. Other spectral ranges also gave similar results. Our results showed that FT-IR spectroscopy can be used to detect the bladder tumors in bladder wash sample with higher sensitivity compared to cytology. In summary, we propose the utilization of the FT-IR spectroscopy for the detection of bladder tumors since specific spectral regions might be used as effective markers for the diagnosis.