Analyse mikrobiologischer Proben auf Basis der Ionenmobilitätsspektrometrie

Die vorliegende Dissertation konzentriert sich zum einen auf experimentelle Anwendungen der Ionenmobilitätsspektrometrie im Bereich der Mikrobiologie und zum anderen auf gezielte instrumentelle Entwicklungen, um die Akzeptanz der Methode zu verbessern. Zunächst konnte gezeigt werden, dass durch Headspace-Analysen unterschiedlicher Bakterienkulturen die Identifizierung verschiedener Spezies und die Überwachung deren bakteriellen Wachstums möglich war. Aufbauend auf diese Arbeiten wurde als neue Ionisierungsmethode das Flexible Microtube Plasma vorgestellt, um damit die üblichen radioaktiven β-Strahlungsquellen mit ihren regulatorischen Einschränkungen zu ersetzen. Nach gezielten Optimierungsschritten konnte Helium durch Stickstoff als Arbeitsgas ersetzt werden ohne Sensitivität einzubüßen. Hierzu wurde unter anderem eine Ionisierungskammer mittels 3D-Druck gefertigt. Darauf aufbauend wurde im Folgenden erstmals ein vollständig, mittels 3D-Druck Verfahren gefertigtes Ionenmobilitätsspektrometer vorgestellt und dessen Performance mit einer Referenz aus PTFE verglichen. Veränderte Materialeigenschaften machten es zunächst notwendig weitere Designoptimierungen durchzuführen. Letztendlich wurde das optimierte, vollständig 3D gedruckte Ionenmobilitätsspektrometer der Referenz qualitativ angeglichen. Somit konnte gezeigt werden, dass sich der 3D-Druck als alternatives Herstellungsverfahren für Ionenmobilitätsspektrometer eignet. Für die Analyse flüssiger Proben wurde abschließend ein miniaturisierter Thermodesorptionschip vorgestellt. Dieser Chip ermöglichte erstmals in Kombination mit dem Flexible Microtube Plasma nicht-flüchtige Stoffe, wie den Autoinducer N-hexanoyl-L Homoserinlacton, direkt aus einer Lösung heraus zu adsorbieren, anschließend zu thermodesorbieren und mittels Ionenmobilitätsspektrometer nachzuweisen.The present thesis focuses on experimental applications of ion mobility spectrometry in the field of microbiology on one hand and on targeted instrumental developments to improve the acceptance of the method on the other. It could be demonstrated that headspace analyses of different bacterial cultures allow early recognition of bacterial growth and rapid pathogen identification. Based on this, a new ionization method, namely the Flexible Microtube Plasma, was presented to replace the commonly used radioactive β-radiation sources with their regulatory restrictions. After targeted optimization steps, helium as working gas could be replaced by nitrogen without losing sensitivity. For this purpose, an ionization chamber was manufactured using 3D-printing. Furthermore, a completely 3D-printed ion mobility spectrometer was presented for the first time and its performance was compared with a reference made of PTFE. Modified material properties made it necessary to optimize the initial design. Overall, comparable analytical response and general performance of the commonly fabricated ion mobility spectrometer was achieved by the completely 3D-printed one. This demonstrates, that 3D printing is a suitable alternative for manufacturing ion mobility spectrometers. Finally, a miniaturized thermal desorption chip was applied for the analysis of liquid samples. For the first time a combination of thermal desorption chip, a Flexible Microtube Plasma and ion mobility spectrometer allowed the adsorption, thermal desorption and detection of non-volatile substances, such as the autoinducer N-hexanoyl-L-homoserine lactone

Hyphenation of a MEMS based pre-concentrator and GC-IMS

A micro-electro-mechanical system (MEMS) based pre-concentrator filled with a standard Tenax TA adsorbent as well as with a synthetic receptor designed to adsorb 3-hydroxy-3-methylhexanoic acid (3H3MHA), a particular metabolite only available from human beings, was adapted to a custom made ion mobility spectrometer with gas-chromatographic pre-separation (GC-IMS). This combination was compared to a traditional sample loop GC-IMS. The application of a pre-concentrator is highly beneficial for the GC-IMS as analysing technique. By variation of the adsorbed sample volume, the system can be adapted to changing sample concentration ranges easily, thus increasing sensitivity significantly. Detection limits of few hundred ppqV could be obtained in this work for eucalyptol and 3 human metabolites (benzaldehyde, 2-ethyl-1-hexanol and decanal) as exemplary analytes. Moreover, the appropriate choice of selective pre-concentration phases in the pre-concentrator enables an adaptation of sampling to the composition of the mixture. Relevant compounds in very low concentrations can be amplified by using specially designed cavitands while interfering substances could be suppressed. This was successfully demonstrated by detecting 3H3MHA, a compound exclusively available in human sweat, which can be used to locate trapped or hidden individuals

Blood Culture Headspace Gas Analysis Enables Early Detection of Escherichia coli Bacteremia in an Animal Model of Sepsis

(1) Background: Automated blood culture headspace analysis for the detection of volatile organic compounds of microbial origin (mVOC) could be a non-invasive method for bedside rapid pathogen identification. We investigated whether analyzing the gaseous headspace of blood culture (BC) bottles through gas chromatography-ion mobility spectrometry (GC-IMS) enables differentiation of infected and non-infected; (2) Methods: BC were gained out of a rabbit model, with sepsis induced by intravenous administration of E. coli (EC group; n = 6) and control group (n = 6) receiving sterile LB medium intravenously. After 10 h, a pair of blood cultures was obtained and incubated for 36 h. The headspace from aerobic and anaerobic BC was sampled every two hours using an autosampler and analyzed using a GC-IMS device. MALDI-TOF MS was performed to confirm or exclude microbial growth in BCs; (3) Results: Signal intensities (SI) of 113 mVOC peak regions were statistically analyzed. In 24 regions, the SI trends differed between the groups and were considered to be useful for differentiation. The principal component analysis showed differentiation between EC and control group after 6 h, with 62.2% of the data variance described by the principal components 1 and 2. Single peak regions, for example peak region P_15, show significant SI differences after 6 h in the anaerobic environment (p < 0.001) and after 8 h in the aerobic environment (p < 0.001); (4) Conclusions: The results are promising and warrant further evaluation in studies with an extended microbial panel and indications concerning its transferability to human samples