Advanced Raman spectroscopy for bioprocess monitoring
The Raman -effect was discovered almost 90 years ago. It took a long time until the importance of Raman spectroscopy was fully understood and accepted for process industrial applications. Still today the usage is limited to a small application area. During the last decades advances in the production of spectroscopic components have reduced the complexity of the instrumentation and further mediated a persistent decline in costs, thus making this technology available for a broader audience. The basic components of a Raman spectrometer are a monochromatic light source, typically laser, a Raman probe with optical fibres, a spectrograph and a detector which is connected to a measurement- and control PC. Raman spectroscopy is increasingly becoming a choice as analytical tool in bioanalytics. Most of biological samples are handled in aqueous form which challenges many other analytical techniques (e.g., infrared spectroscopy). Raman and its enhancement techniques are able to measure quality and quantity of compounds in liquid phase with no or very little interference of water. The quantity of a compound can be determined by the peak-intensity and the quality by the position in the measured spectrum. Another advantage is that Raman spectroscopy does not rely on extensive sample preparation and measurements can be carried out non-invasively by placing an immersion probe with fibre optics directly in the liquid media. A Raman measurement is conducted fast, within milliseconds, and multiple relevant process parameters from the same sample can be measured at the same time. The measurements can be performed in continuous mode, i.e. one after the other or with a delay in-between. The operator can determine the measurement interval and in this way the development of a process can be observed online and in real-time. Further important advantages of spectroscopic methods over many other biochemical and physical measurement tools are the robustness as they do not require assays and they are rather unsusceptible against variations of pH, temperature changes, vibrations and other process parameters. If there is no coating, colour or special treatment of the glass, some Raman set-ups allow measurements directly through the glass into the liquid phase. This option enables real process measurements without the need to disturb or contaminate the analytes. These experimental set-ups are in the focus of this thesis. Despite great advantages of spectroscopic methods, the utilization is often complicated since the threshold values are often above what is required for screening. Besides the lack of sensitivity of conventional Raman in bioprocess applications, the major drawback of this technique so far has been the disturbance of the broad fluorescence background especially in biological samples. The main objective of this thesis was to find solutions for increasing the limit of detection (LOD) for biomolecules, being capable to detect them during the course of the process reliably and being able to diminishing background signals induced by sample- and matrix-related auto-fluorescence. The proposed solutions are mainly surface enhanced Raman spectroscopy (SERS), time-gated (TG) Raman spectroscopy and the combination of both. This thesis had a rather broad scope ranging from biofilm detection on water membrane filtration processes, over low-concentration bacteria detection with various enhancement options to finally follow the course of cell culture media development during cultivations. The aim of the thesis is to show that in challenging bioprocess-environments, Raman spectroscopy can detect weak signals over the background-noise from fluorescence in combination with SERS-enhancement-sensor techniques and with the time-gated Raman technology in particular.
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