Raman based identification of on-chip trapped single micro-organisms

Abstract

An important aspect in increasing our health and safety is the development of new sensors for screening drinking water samples for the presence of microbiological contaminants.The main problem associated with the detection and identification of these microbial contaminants is the long process time. The majority of the time is required fortheir purification and multiplication due to the low concentrations in which they arefound. These steps can be avoided by using spectroscopic identification rather than biochemical techniques. Raman spectroscopy provides a fingerprint based on the vibrational states of molecular bonds from which the bio-particles containing these can be identified. The Raman effect is weak, but rich in information and flexible with respect to its excitation wavelength.When working in an aqueous environment it can take advantage of the absorption minima of water to out perform for instance IR spectroscopy. Optical trapping allows the immobilization of particles without additional preparation steps and provides much added value to, and is highly compatible with Raman spectroscopy. Lab-on-a-chip techniques allow for integration and large-scale parallelization of processes, whichis unavoidable when performing large-scale identification of microbial contaminants on the level of single cells. To take advantage of established CMOS processing techniques for mass production in electronics, a CMOS compatible technology for integrated photonics providing waveguides transparent at a Raman suitable wavelengths, is needed. TripleX is such a waveguide technology. The research presented in this thesis shows the realization of an integrated dual-waveguide optical trap and the feasibility of its use for the identification of micro-organisms based on their Raman spectrum induced by the same on-chip optical beams used for trapping. It does so in fourmain steps. Firstly, laser tweezers Raman spectroscopy is used to classify the closely related yeast species Kluyveromyces lactis and Saccharomyces cerevisiae from measurements at the single cell level. Laser tweezers Raman spectroscopy combines optical trapping of a cell and generation of its characteristic Raman spectrum using one laser-beam focus. This enables fingerprinting of a cell’s molecular composition in a harmless fluidic environment within minutes. Laser tweezers Raman spectroscopy is considerably faster than well-known biological techniques based on streak plating or PCR, and requires far less biological material. For each yeast species a training set and a test set were measured. Visual inspection of the spectra showed intra-species variations obstructing division into two classes by eye. Application of a classification rule based on Fisher’s criterion nevertheless led to the successful blind classification of the test-set cells. Finally, a Kolmogorov-Smirnov test indicated that the difference between the distributions of the species was statistically significant, implying biological origin of the classification. This successful extension of laser tweezers Raman spectroscopy to classification of the aforementioned yeasts underlinesits applicability in microbiology and will hopefully contribute to the process of its adoption in this discipline. Laser tweezers Raman spectroscopy is not limited to rapid classification of single cells, but may also include e.g. study of the cell metabolism. Secondly, a new approach to the dual-beam geometry for on-chip optical trapping and Raman spectroscopy, using box shaped-waveguides microfabricated in TripleX technologyis demonstrated. These waveguides consist of SiO2 and Si3N4, so as to provide alow index contrast with respect to the SiO2 claddings and low signal loss, while retaining the advantages of Si3N4. The waveguides enable both the trapping and Raman functionality with the same dual beams. Polystyrene beads of 1µm diameter can be trapped with this device. In the axial direction discrete trapping positions occur, owing to the intensity pattern of the interfering beams. Interpretation of the trapping events on the basis of simulated optical fields and calculated optical forces indicate that a strong trap is formed by the beams emitted by the waveguides. Furthermore, the acquisition of Raman spectraof a single trapped bead is demonstrated. The spectra obtained in this manner show distinct polystyrene Raman peaks for integration times as short as 0.25 seconds. Thirdly, usual procedure of background subtraction is found to be less effective for Raman spectra obtained with the dual-waveguide trap, due to its specific geometry. The differences in the Raman generating properties between four dual-waveguide traps with varying distances between their waveguide facets are explored using a saturated ascorbic acid solution. Furthermore, the origin of a periodic background observed in the ascorbic acid spectra is investigated. Finally, an alternative method of signal acquisition and processing is presented to deal with the lack of fluidic control in the device and the periodic background and low signal-to-noise ratio observed in the spectra. The 10 µm and 5µm traps are used in trapping and Raman generation experiments with biological relevant particles in the form of Bacillus subtilis spores. These experiments result in noisy spectra for many and few spores in both traps. Using the presented processing the spectra are identified as Bacillussubtilis spore spectra. A comparison of the obtained signal-to-noise values to literature benchmarks shows the feasibility of micro-organisms identification with the dual-waveguide trap.