Modern Raman spectroscopy for investigation of host-pathogen interactions
Ochsenkühn, Michael Andreas
Biomedical sciences are in need of more versatile and more sensitive approaches for research and also for diagnostic purposes. In particular, intracellular detection and imaging of disease relevant proteins is a challenge. Although the state of the art method of intracellular imaging is fluorescence, it suffers from several drawbacks. Raman is an alternative imaging modality and this work investigates the use of different Raman techniques for detection and imaging of cellular constituents. In one aspect of the work, surface-enhanced Raman spectroscopy using gold nanoshells excitable at a wavelength of 780 nm was investigated. Initially the investigation of the uptake of the 150 nm diameter nanoparticles showed that NS are taken up voluntarily by a non-standard en- docytosis mechanism into mammalian fibroblast cells. Furthermore it was shown that internalized particles have no detrimental in uence on cell growth or cell viability. That these nanoparticles are non toxic was further confirmed by testing for markers of apoptosis and necrosis. Preliminary surface-enhanced Raman spectroscopy (SERS) studies produced spectra from intracellular compartments with an enhancement factor of 1010. To yield high specificity of the intracellular Raman protein sensor, two different approaches were studied. The first is based on the application of DNA aptamers which form a stacked G-quadruplex on target protein binding. A SERS sensor based on the well characterized Thrombin binding aptamer (TBA) yielded high reproducibility, high target specificity, and a limit of detection down to 0.1 fM. Further studies on a similar stacked G-quadruplex forming aptamer confirmed that observed detection signal is produced by the aptamer assuming its secondary structure but also showed that the stabilization and formation of the G-quadruplex secondary structure is strongly buffer dependent. A second sensing approach was based on a peptide (a3(IV)NC1) influential in Goodpasture's syndrome, an autoimmune disease. With the help of this peptide we found that an intracellular redoxpotential of -200 mV is necessary to make it accessible for the protease Cathepsin D. We found that SERS sensing has the ability to study the binding of Cathepsin D, its activity and with the help of a synthesized amino-acid SERS library the direct detection of the remaining peptide products. Finally this work concludes with imaging the changes of lipid droplet structure and distribution in fibroblast cells during the infection process of the murine cytomegalovirus (MCMV) in fixed and in living cells by coherent anti-Stokes Raman based on a Synchro-lock phase coupled setup. This showed that CARS imaging is able to non-invasively investigate the changes of lipid structures during different stages of the infection process and therefore promises to be a valuable tool in biological research.
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