Application of vibrational spectroscopy and atomic force microscopy in blood and malaria research
2017-01-16T22:50:39Z (GMT) by
This thesis aimed at developing the applications of Raman and IR spectroscopy as well as atomic force microscopy (AFM) in blood analysis and malaria research. It is shown that vibrational spectroscopy alone or in combination with AFM can be used as a powerful technique for the investigation of the heme environment, hemoglobin conformation, distribution of heme complexes, structural integrity of the red blood cells (RBCs), as well as the interaction of antimalarial drugs with heme both in vitro and in situ. This dissertation is presented in the “thesis by publication” format, with five papers either published, submitted, or pending revision, which are explored in Chapters 3–7. In the first chapter a general introduction to blood and malaria research is presented and the research questions are explained with particular emphasis on the effects of preparation methods on blood analysis, as well as the current knowledge on the mechanism of action of antimalarial drugs which is relevant to our study. The second chapter briefly discusses the analytical techniques and methods used in this thesis along with a short review on the resonance Raman spectroscopy of heme complexes. The third chapter describes a resonance Raman spectroscopic investigation into the effects of fixation and dehydration on heme environment of hemoglobin inside RBCs. The results provide a basis for understanding the effects of ex vivo handling procedures on hemoglobin, which is essential when studying blood samples. Chapter 4 discusses the effect of fixation and dehydration on the structural integrity of the RBCs as well as the spatial distribution of heme species inside the cells, which was investigated using a correlation between Raman mapping and AFM imaging with high resolution. The methodology and information presented in this chapter are very valuable for studying structure-function relationship in both normal and abnormal RBCs. In Chapter 5, the first AFM images of hemozoin crystals in the food vacuole of sectioned malaria parasite infected RBCs are reported along with the first tip-enhanced Raman spectra of malaria pigment within a sectioned erythrocyte at 20 nm spatial resolution. The enhanced Raman spectra clearly show characteristic bands of hemozoin that could be correlated to a precise position on the crystal by comparison with the corresponding AFM image. With the development and optimization of tip-enhanced Raman spectroscopy (TERS) for in situ analyzing of hemozoin crystal within sectioned erythrocytes we foresee this approach paving the way for a detailed investigation of the distribution of antimalarial drugs around hemozoin crystals as well as the drug binding sites on heme complexes, with a resolution in the nanometer range. In Chapter 6, the TER spectra of hematin are reported which was recorded using an AFM–Raman system with top illumination configuration. Moreover, an efficient protocol for the preparation of TERS probes with an apex radius of ca 20 nm is presented which yields a very high fraction of the probes with the capability to provide TERS effect. And the final chapter describes a vibrational study on the interaction site of antimalarial drugs with heme. It is shown that the Ferriprotoporphyrin-IX (FP) in chloroquine–FP complex is not μ-oxo dimeric, ruling out the hypothesis that chloroquine forms a complex with μ-oxo dimer FP with a stoichiometry of 1:2. Moreover, the first vibrational spectroscopy evidence is presented which show that 4-amodiaquine antimalarials (chloroquine and quinacrine) interact with FP via π–π stacking between quinoline and porphyrin rings as well as a hydrogen bonding between the tertiary amino nitrogen of the drug and a carboxylate group of the heme, while arylmethanol antimalarials (quinine and mefloquine) interact with FP through coordination of the heme iron to the 9-hydroxyl group of the drug.