Two-dimensional Correlation Spectroscopy (2D-COS) in the analysis of parasitemia in Raman spectra of red blood cells of patients diagnosed with malaria

Abstract

Abstract This study focuses on finding correlations between Raman bands present in patients infected with Plasmodium falciparum (P. falciparum) malaria at different levels of parasitemia (parasite concentration). The obtained spectra were analysed using two-dimensional correlation spectroscopy (2D -COS) to find out how the Raman bands correlate with each other depending on the level of parasitemia in the patients. A laser line with a wavelength of 442 nm was used to excite the heme resonance, which allowed for obtaining enhanced bands in the heme vibration spectrum. Synchronous auto-peaks were found to be generated by marker vibrations of ν4 and ν37 deoxy-heme structures at position 1354 cm-1 and 1580 cm-1, respectively. The observed cross-peaks were caused by vibrations of deoxyheme structures, often characteristic of hemozoin. For the normal Raman effect, a laser line with a wavelength of 785 nm was used, which allowed obtaining additional information about the structure of infected erythrocytes and enabled the detection of many changes in the structure of proteins and lipids of red blood cells, while the resonance Raman effect focused almost exclusively on changes in the heme structure. Therefore, two-dimensional maps created from these results differed significantly, indicating different spectral regions depending on the laser line used. And thus, many auto-peaks appear for the 785 nm excitation line, which, in addition to the peaks characteristic for heme, also originate from amino acids, protein and lipid structures. Different positions of asynchronous cross-peaks come from different intermediates that characterize the oscillations of ν4 heme structures. Both synchronous and asynchronous 2D correlation maps for laser lines 442 nm and 785 nm, respectively, allow not only to notice changes in the intensities of correlated band, but also phase shifts between bands – i.e. the order of changes occurring between correlated Raman bands. It was observed that 442 nm excitation laser line created conditions for observing specific heme intermediates. The potential possibility of using the relative Raman intensity of the bands representing the oxy- and deoxy-heme structure, I1370/I1354, to correlate with the levels of malarial parasitemia observed in blood samples is discussed.