Spectroscopic detection and quantification of heme and heme degradation products

in: Analytical and Bioanalytical Chemistry (2012)
Neugebauer, Ute; März, Anne; Henkel, Thomas; Schmitt, Michael; Popp, Jürgen
Heme and heme degradation products play critical roles in numerous biological phenomena which until now are only partially understood. One reason for that are the very low concentrations at which free heme, its complexes as well as the partly unstable degradation products occur in living cells. Therefore, powerful and specific detection methods are needed. In this contribution the potential of the non-destructive Raman spectroscopy for the detection, quantification and discrimination of heme and heme degradation products is investigated. Resonance Raman spectroscopy using different excitation wavelengths (413, 476, 532 and 752 nm) is employed to estimate the limit of detection for hemin, myoglobin, biliverdin and bilirubin. Concentrations in the low micromolar range (down to 3 :mol/L) could be reliably detected when utilizing the resonance enhancement effect. Furthermore, a systematic study on the SERS detection of hemin in the presence of other cellular components, such as the highly similar cytochrome c, DNA and the important antioxidant glutathione, is presented. A microfluidic device was used to reproducibly create a segmented flow of aqueous droplets and oil compartments. Those aqueous droplets acted as model chambers, representing a simplified cytosol environment where the analytes have to compete for the colloid. With the help of statistical analysis it was possible to detect and differentiate the pure substances as well as the binary mixtures and gain insights in their interaction.

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