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Spectroscopic detection and quantification of heme and heme degradation products

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Abstract

Heme and heme degradation products play critical roles in numerous biological phenomena which until now have only been partially understood. One reason for this is the very low concentrations at which free heme, its complexes and the partly unstable degradation products occur in living cells. Therefore, powerful and specific detection methods are needed. In this contribution, the potential of nondestructive 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 μmol/L) could be reliably detected when utilizing the resonance enhancement effect. Furthermore, a systematic study on the surface-enhanced Raman spectroscopy (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 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 into their interaction.

Resonance Raman spectrum of met-myoglobin and Raman intensity - concentration - calibration plot.

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Acknowledgments

Financial support by TMWFK PE114-1, DFG (FOR 1738 - Po563/17-1), BMBF (FKZ 01EO1002), European Union (EFRE), the Free State of Thuringia and Thüringer Aufbaubank (2008FE9112) are highly acknowledged. Mathias Köhler and Stefan Bode are highly acknowledged for assisting the resonance Raman measurements, Claudia Beleites for fruitful discussions and Gisela Persch for preparing the colloids.

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Authors and Affiliations

  1. Center for Sepsis Control and Care (CSCC), Jena University Hospital, Erlanger Allee 101, 07747, Jena, Germany

    U. Neugebauer

  2. Institute of Photonic Technology, Albert-Einstein-Str. 9, 07745, Jena, Germany

    U. Neugebauer, T. Henkel & J. Popp

  3. Institute of Physical Chemistry and Abbe Center of Photonics, University of Jena, Helmholtzweg 4, 07743, Jena, Germany

    A. März, M. Schmitt & J. Popp

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  1. U. Neugebauer
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Correspondence to J. Popp.

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Published in the special paper collection Optical Biochemical and Chemical Sensors with guest editor Laura M. Lechuga.

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Neugebauer, U., März, A., Henkel, T. et al. Spectroscopic detection and quantification of heme and heme degradation products. Anal Bioanal Chem 404, 2819–2829 (2012). https://doi.org/10.1007/s00216-012-6288-9

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  • DOI: https://doi.org/10.1007/s00216-012-6288-9

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