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Characterization of bio-molecular surfaces and liquids by means of reference-free X-ray spectrometry
(2016)
An increasing field of application for X-ray spectrometry and related analysis techniques is the investigation of tailor made functional surfaces modified by organic molecules. For a detailed understanding and further development of such functionalized surfaces, a quantitative determination of the surface density of molecular species or even specific functional groups is required. By means of reference-free X-Ray Fluorescence (XRF) spectrometry such customized surfaces can be analyzed quantitatively, when using specific marker elements. A chemical analysis of molecular bonds can be accomplished by X-Ray Absorption Spectroscopy in the Near-Edge region (NEXAFS). Especially in the soft X-ray range an access to relevant light elements like carbon C, nitrogen N and oxygen O is possible. Especially, the characterization of liquids and solid-liquid interfaces aiming at the determination of bio-molecular moieties (functional groups) is of great interest. The characterization of such systems requires the analysis of bio-molecules in their liquid environment. For the understanding of (biomolecular) reactions and binding processes the verification and quantification of biomolecule immobilization is essential. However, the analysis in the soft-X-ray range involves a higher experimental effort. In this contribution various successful applications of the characterizations of organofunctionalized surfaces, solid-liquid interfaces and liquids containing bio-molecular species by means of reference-free X-ray spectrometry will be presented. The experiments were performed at the plane-grating monochromator beamline (PGM-U49) in the soft X-ray range at the synchrotron radiation facility BESSY II. Using calibrated instrumentation and a quantification approach based on atomic fundamental parameters a quantitative access without any calibration sample or reference material is possible. In a complementary analysis by X-Ray Photoelectron Spectroscopy (XPS) and fluorescence measurements based on laserexcitation in the optical light spectrum the functional-group density of silane monolayers were determined. Here, the nitrogen atoms of the head-group of the label molecule employed could be used as specific marker for the reference-free quantitative XRF analysis and is used for traceable calibration of XPS and fluorescence spectroscopy. Furthermore, a novel UHV-compatible liquid-cell developed to investigate liquids and solidliquid interfaces will be presented. Protein attachment at the solid-liquid interface was investigated by detection of the characteristic N1s→π*(O=C-N) amide bond resonance in the N K-edge NEXAFS by using ultra-thin epoxy-functionalized SiC-windows.