Label-Free Mapping of Osteopontin Adsorption to Calcium Oxalate Monohydrate Crystals by Tip-Enhanced Raman Spectroscopy

Abstract

In the ectopic biomineralization of calcium oxalate kidney stones, the competition between calcium oxalate monohydrate (COM) formation and its inhibition by the phosphoprotein osteopontin (OPN) plays a key role in COM stone-forming processes. To get more insights into these processes, tip-enhanced Raman spectroscopy (TERS) was used to provide surface-specific information about the adsorption of OPN to faces of COM crystals. In TERS, the surface plasmon resonance of a metallic AFM tip is locally excited when the tip is placed in the optical near-field of a laser focused on the crystal surface. Excitation of this localized surface plasmon resonance allows the enhancement of the Raman signal as well as the improvement of the spatial resolution beyond the diffraction limit of the light. As TERS works label free and noninvasively, it is an excellent technique to study the distribution of adsorbed proteins on crystal faces at the submicrometer scale. In the present work, we generated Raman intensity maps indicating high spatial resolution and a distinct variation in relative peak intensities. The collected TERS spectra show that the OPN preferentially adsorbs to edges and faces at the ends of COM crystals (order: {100}/{121} edge > {100} face > {100}/{010} edge ≈ {121}/{010} edge > {010} face) providing also relevant information on the inhibition of crystal growth. This study demonstrates that TERS is an excellent technique for detailed investigations of biomolecules adsorbed, layered, or assembled to a large variety of surfaces and interfaces.

Raman spectroscopy: techniques and applications in the life sciences

“…Opilik et al demonstrated imaging of two-phase lipid domains with STM-TERS (Figure 29 (b)) [368]. Kazemi-Zanjani et al were able to image phosphoprotein osteopontin on calcium oxalate monohydrate crystal facets (relevant to the breakup of kidney stones), with sufficient resolution to observe preferential adherence to the edge facets of the crystal [369].…”

Hydroxyapatite Growth Inhibition Effect of Pellicle Statherin Peptides

“…1.5; diameter: 10 mM ), using 200 µl of 50 mM HEPES, 150 mM NaCl, pH 7.4, at 37 °C. Raman spectra were taken directly from the in situ-grown crystals as previously described (Hug et al 2012;Kazemi-Zanjani et al 2012). To this end, a LabRAM HR spectrometer (Horiba-Jobin-Yvon Scientific, Edison, NJ, USA) provided with a 632.8 nm excitation laser was connected to an Olympus IX71 inverted optical microscope (Olympus Corporation, Tokyo, Japan).…”

The osteopontin-controlled switching of calcium oxalate monohydrate morphologies in artificial urine provides insights into the formation of papillary kidney stones

“…rat bone, rat kidney, bovine milk) have shown a strong and irreversible adsorption to COM crystal faces [10,[22][23][24], which resulted in the hindrance of propagating crystals steps [25], in the inhibition of three-dimensional growth [10,26] and in the incorporation of the protein into the crystal structure [23,27]. Nene et al have reported that an OPN phosphopeptide adsorbs at the crystal face in front of a growing step and hinders its growth, before it is integrated into the crystal structure and overgrown (after exchanging the protein containing solution by a control solution) by freshly crystallizing monohydrate phase [28].…”