Structure of the ring in drop coating deposited proteins and its implication for Raman spectroscopy of biomolecules Vladimı ´r Kopecky ´ Jr., Vladimı ´r Baumruk * Institute of Physics, Faculty of Mathematics and Physics, Charles University in Prague, Ke Karlovu 5, CZ-12116 Prague 2, Czech Republic Available online 9 June 2006 Abstract Drop coating deposition Raman spectroscopy represents a new technique that enables nondestructive measurements of solutions with concentration of biomolecules down to 1 mM. It has been demonstrated that the solution structure is preserved even after drying and Raman spectra taken from the glass-like dried deposit and a solution are virtually identical. Here, we report for the first time measurements of the structure of a drop coating ring. Proteins deposited at the outer part of the ring perimeter are affected by desiccation and the spectra differ significantly from those taken in solution. Reproducible measurements of biomolecules by means of drop coating deposition Raman spectroscopy must therefore be obtained from central or slightly inward-located parts of the coating ring. The structure as well as mechanisms participating in the formation of the coating rings is explained on the basis of recently published physical theories of droplets desiccation. Formation of the final shape of the ring is analogous to processes that give rise to desiccated droplets, whereas the coating ring behaves as an ‘‘independent ring droplet’’ in final stages of desiccation of an ancestral droplet. Its structure is dominated by a dip or plateau in the upper part. Oscillation of the ancestral droplet contact line is probably responsible for complete desiccation of the proteins at the outer perimeter of the coating ring. It seems plausible the arrangement of a glassy ‘‘skin’’ at the coating ring surface caused by the accumulation of the biomolecules near this region plays an important role in preservation of ‘‘solution-like’’ spectral shape. # 2006 Elsevier B.V. All rights reserved. Keywords: Raman spectroscopy; Drop coating deposition Raman; Proteins; Droplet evaporation; Structure of coatings; Coating rings 1. Introduction Raman spectroscopy can be used as a powerful probe into the structure and function of biomolecules. However, its usage, particularly in biochemistry, is often limited by the amount and concentration of samples. A recently reported new technique of non-enhanced Raman spectroscopy – the drop coating deposition Raman (DCDR) method [1] – based on a coffee ring effect [2], enables nondestructive measurements of solutions with concentration of biomolecules down to 1 mM (ca. 0.01 mg/mL). The method is based on deposition of a small amount of sample solution (typically units of mL) on a Teflon- coated stainless steel surface with almost no Raman signal [1]. The hydrophobic surface enables drying of a sample by the coffee ring effect [2], which can be described as follows. The evaporating droplet has a pinned contact line which causes that liquid evaporating from the contact line is replenished by liquid from the interior. The resulting flow can carry virtually all dispersed material to the edge where a ring of sample molecules is formed. It has been demonstrated that even after drying the sample solution structure is preserved and Raman spectra taken from the dried deposit and a (concentrated) solution are virtually identical [1]. An increasing number of papers have demonstrated the usefulness of DCDR method. It has been shown that DCDR can serve as an analytical tool in identification of insulin variants [3] or as a detection method of peptide tyrosine phosphorylation [4] and of amino acid and peptide phosphate protonation [5]. DCDR is useful also in chemometric analysis, as demonstrated by oligosaccharide identification and quantification in mixtures [6]. The ability of DCDR to segregate fluorescence from sample impurities [7] as well as possible coupling of DCDR with HPLC analysis and extension to proteomic analysis in general can bring new quality to scientific research. www.elsevier.com/locate/vibspec Vibrational Spectroscopy 42 (2006) 184–187 * Corresponding author. Tel.: +420 221 911 473; fax: +420 224 922 797. E-mail address: baumruk@karlov.mff.cuni.cz (V. Baumruk). 0924-2031/$ – see front matter # 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.vibspec.2006.04.019