Published on Web Date: July 12, 2010 r2010 American Chemical Society 2339 DOI: 10.1021/jz100742j | J. Phys. Chem. Lett. 2010, 1, 2339–2342 pubs.acs.org/JPCL In Vitro Characterization of Surface Properties Through Living Cells Mark-Oliver Diesner, †,‡ Caitlin Howell, z,‡ Volker Kurz, z,‡ Dominique Verreault, z,‡ and Patrick Koelsch* ,z,‡ † Faculty of Biosciences, Universityof Heidelberg, D-69120 Heidelberg, Germany, ‡ Institute of Toxicology and Genetics, Karlsruhe Institute of Technology, D-76344 Eggenstein-Leopoldshafen, Germany, and z Department of Applied Physical Chemistry, University of Heidelberg, D-69120 Heidelberg, Germany ABSTRACT The ability to probe an interface beneath a layer of living cells in situ without the need for labeling and fixation has the potential to unlock some of the key questions in cell biology and biointerfacial phenomena. Here, we show that vibrational sum frequency generation (SFG) spectroscopy can be used to detect alkanethiol self-assembled monolayers (SAMs) buried underneath a layer of living erythrocytes (ECs). SFG spectra with and without ECs showed the spectral signatures typical of these SAMs, indicating that the signal was being generated solely by the SAM and was not influenced by the presence of cells. Direct comparison of infrared spectroscopy to SFG measurements of cells adhered on a fibronectin layer showed that the SFG signal emanated solely from this layer. These results have important implications for the characterization of surfaces in biomedical, environmental, and industrial applications. SECTION Surfaces, Interfaces, Catalysis T he determination of surface properties at solid/liquid interfaces such as chemical composition, molecular ordering, dynamics, and conformation is central to the understanding of the development of biofilms and the interac- tion of cells with a given substrate. 1-3 In particular, the in situ monitoring of the interphase between a substrate and the cellular layer is of great interest as it allows determination of changes in surface properties upon cell adhesion. Typically, this is accomplished by labeling and fixing the cell samples, which may result in their disruption and in the loss of valuable infor- mation. In this work, we show that the surface specificity of vibrational sum frequency generation (SFG) spectroscopy per- mits the in situ characterization of an interfacial layer through living cells, without the need for fixation or labeling. These results will be useful for those seeking to apply this system to investigate layers under living cells with high surface specificity. SFG spectroscopy has proven well-suited to probing various interfaces. 4,5 In recent years, this technique has been applied to the in situ investigation of biomolecules, 6 including peptides, 7,8 proteins, 9 and DNA. 10,11 Previous work in our lab has demon- strated the ability of SFG spectroscopy to detect a substrate through a layer of fixed cells. 12 In order to test the ability of this technique to probe a substrate through living cells, a well- characterized self-assembled monolayer (SAM) was probed underneath of a layer erythrocytes (ECs). Highly ordered SAMs of deuterated dodecanethiol (CD 3 (CD 2 ) 11 -SH, d-DDT) were chosen as they have a distinctive spectral signature in the CD stretching vibrational region (2000-2300 cm -1 ). These deute- rated layers give little to no signal in the CH stretching region (2800-3000 cm -1 ), allowing us to detect any contribution from the cells or cellular debris. In addition, we investigated undeuterated dodecanethiol (CH 3 (CH 2 ) 11 -SH, DDT) in the CH region to confirm that the typical spectral signatures of an alkanethiol SAM remained unchanged even in the presence of cells. Living ECs were selected as the biological barrier since they contain the majority of molecule types encountered in most biological systems. These cells are known to have no direct interaction with the surface offered to them and will lay flat on it if given enough time to settle (Figure 1). 13 ECs do not deposit an extracellular matrix (ECM) of their own or modify surface chem- istry, allowing them to act as biological barriers without altering surface properties. 14 Additionally, these cells contain a large amount of hemoglobin, which makes them optically dense in comparison to most other thin biological layers. If these cells pose no obstacle to the generated SFG signal, many biological barriers, either pro- or eukaryotic, become eligible for use with this technique. A scheme of the SFG setup and the measuring cell has been previously described. 15 A crudely purified solution of ECs in phosphate-buffered saline (PBS) was placed in between the SAM substrate and an optically transparent prism. All samples were measured using a femtosecond broad-band SFG spectro- meter in ppp polarization. A ppp configuration (both input beams as well as the SFG beam are p-polarized) was chosen as it gives a higher SFG response and it probes more χ (2) tensor ele- ments simultaneously than other polarization configurations. It is thus more likely to probe additional contributions from the Received Date: June 2, 2010 Accepted Date: July 7, 2010