Orientation changes in surface-bound hybridized DNA undergoing preparation for ex situ spectroscopic measurements Caitlin Howell a,b , Hicham Hamoudi b , Stefan Heissler c , Patrick Koelsch a,b,⇑ , Michael Zharnikov b,⇑ a Institute of Toxicology and Genetics, Karlsruhe Institute of Technology, Hermann-von-Helmholtz-Platz 1, 76344 Eggenstein-Leopoldshafen, Germany b Angewandte Physikalische Chemie, Universität Heidelberg, Im Neuenheimer Feld 253, 69120 Heidelberg, Germany c Institute of Functional Interfaces, Karlsruhe Institute of Technology, Hermann-von-Helmholtz-Platz 1, 76344 Eggenstein-Leopoldshafen, Germany article info Article history: Received 1 June 2011 In final form 30 July 2011 Available online 4 August 2011 abstract Films of surface-bound homo-oligonucleotides on gold were hybridized and subjected to rinses using various volumes of water in preparation for ex situ spectroscopic analysis performed by high-resolution X-ray photoelectron spectroscopy, near-edge X-ray absorption fine structure spectroscopy, and infrared reflection–absorption spectroscopy. It was found that films rinsed with 0.5 mL of water contained well- ordered DNA hybrids, while films rinsed with greater volumes (1 and 2 mL) showed a decreased number of hybrids and a significant disruption of their intactness and order. These results illustrate the impor- tance of taking into account preparation procedures when interpreting ex situ spectroscopic data of such systems. Ó 2011 Elsevier B.V. All rights reserved. 1. Introduction DNA holds great promise as the basis for new biotechnologies due to its unique recognition and coupling properties [1]. In partic- ular, surface-bound DNA has been exploited in such diverse appli- cations as DNA computing [2], nanoparticle assembly [3], and the directing of cell settlement [4], in addition to the more standard microarray applications that have proven valuable for high- throughput genetic screening [5]. However, the efficient function- ing of these systems is highly dependent on a variety of parame- ters. Of these, packing density and molecular orientation of the surface-bound strands are probably the most important factors in determining the effectiveness of the hybridization process on which these technologies are based [6–8]. To monitor the effect of these parameters and hybridization processes in detail one needs efficient experimental tools which give not only non-specific information on the amount of the surface-bound DNA but also spe- cific information on the intactness of the hybrids and orientation of the DNA strands. Only on this molecular-level basis can a more thorough understanding of DNA hybrids and hybridization at sur- faces be achieved. The use of various types of spectroscopy has recently proven to be advantageous in obtaining molecular-level information about surface-bound DNA systems [1,9–15]. Among these techniques, those which operate under ex situ conditions have been shown to yield valuable information about the composition and molecular density of DNA films [16,17], the orientation of the molecules [9,12,13,18], and changes that can occur between ex situ and in situ conditions [10,19,20]. However, for all of these techniques the question of sample preparation is essential, as the relatively high amount of salt necessary for successful DNA deposition and/ or hybridization must be removed to some degree in order to be able to perform ex situ measurements accurately [21,14]. It is known that salt removal by solutions containing little to no salt re- sults in the destabilization of surface-bound hybrids [14]. In this work, we provide evidence that such treatment can have the fur- ther effect of significantly disrupting the intactness of the remain- ing hybrids as well as orientation of the surface-bound DNA probe molecules. 2. Materials and methods HPLC-purified thymine homo-oligonucleotides [oligo(dT) 25 S] modified with a C6 linker and thiol group on the 5 0 end were pur- chased from Sigma–Aldrich. The thiol linkers were deprotected using DTT by the vendor and used as-received. Films of these mol- ecules immobilized on gold (T25S/Au) were prepared in 1 M CaCl 2 solutions containing 10 lM TRIS and 1 lM EDTA (CaCl 2 –TE) according to previously described procedures [14], followed by hybridization with adenine (dA) 25 for 8 h in 1 M TE–NaCl buffer (T25S/Au:A25) at room temperature. After hybridization, samples were first rinsed for 1 min under flowing 1 M NaCl buffer to remove excess DNA strands, then were rinsed with 0.5, 1, or 2 mL of Milli-Q water (resistiv- ity > 18.2 MX cm). The samples were held vertically and the water was directed at the surface at an angle of 45° by a pipette. The flow 0009-2614/$ - see front matter Ó 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.cplett.2011.07.096 ⇑ Corresponding authors. E-mail addresses: patrick.koelsch@kit.edu (P. Koelsch), Michael.Zharnikov@ur- z.uni-heidelberg.de (M. Zharnikov). Chemical Physics Letters 513 (2011) 267–270 Contents lists available at SciVerse ScienceDirect Chemical Physics Letters journal homepage: www.elsevier.com/locate/cplett