DNA Nanostructures DOI: 10.1002/anie.201204245 Fabrication of ssDNA/Oligo(ethylene glycol) Monolayers and Complex Nanostructures by an Irradiation-Promoted Exchange Reaction** M. Nuruzzaman Khan, Vinalia Tjong, Ashutosh Chilkoti, and Michael Zharnikov* The macromolecular structure of DNA, its ability to hybrid- ize, the diversity of non-natural nucleotides with unique functional groups that are available to perform chemistry on it, and enzymes that are capable of manipulating its sequence, structure, and topology provide rich opportunities for its use in clinical diagnostics, biosensors, gene therapy, and drug delivery. [1–3] Some of these applications rely on the immobi- lization of single-stranded DNA (ssDNA) onto a solid sup- port, which is subsequently used for binding and detection of its complementary ssDNA target or for the recognition of DNA binding proteins. [4–6] A commonly used method for immobilization of ssDNAs is to functionalize them with a terminal reactive group that is selective for the surface of interest. [4, 5] Depending on the particular application, immo- bilization can be performed either homogeneously over the entire surface or lithographically, resulting in an array of ssDNA spots. [4, 7–9] The hybridization activity of supported ssDNA depends on the packing density and molecular organization, which can be manipulated by diluting ssDNA with other molecules that have the same reactive group. [4, 10–13] For a gold substrate, the diluent molecules of choice are short-chain alkanethiols (ATs) [4, 12–16] or thiolated oligo(ethylene glycol)s (OEG- ATs). [8–11, 17–21] OEG-ATs are especially attractive for applica- tions in which the DNA comes into contact with complex biological fluids because of their ability to resist the adsorp- tion of proteins. Strategies to prepare such mixed films include co-deposition, [8, 11, 17–19, 21] backfilling, [9, 19] and post- deposition by substitution. [20] In an alternative approach, ssDNA can be covalently conjugated to a terminal reactive functional group presented by the monolayer. [6, 22, 23] OEG- ATs are also frequently used to provide a protein-repelling background to ssDNA patterns. These patterns are usually prepared by one of the standard techniques such as micro- contact printing, UV lithography, or drop casting by a syringe or microarrayer. [7–9] The preparation of the OEG-ATs back- ground typically occurs by backfilling after deposition of the ssDNA on the surface. Herein, we present a new and potentially universal approach to prepare both mixed ssDNA/OEG-AT films in a broad range of compositions and ssDNA/OEG-AT patterns of arbitrary form. We demonstrate the strength of the approach by combining it with surface-initiated enzymatic polymerization (SIEP) and sculpting complex DNA nano- structures. This approach is based on an irradiation-promoted exchange reaction (IPER) and electron-beam lithography (EBL). Generally, IPER gives control over the extent and rate of the molecular exchange between the primary mono- layer and a potential substituent by electron irradiation of the monolayer with a suitable dose. [24] It works well with a monolayer comprised of methyl-terminated, short-chain ATs, [24, 25] but is difficult to apply to OEG-ATs films, because of the inefficiency of promoted exchange (the molecules are too long) and contrast deterioration owing to non-promoted exchange occurring frequently in these systems. [26, 27] However, IPER works well with thiolated ssDNA as the substituent. The procedure is illustrated in Figure 1a. A primary monolayer of a test OEG-AT compound, HO- (CH 2 CH 2 O) 3 (CH 2 ) 11 SH (termed EG3; see Refs. [28] and [29] for its protein-resistance) was homogeneously irradiated with electrons or using EBL, resulting in preferential damage of the OEG chain parts and cleavage of thiolate–gold bonds. [27, 29] Next, the film was incubated with a solution of a model thiolated homo-oligonucleotide, 5’-SH-(CH 2 ) 6 - d(A) 25 -3’ (termed A25SH) for the exchange reaction pro- moted by the irradiation defects. We used EBL to visually demonstrate the efficiency of IPER in substituting A25SH in an EG3 matrix. AFM clearly shows the formation of a nano- scale A25SH pattern against a background of the EG3 that spells “DNA” (Figure 1c and Supporting Information, Fig- ure S1). The proportion of the A25SH component in the mixed A25SH/EG3 monolayer can be precisely controlled by selection of the irradiation dose. As shown in Figure 2a, N1s photoemission (PE) spectra of the one-component A25SH monolayer and mixed A25SH/EG3 films prepared by IPER exhibit the characteristic two-peak signature of adenine at 399.3 and 401.1 eV [5, 30] and the intensity of this signal increases with increasing irradiation dose, demonstrat- [*] M. N. Khan, Prof. Dr. M. Zharnikov Angewandte Physikalische Chemie, Universität Heidelberg Im Neuenheimer Feld 253, Heidelberg (Germany) E-mail: Michael.Zharnikov@urz.uni-heidelberg.de Homepage: http://www.pci.uni-heidelberg.de/apc/zharnikov/ V. Tjong, Prof. A. Chilkoti Department of Biomedical Engineering, Duke University Box 90300, Durham, NC 27708-0300 (USA) [**] This work has been financially supported by grants from DFG (ZH 63/9-3 and EC 152/4-1), DAAD scholarship to M.N.K., NSF grant CBET-1033621 to A.C., and by a Duke University Nanoscience Graduate Fellowship to V.T. A.C. also acknowledges support of the NSF through the Research Triangle MRSEC (NSF DMR-11-21107). We thank M. Grunze for support of this work and the Max-lab and BESSY II staff for their assistance. Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/anie.201204245. A ngewandte Chemi e 10303 Angew. Chem. Int. Ed. 2012, 51, 10303 –10306  2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim