Bifunctional DNA–gold nanoparticle conjugates as building blocks for the self-assembly of cross-linked particle layers Christof M. Niemeyer, a, * Buelent Ceyhan, a Michael Noyong, b and Ulrich Simon b a Universit€ at Dortmund, Fachbereich Chemie, Biologisch-Chemische Mikrostrukturtechnik, Otto-Hahn Str. 6, Dortmund D-44227, Germany b Institut f€ ur Anorganische Chemie der RWTH Aachen, Prof.-Pirlet-Str. 1, Aachen D-52056, Germany Received 13 October 2003 Abstract The DNA-directed self-assembly of surface-bound layers of gold nanoparticles offers a broad range of applications in biomedical analyses as well as in materials science. We here describe a new concept for the assembly of substrate-bound nanoparticle mono- layers which employs bifunctional nanoparticles as building blocks, containing two independently addressable DNA oligomer se- quences. One of the sequences was utilized for attaching the particle at the solid support, while the other sequence was used to establish cross-links between adjacently immobilized particles. AFM analyses proved the functionality of inter-particle cross-links leading to enhanced surface coverages and the formation of monolayered supramolecular aggregates attached to the substrate. We anticipate that further refinement of this approach will enable applications, for instance, the assembly of ordered layers useful as transducers in biosensing. Ó 2003 Elsevier Inc. All rights reserved. Motivated by a wide variety of applications ranging from life sciences to materials- and nano-sciences, there is currently a great interest in the rational self-assembled fabrication of hybrid materials from inorganic nano- particles and biomolecules, such as proteins and nucleic acids [1]. DNA molecules, in particular, play an im- portant role in this approach due to their tremendous molecular recognition capabilities. For example, DNA has already been used to functionalize gold nanoparti- cles, thus enabling the bottom-up fabrication of nano- structured materials [2] as well as highly sensitive assays for detecting nucleic acids [3] and proteins [4]. In all of these studies [5–14], DNA–nanoparticle conjugates were used which contained just a single recognition site, i.e., oligonucleotide sequence, and thus, the conjugates can be considered as monofunctional with respect to their recognition capabilities for complementary nucleic acids. To extend the spectrum of applications of DNA- functionalized nanoparticles, we have recently devel- oped oligofunctional gold nanoparticle conjugates containing different DNA sequences (D n –Au) [15]. The index n in D n –Au denotes the number of different se- quences attached to the nanoparticle, ranging from one (monofunctional) up to seven (heptafunctional). The D n –Au nanoparticles reveal almost unaltered hybrid- ization capabilities as compared to monofunctional conjugates. Moreover, due to the extraordinary speci- ficity of Watson–Crick base pairing, the various oligo- nucleotide sequences can be individually and selectively addressed as members of an orthogonal coupling system present on the particle’s surface. We here report on the use of bifunctional DNA–nanoparticle conjugates D 2 – Au, containing two different DNA oligomers, in the self- assembly of surface-bound layers comprised of closely packed, cross-linked nanoparticles (Fig. 1). Such kind of particle layers have a huge potential in materials science as well as transducers in biomedical analyses [5–14]. We reasoned that the two different sequences of D 2 – Au might be utilized such that, for instance, oligomer 1 is used for immobilization purposes by connecting the particles to capture oligomers 4 and linker oligomers 5 attached to a solid support, while particle-bound olig- omers 2, 3 of D 2 –Au are utilized, by means of linker 6, for the cross-linking of particles immobilized to adjacent * Corresponding author. Fax: +49-231-755-7082. E-mail address: cmn@chemie.uni-dortmund.de (C.M. Niemeyer). 0006-291X/$ - see front matter Ó 2003 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2003.10.103 Biochemical and Biophysical Research Communications 311 (2003) 995–999 BBRC www.elsevier.com/locate/ybbrc