The Peptide Route to Multifunctional Gold Nanoparticles Zhenxin Wang, † Raphae ¨l Le ´vy, †,‡ David G. Fernig, ‡ and Mathias Brust †, * Centre for Nanoscale Science, Department of Chemistry and School of Biological Sciences, The University of Liverpool, Liverpool, UK. Received February 21, 2005; Revised Manuscript Received April 8, 2005 Extremely stable, peptide-capped gold nanoparticles with two different biomolecular recognition motifs expressed on their surface have been prepared, and their specific and selective binding to artificial, DNA-modified target particles and to DNA and protein microarrays has been demonstrated. Stabilization and biofunctionalization has been achieved in a single preparative step starting with citrate-stabilized gold hydrosols and a derivatization cocktail of peptide-capping ligands, which carry the functionalities of choice. Exploiting the optical properties of nanoparticles for the development of ultrasensitive detection and imaging methods in the biomedical sciences is becoming increas- ingly important (1-3). Particularly attractive is the use of Au and Ag nanoparticles in resonant light scattering (RLS) and photothermal microscopy, which are able to image single particles (4-7). The latter technique is capable of detecting particles at least as small as 2 nm in complex biological environments. For these applica- tions, attaching the biomolecular recognition motif of interest to the nanoparticles has to be readily achieved, and, most importantly, the probes must not bind non- specifically to each other or to anything else present in the system under investigation. In addition, introducing multiple functionalities would be of great value, as it provides more flexibility for multiplexing in bioanalytical applications and new tools to control the bottom-up assembly of nanostructures. Here we demonstrate that multiply functional peptide- stabilized gold nanoparticles are readily obtained in a one-step surface coating procedure and that the respec- tive surface functionalities can be selectively addressed on a microarray. The particles are of the stability typical for peptide capping (8) and show no indication of non- specific binding as established by transmission electron microscopy (TEM), UV-vis spectroscopy, and microarray imaging even under testing conditions of ionic strength (see Supporting Information). In contrast to most previ- ously reported approaches, stabilization and functional- ization of the particles are independent of each other, while both are achieved in a single step. Functionality is simply introduced by including a proportion of stabiliz- ing peptide to which a functionality of choice has been attached. Therefore, the number of recognition functions present on each particle could be altered and even reduced to a single or very few moieties without compro- mising the stability of the particles. We have previously reported the preparation of pep- tide-capped gold nanoparticles and their functionalization with biotin (see also Supporting Information) (8). We now take advantage of the generality of the peptide route and demonstrate that it provides a fast and simple approach to DNA functionalization and to bifunctional nano- particles carrying both DNA and biotin moieties. These functionalities were chosen for being already well estab- lished in nanoparticle systems and thus predictable in their recognition properties (1-7, 9-26). Thirteen nanometer gold nanoparticles were prepared via the classical citrate reduction route (27, 28). Stabi- lization and DNA functionalization was achieved by adding an aqueous mixture of the CALNN (cysteine, alanine, leucine, asparagine, asparagine) capping peptide and a CALNN-DNA conjugate (18 base single-stranded DNA) followed by centrifugation to purify the material. * Corresponding author. E-mail: M.Brust@liv.ac.uk; Fax: (+44) 151-794-3588. † Centre for Nanoscale Science, Department of Chemistry. ‡ Centre for Nanoscale Science, School of Biological Sciences. Figure 1. Nanoparticle binding scenarios and sequences of DNA and functionalized peptides. (a) 13 nm DNA-functionalized small particles with high DNA loading (>3%) binding to 40 nm DNA-stabilized large particles to form extended aggregates. (b) 13 nm DNA functionalized small particles with intermediate DNA loading (0.3-1%) binding to DNA-stabilized 40 nm large particles at a small particle to large particle ratio of (i) 10:1, (ii) 30:1, and (iii) 100:1. (c) Sequences of DNA and functionalized peptides used. Peptides are in the conventional N- to C-terminal orientation. 497 Bioconjugate Chem. 2005, 16, 497-500 10.1021/bc050047f CCC: $30.25 © 2005 American Chemical Society Published on Web 04/26/2005