Supplemental material

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1 Supplemental material Self-assembly of Small Gold Colloids on Functionalized Gold Nanorods Sebastien Pierrat, Inga Zins, Aaron Breivogel, Carsten Sönnichsen* Institute for Physical Chemistry, University of Mainz, Jakob-Welderweg 11, Mainz, Germany. Detailed experimental protocols The gold nanorods are functionalized by reacting thiol-terminated polyethylene glycol (PEG) on the gold surface of the rods. A mono-functional PEG, Methoxy-PEG-Thiol (mpeg-sh, MW=5682, Nektar Therapeutics) is used to stabilized the gold nanorods and is mixed with a bi-functional PEG, PEG-bis-Thiol (SH-PEG-SH, MW=3878, Nektar Therapeutics) to allow further chemical functionalization. The gold nanorods are prepared according the seed growth method [1, 2]. Seeds are prepared by reducing 10ml of an aueous solution containing 0.25mM gold tetrachloride (HAuCl 4, Aldrich) in 0.1M Hexadecyltrimethylammoniumbromide (CTAB, Sigma) adding 0.6ml of 0.01M sodium borohydride (NaBH 4, Sigma-Aldrich). 15µl of this seeds solution is added to 10ml of a growth solution consisting of 0.5mM HAuCl 4 and 0.08mM silver nitrate (Sigma- Aldrich) in 0.1M CTAB mixed with 70µl of M ascorbic acid (Sigma). A strong color change indicates the formation of the gold nanorods. PEGylation of the gold nanorods Degassed water is prepared by sonicating 250ml of deionized water (Millipore, 18MΩ.cm) for 1h (200W, Transsonic Digital T490DH, Elma, Germany). Freshly degassed water is used in the following procedure to prevent oxidation of the thiol group. Cleaning of the gold nanorods To be able to chemically modify the surface of the gold nanorods, excess of CTAB has to be removed. This is completed by several centrifugation cycles. Typically, 1ml of the growth solution in 1.5ml microcentrifuge tube is centrifuged at 6000g for 20min. The pellet is resuspended in 1ml of water and centrifuged a second time (6000g, 20min). The gold nanorods are resuspended in 900µl of water. PEGylation of the rods Fresh stock solutions (2mM in water) of mpeg-sh and SH-PEG-SH are prepared just before use. Both solutions are mixed together to prepare solutions of the desired ratio (1-x):x. 100µl of the mixed solution [(1-x) mpeg-sh / (x) SH-PEG-SH] is then added to the 900µl of the

2 cleaned gold nanorods solution. After vortexing, the reaction mix is incubated and shaken at room temperature for 1h30min. Excess of unreacted products is removed by centrifugation (6000g, 20min). The gold nanorods are rinsed with 1ml of water. After centrifugation (6000g, 20min), the pellet is resuspended in 100µl of water. Attachment of small gold spherical particles to the functionalized gold nanorods Reacting the particles to the PEGylated nanorods 100µl of gold colloid suspension (5nm particles, 80nM, Sigma) is added to the 100µl PEGylated nanorods solution. After vortexing, the reaction mix is incubated and shaken overnight at room temperature. Excess of small spherical particles is removed by adding 200µl of water and centrifuging once the solution (500g, 8min). The pellet is resuspended in 50µl of water. TEM preparation A TEM grid (Formvar/Carbon, 200mesh, Copper, Plano) is cleaned and charged using plasma treatment under oxygen (20W, 30s). A drop of 5µl of the sample is deposed on the TEM grid and evaporated at room temperature. About one hundred of individual gold nanorods are investigated at 125kx magnification in a CM12 electron microscope (Phillips). Biotinylation of the PEGylated gold nanorods The gold nanorods are biotinylated using a thiol reactive biotin derivative (EZ-Link TM Biotin- HPDP, Pierce). A 4mM of Biotin-HPDP stock solution in DMF is prepared, aliuoted and stored frozen. 10µl of this stock solution is added to the 100µl water solution of PEGylated gold nanorods. After vortexing, the reaction mix is incubated and shaken overnight. Unreacted Biotin-HPDP is removed by adding 500µl of water and centrifugation (6000g, 20min). The pellet is resuspended in 1ml of water and centrifuged (6000g, 20min). After removing the supernatant, the pellet is resuspended in 500µl of PBS. Gel electrophoresis The particles are prepared as described above and concentrated by centrifugation. 5µl are loaded with 5µl loading buffer (30% D(+)-Sucrose, Fluka, in 0.5x TBE-buffer, prepared by diluting 10x stock solutions, Fluka) into the pockets of a 0.25 % agarose gel (Agarose, for routine use, Sigma). The gel is prepared with and immersed in 0.5x TBE. The gels are run in a horizontal electrophoresis system (Mini-Sub Cell GT, Biorad) at 130 V (electrode spacing 15 cm) for 1-3 hours. The images are recorded with a consumer digital camera (Canon PowerShot A95), converted to gray scale with some linear contrast adjustment.

3 Micropatterned subtrates Glass coverslips are first boiled in a sodium hydroxide solution (H 2 O:NaOH:H 2 O 2 10:0.5:1) for 10 min and rinsed with water and methanol. The glass surface is then aminofunctionalized with a silanizing agent by incubating for 1 h in 94% methanol containing 0.15M acetic acid, 4% water and 2% N-[3-(Trimethoxysilyl)-propyl]-ethylenediamine. The amino-functionalized glass substrates are washed with methanol and cured for 20 min at 100 C. A microstructured PDMS pad is put into contact with the slide and the channels are filled by capillarity action with a drop of NHS-terminated biotin PEG (2mg/ml in water). After 1 h incubation, the pad is uickly removed from the surface. The glass surface is rinsed with water and dried under a stream of argon. Passivation of the remaining substrate is achieved by incubation of methoxy-peg-spa (2mg/ml in water) for 1h. After rinsing with water and drying with argon, the slides are incubated in a streptavidin solution (1mg/ml in PBS) for 30 min and rinsed with PBS. [1] N. R. Jana, L.G., C. J. Murphy. Seed-Mediated Growth Approach for Shape-Controlled Synthesis of Spheroidal and Rod-like Gold Nanoparticles Using a Surfactant Template. Advanced Materials (18). p [2] Nikoobakht, B., El-Sayed, M. A. Preparation and growth mechanism of gold nanorods (NRs) using seed-mediated growth method. Chemistry of Materials (10). p

4 Supplemental Figures Figure S1: Images of individual gold nanorods investigated by Transmission Electron Microscopy at 125k magnification. The gold nanorods have been PEGylated with monofunctional PEG.

5 Figure S2: Images of individual gold nanorods investigated by Transmission Electron Microscopy at 125k magnification. The gold nanorods have been PEGylated with 0.01 of bifunctional PEG.

6 Figure S3: Images of individual gold nanorods investigated by Transmission Electron Microscopy at 125k magnification. The gold nanorods have been PEGylated with 0.02 of bifunctional PEG.

7 Figure S4: Images of individual gold nanorods investigated by Transmission Electron Microscopy at 125k magnification. The gold nanorods have been PEGylated with 0.05 of bifunctional PEG.

8 Figure S5: Images of individual gold nanorods investigated by Transmission Electron Microscopy at 125k magnification. The gold nanorods have been PEGylated with 0.10 of bifunctional PEG.

9 Figure S6: Images of individual gold nanorods investigated by Transmission Electron Microscopy at 125k magnification. The gold nanorods have been PEGylated with 0.20 of bifunctional PEG.

10 Figure S7: Tilting images of an individual gold nanorod from -45 to +25. The colloidal particles sitting in the sides of the rods on the non-tilted image are visible on the sides and are projected on the surface of the rods (visible as shadows) in the tilted images. This result together with the observed absence of shadows of small colloids on the rods in Fig. S1-6 suggests that the 2-D projection of such a self-assembly allows a correct estimation of the number of colloids attached. Presumably, the colloids are pushed to the sides during the TEM grid preparation process upon drying of the solvent.

11 * * + ] [ K 1 ] [ K x x N N sat + = * * + ] [ K 1 ] [ K x x N N sat + = Figure S8: Average number of small gold particles attached per gold nanorod as function of the fraction of bi-functional PEG as extracted from approximately 100 TEM images per point (Fig. 3). The standard deviation is indicated by the bars. The red line is a fit using the Langmuir absorption model (inset upper left) with the parameters shown (inset lower right).

12 (a) (b) (c) Figure S9: Absorbance spectra of a suspension of gold nanorods PEGylated with (a) monofunctional PEG and (b) 20% of bi-functional PEG and subseuently incubated with small gold colloids. (c) The resonant wavelength as a function of the fraction of bi-functional PEG. The black dots are experimental data whereas the red line is guide for the eyes. In these experiments, the small colloids are removed after 12hrs of incubation by a centrifugation step. Since each centrifugation step results in a loss of particles and some aggregates, the linear relationship shown in (c) is not always clearly observed unless great care is employed.