Supporting Information for: A New Peptide-Based Method for the Design and Synthesis of Nanoparticle Superstructures: Construction of Highly-Ordered Gold Nanoparticle Double Helices Chun-Long Chen, Peijun Zhang, and Nathaniel L. Rosi * Department of Chemistry, University of Pittsburgh, 219 Parkman Ave. and Department of Structural Biology, University of Pittsburgh School of Medicine, 3501 5 th Ave., Pittsburgh, PA 15260 E-mail: nrosi@pitt.edu Materials and Methods All solvents and chemicals were obtained from commercial sources and used without further purification. 0.1M HEPES Buffer (HEPES = 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) was made by directly diluting 1.0M HEPES buffer (ph = 7.3 ± 0.1; Fisher Scientific) with water (NANOpure, Barnstead Diamond TM System.; 18.2 MΩ). Peptide with sequence of AYSSGAPPMPPF (PEP Au ) was synthesized and purified by Sigma-Aldrich with final purity of 99%. Reverse-phase high-pressure liquid chromatography (HPLC) was performed at ambient temperature with an Agilent 1200 liquid chromatographic system equipped with diode array and multiple wavelength detectors using a Grace Vydac protein C4 column (214TP1010, 1.0 cm 25 cm). Matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectra were obtained on an Applied Biosystem Voyager System S-1
6174 MALDI-TOF mass spectrometer using α-cyano-4-hydroxy cinnamic acid (CHCA) as the matrix. Transmission electron microscopy (TEM) samples were prepared by pipetting one drop of solution onto a 3-mm-diameter copper grid coated with carbon film; 2% aqueous phosphotungstic acid was used for negative staining. TEM was conducted on a JEOL 200CX instrument operated at 200 kv and images were collected using a Gatan CCD image system. Samples for atomic force microscopy (AFM) were prepared on freshly peeled MICA substrates. Tapping-mode AFM was performed on a Veeco Dimension V SPM. Circular dichroism (CD) spectra were recorded on an Aviv CD spectrometer (Model 202). Transmission fourier transform infrared (FT-IR) spectroscopy measurements were recorded on a Nicolet Avatar 360 FT-IR spectrometer in the range of 4000 cm -1 to 500 cm -1 at about 1 cm -1 resolution. The projection images from electron tomography studies were recorded with a Gatan 4K 4K charge-coupled device (CCD) camera mounted on a Tecnai F20 electron microscope (FEI Corporation, Hillsboro, Oreg.) equipped with a field emission gun (FEG) operating at 200 kv. For electron tomography, a series of images were recorded at room temperature with the Gatan 4K 4K CCD camera at a nominal magnification of 50,000 by tilting the specimen from 70 to 70 in increments of 1. Images were recorded at underfocus value around 2μm along the tilt axis. A back-projection algorithm, as implemented in the IMOD reconstruction package, 1 was used to convert the information present in the series of tilted projection images into three-dimensional density maps. The surface rendering was generated using the Chimera software. 2 Citrate-stabilized Au nanoparticles (~13 nm) were prepared by the citrate reduction of HAuCl 4. 3 Preparation of N-hydroxyl-succinimide esters. Dodecanoic acid (1.2 g, 6 mmol) and N-hydroxysuccinimide (0.73 g, 6.3 mmol) were dissolved in 12.5 ml dry DMF under an S-2
argon atmosphere. After addition of dicyclohexyl carbodiimide (DCC) (1.35g, 6.5 mmol) at 0ºC, the solution was stirred overnight at room temperature. The reaction mixture was processed by removing the precipitate via filtration. The solvent was removed under reduced pressure and the crystalline residue recrystallized from EtOH to yield the N-hydroxyl-succinimide ester (0.75 g, 2.5 mmol, 42.0%). Preparation of C 12 -PEP Au. AYSSGAPPMPPF (1.20 mg, 9.82 10-7 mol) was dissolved in 0.1 ml dry DMF. After addition of 1.2 equivalents of dodecanoic N-hydroxyl-succinimide ester (0.350 mg, 1.18 10-6 mol) in 35.0 μl DMF and 1 μl Et 3 N under stirring, the solution was stirred at room temperature for 12 hours. Pure C 12 -PEP Au was obtained by conducting reversed-phase HPLC eluting with a linear gradient of 0.05% formic acid in CH 3 CN and 0.1% formic acid in water (5/95 to 95/5 over 30 mins ) (isolated yield: 91.1%, based on integration of HPLC peaks). The molecular weight for C 12 -PEP Au was confirmed by MALDI-TOF mass spectrometry. The purity of the peptide was >95% as determined by analytical HPLC. Concentration of the peptide was determined spectrophotometrically in water/acetonitrile (1:1) using a molar extinction coefficient of tyrosine (1280 M -1 cm -1 ) at 280 nm. Preparation of Gold Nanoparticle Double Helices. Lyophilized C 12 -PEP Au (~ 7.49 10-8 mol) was completely dissolved in 0.5 ml 0.1 M HEPES buffer in a plastic vial. After 30 mins, 2 μl of freshly prepared 0.1M chloroauric acid (HAuCl 4 ) in 1.0 M triethylammonium acetate (TEAA) buffer solution was added to the above clear Peptide-HEPES solution or to a clear filtrate obtained by filtering above Peptide-HEPES solution with 0.2 μm centrifuge filter. The above mixture was vortexed for a few seconds and then left undisturbed at room temperature. S-3
Supplementary Figures S1-S14 with Legends Figure S1. The reverse-phase HPLC chart for the coupling reaction of AYSSGAPPMPPF with dodecanoic N-hydroxyl-succinimide ester. 100 1425.99 M+Na + Voyager Spec #1[BP = 1426.0, 11011] 1.1E+4 90 80 70 % Intensity 60 50 40 M+H + 1441.83 M+K + 1404.58 30 1402.87 20 1448.33 1419.08 10 1355.65 1460.82 1562.12 0 0 1275 1488 1701 1914 2127 2340 Mass (m/z) Figure S2. MALDI-TOF mass spectrum of purified C 12 -PEP Au. S-4
a b Figure S3. TEM images of C 12 -PEP Au fibers obtained directly after addition of HAuCl 4 /TEAA solution. a b c d Figure S4. Additional AFM data that reveal the morphology of the twisted nanoribbons. S-5
a b Figure S5. AFM height images of peptide C 12 -PEP Au nanoribbons with pitch measurements (A) and height measurements (B). [θ] x 10-3 (deg x cm 2 x dmol -1 ) 20 15 10 5 0-5 -10 C 12 -PEP Au dissolved in HEPES buffer C 12 -PEP Au dissolved in HEPES buffer after addition of HAuCl 4 210 220 230 240 250 260 270 280 Wavelength (nm) Figure S6. Circular dichrosm (CD) spectra of clear solution of C 12 -PEP Au in 0.1 M HEPES buffer (red) and solution of C 12 -PEP Au in HEPES buffer immediately after addition of HAuCl 4 (black). We note that the features in the spectra (~227 nm in red and ~230 nm in black) could correspond to β-sheet structures. S-6
Figure S7. The FT-IR Spectrum of a thin film of C 12 -PEP Au fibers on a sodium chloride disk. The peak occurring at 1728 cm -1 is characteristic of the C=O stretching mode for the carboxylic acid at the C-terminus of PEP Au. The N-H stretching mode of the amide groups occurs around 3275 cm -1 and it overlaps with the strong O-H band from hydroxyl-containing amino acid residues such as tyrosine and serine. Other relevant bands are discussed in the text. S-7
Figure S8. The FT-IR Spectrum of a thin film of C 12 -PEP Au -Gold nanoparticle superstructures on a sodium chloride disk. The strong peak at 1631 cm -1 occurs in the amide I region (1600 1700 cm -1 ) and the amide II band occurs at 1538 cm -1 ; both can be identified and attributed to β-sheet conformations. The C-H vibration bands at 2918 cm -1 and 2850 cm -1 indicate ordered packing of the aliphatic chains. S-8
Figure S9. The UV-Vis spectrum of the nanoparticle double helices in solution. The absorbance maximum is observed at 556 nm. The peak is significantly red-shifted and broadened compared to what is typically observed for monodisperse colloidal solutions of gold colloids having particles similar in size to what is observed for the nanoparticle double-helices. 4 This is expected due to the plasmonic coupling between the assembled nanoparticles. S-9
a b S-10
c S-11
d Figure S10. Additional TEM images (a-d) of gold nanoparticle double helices at different magnifications. S-12
a b c d e f Figure S11. (a) Width distribution of the C 12 -PEP Au fibers (based on 60 counts from TEM images of stained fibers; width = 6.1 ± 0.6 nm). (b) Distribution of the pitch of the twisted nanoribbons (based on 60 counts from AFM height images; pitch = 84.1 ± 4.2 nm). (c) Distribution of the maximum inner-distances between particles along the width of the gold nanoparticle double helices (based on 80 counts from TEM images; distance = 6.0 ± 0.8 nm). (d) Distribution of the pitch of the gold nanoparticle double helices (based on 80 counts from TEM images; pitch = 83.2 ± 4.4 nm). (e) Distribution of the number of gold nanoparticles per pitch of the double helix (based on 40 counts from TEM; images, number = 22 ± 2). (f) Distribution of the edge-to-edge distance between nanoparticles along the longitudinal dimension of the gold nanoparticle double helices (based on 60 counts from TEM images; distance = 1.5 ± 0.8 nm). S-13
a b Figure S12. C 12 -PEP Au. TEM images of gold nanoparticles formed using PEP Au instead of a b Figure S13. TEM images of (a) stained assemblies of C 12 -FPPMPPAGSSYA and (b) gold nanoparticle aggregates formed using C 12 -FPPMPPAGSSYA instead of C 12 -PEP Au. S-14
Figure S14. TEM image of citrate-stabilized particles after they were mixed a HEPES solution containing C 12 -PEP Au nanoribbons. No double-helical nanoparticle assemblies were observed. The aim of this experiment was to determine whether the C 12 -PEP Au nanoribbons could template the assembly of pre-formed gold nanoparticles. The citrate particles used in this experiment were prepared using the method reported in Reference 3. References (1) Kremer, J. R.; Mastronarde, D. N.; McIntosh, J. R. J Struct Biol 1996, 116, 71-6. (2) Pettersen, E. F.; Goddard, T. D.; Huang, C. C.; Couch, G. S.; Greenblatt, D. M.; Meng, E. C.; Ferrin, T. E. J Comput Chem 2004, 25, 1605-12. (3) Storhoff, J. J.; Elghanian, R.; Mucic, R. C.; Mirkin, C. A.; Letsinger, R. L. J. Am. Chem. Soc. 1998, 120, 1959-1964. (4) Mafune, F. Chem. Phys. Lett. 2004, 397, 133-137. S-15