Supporting Information for : Optical and topological. characterization of gold nanoparticle dimers linked. by a single DNA double-strand

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1 Supporting Information for : Optical and topological characterization of gold nanoparticle dimers linked by a single DNA double-strand Mickaël P. Busson, Brice Rolly, Brian Stout, Nicolas Bonod, Eric Larquet, Albert Polman, and Sébastien Bidault sebastien.bidault@espci.fr Synthesis of gold nanoparticle dimers General Citrate coated 27 nm and 36 nm diameter gold nanoparticles (AuNPs) were purchased from BBInternational (UK). Bis(p-sulfonatophenyl)phenylphosphine (BSPP) was obtained from Strem Chemicals Europe (France). Thiolated, methyl terminated and biotin conjugated dissufilde ethylene glycol oligomers (n=6 and 8 respectively) were purchased from Polypure (Norway). NeutrAvidin and Superblock buffer were purchased from Thermo Scientific (USA). Solvents, other buffer solutions and BSA biotin were obtained from Sigma-Aldrich (USA). PAGE purified trithiolated DNA sequences were purchased from Fidelity Systems, Inc (USA). PAGE purified unmodified DNA sequences were obtained from Integrated DNA Technologies Europe (Belgium). All chemicals were used as received without further purification. The different DNA sequences, designed to minimize non-specific interactions, are the following: To whom correspondence should be addressed 1

2 30b-trithiol: 5 -trithiol-gcacgaaacctggaccagatgggaacagca-3 30b-trithiol-comp: 5 -trithiol-tgctgttcccatctggtccaggtttcgtgc-3 50b-trithiol: 5 -trithiol-gcacgaaacctggacacccctaagcaactccgtatca GATGGGAACAGCA-3 50b-trithiol-comp: 5 -trithiol-tgctgttcccatctgatacggagttgcttagg GGTGTCCAGGTTTCGTGC-3 50b-3 -trithiol-comp: 5 -TGCTGTTCCCATCTGATACGGAGTTGCTTAGGGGTGTCC AGGTTTCGTGC-trithiol-3 L1: 5 -ATCCTGACATCGGCATTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT TTTTTTTTTTTTTTTTTTTTTTTTTTGTGCTGTTCCCATCTG-3 L2: 5 -TGCCGATGTCAGGATTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT TTTTTTTTTTTTTTTTTTTTTTTTTTTGTAACGGTAGGCTGG-3 L3: 5 -TCCCCAGTCTGAACATTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT TTTTTTTTTTTTTTTTTTTTTTTTTTTCCAGCCTACCGTTAC-3 L4: 5 -TGTTCAGACTGGGGATTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT TTTTTTTTTTTTTTTTTTTTTTTTTTTGACCTCGCACTTAGT-3 L5: 5 -ACTGCGTTCATGGTGCCTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT TTTTTTTTTTTTTTTTTTTTTTTTTTTACTAAGTGCGAGGTC-3 L6: 5 -CACCATGAACGCAGTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT TTTTTTTTTTTTTTTTTTTTTTTTTTTGGAGGACAATCGCTG-3 L7: 5 -ACGAGCACCGAAGAATTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT TTTTTTTTTTTTTTTTTTTTTTTTTTTCAGCGATTGTCCTCC-3 L8: 5 -TTCTTCGGTGCTCGTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT TTTTTTTTTTTTTTTTTTTTTTTTTTTGCACGAAACCTGGAC-3 L3 -comp: 5 -CAGATGGGAACAGCACTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTGTAACGGTAGGCTGG-3 The 30b-trithiol and 50b-trithiol sequences hybridize over 15 base pairs on their 3 ends with 2

3 the L1 strand while 30b-trithiol-comp and 50b-trithiol-comp hybridize similarly with the L8 strand. The target trithiolated DNA sequence is mixed with an excess of lengthening strands. 50b-3 - trithiol-comp hybridizes on its 5 end with the L3 -comp strand. The Li strand hybridizes with the L(i + 1) strand over 15 base pairs. L3 -comp hybridizes with L3. To lengthen the 30b-trithiol and 50b-trithiol sequences with 5 extra 100 bases long strands, they are mixed with L1, L2, L3, L4 and L5. In the same conditions, 30b-trithiol-comp and 50b-trithiol-comp are mixed with L8, L7, L6, L5 and L4. For 50b-3 -trithiol-comp, the order is L3 -comp, L3, L4, L5 and L6. Synthesis of gold nanoparticle / DNA conjugates AuNPs are coated with BSPP following published procedures. 1,2 In brief, 50 ml citrate coated particles are left to incubate overnight with 10 mg BSPP. Particles are then centrifuged to remove the supernatant before being rinsed and finally resuspended in a 0.5 mm BSPP solution. The final AuNP concentrations are of the order of respectively 0.05 µm and 0.02 µm for 27 nm and 36 nm particles. The target thiolated DNA sequence is mixed with an excess of lengthening strands following the order described above in a 10 µl solution with a 40 mm NaCl final concentration. The first lengthening strand is added with a three-fold excess and the following strands are added with 1.5X increase in concentration. This solution is heated at 85 C and left to cool to room temperature overnight in order to favor the hybridization of all target DNA strands with the same number of lengthening strands. The concentration of lengthening strands was optimized to obtain the thinnest bands in electrophoresis for the lowest amount of added DNA molecules. 0.1 pmol of 27 nm AuNPs or 0.06 pmol of 36 nm AuNPs are mixed with about 2 pmol of thiolated DNA strands already hybridized with the chosen number of lengthening strands and left to incubate overnight at room temperature. The final volume is around 15 µl with 30 mm NaCl and 1 mm BSPP final concentrations. The added BSPP is used as a reducing agent to minimize oxidation of the thiol moieties. If the number of added lengthening strands is lower than 3, 100 pmol of an unmodified 100 bases long DNA strand that does not hybridize to any of the involved 3

4 oligonucleotides is added. For instance, in figure 1-a where the 30b-trithiol-comp target sequence is used with no lengthening strand or just L8 and L7, we added 100 pmol of L1 to avoid NaCl induced aggregation of the AuNPs. Surface passivation of the gold particle surface is performed using published procedures with thiolated ethylene glycol oligomers. 2 Methyl terminated thiolated PEG is added to the AuNP suspensions in a excess for 36 nm particles and left to incubate for 30 mins. If the particles need to be conjugated to biotin, biotin terminated disulfide PEG is left to react for two hours with a 20x excess of BSPP to cleave the disulfide bound. This solution is added to the methyl PEG solution with a methyl PEG / biotin PEG ratio of 16 before incubation with the AuNPs. The solutions are loaded in 1.5 % agarose gels (0.5x Tris-Borate-EDTA as running buffer) after adding one volume of ficoll loading buffer for four volumes of loaded sample. The gels are run at 8 V/cm for 1 hour before obtaining results shown on figure 1-a of the manuscript or figure S1. The extraction procedure is done following published protocols 1,3 and the sample is concentrated by centrifugation. Figure S1 shows the result of the electrophoretic separation of 27 nm and 36 nm particles linked to a 30 bases long DNA strand hybridized to, respectively, 4 and 5 lengthening molecules. The different lanes correspond to increasing DNA / AuNP ratios compared to unconjugated PEG passivated particles. Since the same amount of particles is used in each lane of figures 1-a and S1, the AuNP density in each band depends on the number of observed bands and the DNA / AuNP ratio. Synthesis and purification of particle dimers Concentrated samples of DNA mono-conjugated passivated AuNPs with complementary sequences are mixed at 10 nm concentrations with 40 mm NaCl in a 15 µl volume before being heated up to 55 C and left to cool overnight. The samples are then loaded in 1.5 % agarose gels in a manner similar to the previous paragraph. Agarose gels obtained for symmetric and asymmetric dimers are given in figure S2. The second band in lanes 2 and 3 of figure S2-a and third band in lanes 3 and 4 of figure S2-b correspond to dimer suspensions as verified by electron microscopy. A weak 4

5 Figure S1: Agarose gel purification of 27 nm and 36 nm particles after incubation with 30b-trithiol and 30b-trithiol-comp target DNA strands respectively. The 30b-trithiol sequence was hybridized to L1-L4 prior to incubation with 27 nm AuNPs. The 30b-trithiol-comp strand was hybridized to L8-L4 prior to incubation with 36 nm AuNPs. Lanes 1 and 5 : reference pegylated 27 nm and 36 nm AuNP samples. Lanes 2-4 : the DNA/AuNP ratio is respectively 20, 40 and 80. Lanes 6-8 : the DNA/AuNP ratio is respectively 35, 75 and 150. band of hybridized trimers is also visible (third band in lanes 2 and 3 of figure S2-a and fourth band in lanes 3 and 4 of figure S2-b). By mixing bi-conjugated and complementary mono-conjugated AuNPs, the trimer band becomes relatively stronger indicating again that the different bands in figure 1 of the manuscript correspond to increasing numbers of grafted DNA strands. The bands are cut from the gel and the groupings are eluted in running buffer. Dimer suspensions are stored in running buffer for up to a month and are re-purified by electrophoresis prior to EM or optical measurements. The end result of this purification shows a strong dimer band next to a faint single particle band which could originate from either incomplete separation during the first electrophoresis step or slow degradation of the dimers. 5

6 a) b) Figure S2: Agarose gel purifications of symmetric 36 nm AuNP dimers (a) and asymmetric 27 nm - 36 nm dimers (b). (a) : lane 1 is a reference 36 nm sample and lanes 2-3 correspond to the hybridized complementary strands. (b) lanes 1 and 2 correspond to reference 27 nm and 36 nm samples respectively while lanes 3 and 4 correspond to 27 nm and 36 nm particles linked to complementary strands. Cryo-EM characterization Experimental procedures The samples are observed with transmission electron microscopy in a cryogenic environment. Concentrated AuNP dimer suspensions (1-10 nm) are deposited on 200 mesh holey-carbon-coated grids (QUANTIFOILő R 2/2). After being blotted with filter paper, the grids are frozen by being rapidly plunged into liquid ethane and are mounted and inserted in the microscope using a nitrogen-cooled side entry Gatan 626 DH cryoholder. Observations are carried out at a temperature of -180 řc in a JEOL 2100 LaB6 electron microscope equipped with a CRP objective lens (Cs,2.0 mm; Cc, 2.1 mm; focal length, 2.8 mm) using an accelerating voltage of 200 kv with the following illumination conditions: alpha 3, spot 3, 100 µm condenser aperture, without objective aperture. Images are recorded using the MDS minimum electron dose system (10 electrons / A 2 / s) at magnifications between to x on a Gatan 2k x 2k Ultrascan US1000 CCD caméra. For each field, images are recorded using a different defocus value (-1.2 µm to -1.5 µm) for good visualization of the particles. Figures S3, S4 and S5 provide several complementary cryo-em images to figure 1 of the manuscript for the three different sample geometries. 6

7 Figure S3: Cryo-EM images of the 27 nm - 36 nm AuNP dimers with a 50 bp DNA linker perpendicular to the dimer axis at different magnifications. 7

8 Figure S4: Cryo-EM images of the 27 nm - 36 nm AuNP dimers with a 50 bp DNA linker parallel to the dimer axis at different magnifications. 8

9 Figure S5: Cryo-EM images of the 27 nm - 36 nm AuNP dimers with a 30 bp DNA linker parallel to the dimer axis at different magnifications. 9

10 Analysis of cryo-em images The measured distributions of interparticle distances are obtained by analyzing automatically the cryo-em images and estimating the center to center distance between two circles that best fit the AuNPs. The widths of the distributions arise from three contributions : distributions of particle diameters, distribution of interparticle gaps and projection of the dimer axis on the plane perpendicular to the electron beam. This last factor cannot be neglected as shown on figures 1 and S3 : partial superposition of the two particles is observed on several cryo-em images and would correspond to negative gaps if measured directly. The distributions of particle diameters are given in figure 3-a-iv by fitting with circles the AuNPs in all cryo-em images. The distributions of interparticle gaps, s, need to be estimated to understand the geometries of the hybridized DNA linkers. If l is the interparticle distance (sum of s and the respective radii of the AuNPs) and l the measured projected distance, then the distribution of l can be expressed as: D l ( l)= + l D l (l)d θ (l, l) l 1 dl (1) l l 2 l2 D l (l) is the convolution of the unknown gap distribution with the diameter distributions given in figure 3-a-iv and readily fitted by Gaussian distributions. The diameter distributions of the smaller and larger particles are respectively centered on 27.3 nm and 36 nm with 1.65 nm and 2.8 nm standard deviations. D θ (l, l) is the distribution of the angle θ between the dimer axis and the electron beam. For thick cryogenic samples, D θ (l, l) would be equal to 1/π corresponding to an isotropic distribution of dimer orientations. However, only parts of the sample where the vitreous water layer is a few tens of nanometers thick are transparent enough for the electron beam to allow proper imaging. 4 Since the dimers are close to 100 nm in length, the probability of finding particle pairs parallel to the beam axis is essentially zero. It is therefore impossible to provide an accurate gap distribution from cryo-em images without knowing the angle distribution. Figure S6 shows the evolution of a Gaussian distribution of l centered on 45 nm with a 3 nm standard deviation when taking into account the angle distribution. 10

11 Probability Distance (nm) Figure S6: Comparison between the initial Gaussian distribution D l (l) (solid black line) and the projected distributions D l ( l) for D θ (θ)=1/π (solid blue line), D θ (θ)=(2/π)sin 2 (θ) (solid red line) and D θ (θ)=(8/3π)sin 4 (θ) (solid green line). The black curve corresponds to the initial Gaussian distribution. The blue curve corresponds to an isotropic distribution of dimer orientations D θ (θ)=1/π. The blue curve stretches all the way to l= 0, meaning that the probability of finding dimers nearly parallel to the electron beam is non-zero which is never observed in our samples. Our experimental data are intermediate and correspond to angle probabilities that reach 0 for θ = 0 and are maximum for θ = π/2. The red and green curves are obtained for angle distributions D θ (θ) = (2/π)sin 2 (θ) and D θ (θ) = (8/3π)sin 4 (θ). These distributions correspond to D θ (l, l) =(2/π)( l/l) 2 and D θ (l, l) =(8/3π)( l/l) 4 respectively. The projected distributions have similar centers and width and their shapes agree well with the distance distributions experimentally observed. The uncertainty is of the order of 1 nm for the distribution center and 0.5 nm for the standard deviation when switching from one angle distribution function to the other. Even though the experiments cannot be properly fitted without knowing the projection function, using an intermediate angle distribution can provide a good estimation of the 3D interparticle spacings. We thus obtained the solid curves in figure 3-a using a D θ (l, l) = (2/π)( l/l) 2 angle distribution. 11

12 Optical measurements Sample preparation Home-made microfluidic chambers are prepared using modified published procedures: 5 freshly cleaned 200 mm and 250 mm square glass slides are stacked together with two layers of parafilm that provide a 200 µm spacer with a few millimeters wide channel. The sandwich structure is heated up to 100 C to glue the glass slides together. The channel is used as a µl flow chamber. Following protocols from the literature, 2 the glass slides are functionalized with BSAbiotin and NeutrAvidin before introducing the dimer suspension at a sub-nm concentration. The different functionalization and rinsing steps are performed in 50 mm NaCl / 10 mm Tris buffer solutions (ph=8) but the sample is rinsed with water before sealing the micro-chamber with hot wax. Optical setup In our experimental setup based on an inverted microscope (IX71, Olympus), unpolarized white light from a 100 W halogen lamp is focused on the micro-chamber using an oil immersion darkfield condenser (NA= ). Light scattered from the bottom slide of the chamber is collected with an oil-immersion objective with a numerical aperture smaller than the minimum NA of the condenser (NA=0.6). A color CCD camera (Qicam, Roper) is used to observe AuNP dimers before spatially filtering the scattered light using the entrance slit of an imaging spectrometer (ImSpector V8E, Specim) which projects the dispersed spectrum on a back-illuminated EMCCD camera (ixon+ 512x512, Andor). The CCD image provides both the grouping scattering spectrum and background signal from the same sample area. The background is subtracted to the measured spectra before correction according to the wavelength dependent white light illumination. The obtained scattering spectra are not normalized and therefore sensitive to varying cross-sections of the different dimers as observed on figure 2-b of the manuscript. The integration time for all spectra is 3 s with an EMCCD gain 12

13 of 30. However, the resonance wavelength is the only parameter that is insensitive to alignment errors and was therefore used in figure 3-b to analyze plasmon coupling effects. References (1) Zanchet, D.; Micheel, C. M.; Parak, W. J.; Gerion, D.; Alivisatos, A. P. Nano Lett. 2001, 1, (2) Reinhard, B. M.; Sheikholeslami, S.; Mastroianni, A.; Alivisatos, A. P.; Liphardt, J. Proc. Natl. Acad. Sci. U.S.A. 2007, 104, (3) Bidault, S.; de Abajo, F. J. G.; Polman, A. J. Am. Chem. Soc. 2008, 130, (4) Dubochet, J.; Adrian, M.; Chang, J. J.; Homo, J. C.; Lepault, J.; Mcdowall, A. W.; Schultz, P. Quart. Rev. Biophys. 1988, 21, (5) Dulin, D.; Le Gall, A.; Perronet, K.; Soler, N.; Fourmy, D.; Yoshizawa, S.; Bouyer, P.; Westbrook, N. Proceedings of the Tenth International Meeting on Hole Burning, Single Molecule and Related Spectroscopies 2010, 3,