Gold Nanostar-coated Polystyrene Beads as Multifunctional Nanoprobes for SERS Bioimaging

Transcription

1 Supporting Information Gold Nanostar-coated Polystyrene Beads as Multifunctional Nanoprobes for SERS Bioimaging Ana B. Serrano-Montes a, Judith Langer a, Malou Henriksen-Lacey a,f, Dorleta Jimenez de Aberasturi a,f, Diego M. Solís b, José M. Taboada c, Fernando Obelleiro b, Kadir Sentosun d, Sara Bals d, Ahmet Bekdemir e, Francesco Stellacci e and Luis M. Liz-Marzán a,b,f,* a CIC biomagune, Paseo de Miramón 182, 20009, Donostia - San Sebastian, Spain b Ikerbasque, Basque Foundation for Science, Bilbao, Spain b Dept. Teoría de la Señal y Comunicaciones, University of Vigo, Vigo, Spain c Dept. Tec. Computadoras y Comunicaciones, University of Extremadura, Cáceres, Spain d EMAT-University of Antwerp, Groenenborgerlaan 171, B-2020 Antwerp, Belgium e Institute of Materials, Ecole Polytechnique Fédérale de Lausanne, Lausanne, Switzerland f Biomedical Research Networking Center in Bioengineering, Biomaterials, and Nanomedicine, CIBER-BBN, Spain * llizmarzan@cicbiomagune.es S1

2 Synthesis and assembly of citrate gold nanoparticles (Au NPs) Small citrate gold nanoparticles (Au NPs) displaying LSPR maximum at 519 nm and average size around 14 nm were prepared by adding 5 ml of 1% citrate solution to 95 ml of boiling 0.5 mm HAuCl 4 solution under vigorous stirring. After 15 min of boiling, the solution was cooled down to room temperature and then kept at 4 C for longterm storage. 1 1mL of the as-synthesized AuNPs was added onto 0.1mL of PS beads (2.5% in solids). The unbounded Au particles were removed by two centrifugation steps (1770 g, 5 min, 20 C) and the precipitate was redispersed in 1 ml of milli-q water. 100 µl of these Au NP-coated PS beads (Figure S1a) were used in a typical 10 ml nanostar synthesis as seeds to growth Au tips. 2 Figure S1. TEM images of gold nanoparticle-coated PS bead used as seeds (a), spiky particles obtained after the tip growth (b) and COOH-functionalized PS beads showing the no attachment of Au NSs after reaction (c). Synthesis and assembly of PVP-coated Au NSs PVP-coated Au NSs were synthesized as previously reported. 3 In a typical synthesis 82 μl of an aqueous solution of 50 mm HAuCl 4 was mixed with 15 ml of 10 mm PVP solution in DMF. After 20 minutes, 43 μl of a preformed dispersion of 15 nm PVPcoated Au seeds in ethanol ([Au 0 ] = 4.2 mm) was added and within five minutes the solution color changed from pink to blue, indicating the formation of gold nanostars. The particles were purified by centrifugation (4 times, 1770 g, 60min, 20 C) and redispersed in water. The concentration of Au 0 was adjusted to 0.5 mm and the assembly onto the PS surface was done as explained. S2

3 Synthesis and assembly of silver nanoparticles (Ag NPs) Ag NPs with LSPR located at 420 nm were synthesized following a reported method. 4 A 100 ml volume of aqueous solution containing sodium citrate (5 mm) and tannic acid (TA) 2 mm was prepared and heated with a heating mantle in a three-neck round bottomed flask for 15 min under vigorous stirring. A condenser was used to prevent the evaporation of the solvent. After boiling had commenced, 1 ml of AgNO 3 (25 mm) was injected into this solution. The solution became bright yellow immediately. The particles were washed by centrifugation (2 times, 6000 rpm, 30, 20 C) in order to remove the excess of TA and further redispersed in sodium citrate 1.1 mm. 1mL of the as-purified AgNPs was added dropwise onto the 100 µl of PS beads (0.15 % in solids) under sonication and the samples were incubated overnight. The unbounded Ag NPs were removed by two centrifugations (1770 g, 5min, 20 C). Figure S2. Vis-NIR spectra (left) and TEM images (right) of PS assembled with PVPcoated Au NSs (top) and Ag NPs (bottom). The Vis-NIR spectra in black correspond to the plasmonic particle before the assembly process. S3

4 Figure S3: TEM images of the Au NS-PS assemblies obtained when the concentration of Au NS is increased up to 2.75 mm. Figure S4. TEM images (a-c) showing Au NS-PS assemblies (low, medium and high Au NS coverage), prepared from at least four different batches. d) Average number of Au NS per PS bead as a function of coverage. S4

5 Figure S5. Vis-NIR spectra of Au NS-PS particles before (black) and after 24h incubation with cdmem (red). Inset TEM image of Au NS-PS beads after 24h incubation with cdmem, centrifugation and redispersion in water. Transmission Electron Microscopy of cells Cells (ca cells) were plated in 60 cm diameter tissue culture treated dishes and allowed to adhere. NP formulations were added at (% PS in solids, [Au 0 ] = 0.25 mm) in 3 ml of media and incubated overnight (at least 12 h) for uptake. The following day the excess of NPs was removed by washing with warmed media, followed by PBS and finally trypsin-edta to bring cells into suspension. Cells were washed 3-fold in Sorensens buffer (0.1 M, ph 7.4) at 300 g, 4 C, for 5 min. Cells were fixed for 10 min at room temperature using a mixture of formaldehyde and glutaraldehyde in Sorensens buffer (final concentrations 2% and 2.5%, respectively). The fixative was replaced with new fixative and samples left at 4 C for 2 h. Fixative was removed in 3-fold 5 min washes using Sorensens buffer (300 g, 4 C, 5 min). Cell pellets were transferred to solubilized agar; a 2% w/v solution of low melting weight agar in Sorensens buffer was made and 160 µl (approximately 4/5 th of the cell volume) added directly to the cell pellet. Samples were flicked and immediately centrifuged at 1700g for 5 min at 30 C. Cell pellets could be visualised in the lower part of the agarose. Samples were placed on ice for approximately 1 h in order to harden. S5

6 Pieces of cell-containing agarose were cut, approximately 1 mm 3 in size, and transferred to 1 ml glass vials (Thermo Fisher) for the addition of a 1% solution of OsO 4 in Sorensens buffer (300 µl/sample). Samples were left lightly capped on ice, in the dark, for 1 h, followed by 3 washes with Sorensens buffer. A dehydration series of sorensens:ethanol (50, 70, 90 and 100 % ) was carried out, 10 min each, followed by two final steps in 100% acetone. Spurrs resin (Pelco) was made as stated in the data sheet with an increased proportion of softener. Samples were embedded beginning with a 1:1 acetone:spurrs mixture, followed by 1:2 acetone:spurrs, and then a 100 % Spurrs, for 30 min each. Samples were finally transferred to BEEM capsules and pure Spurrs resin added. Samples were cured at 60 C overnight (approximately 12 h) before 100 nm slices were cut using glass knives on a Leica Ultramicrotome. Slices were transferred to carbon coated grids and imaged with a JEOL JEM-1400PLUS TEM operating at 120 kv. S6

7 Figure S6. TEM images showing the cellular uptake of 4-MBA labeled Au NS-PS (a) and Au NS particles (b). S7

8 Estimation of SERS enhancement factors 1. n bulk,mba n "#$%,'() = ρ '()V./0 M '() where n "#$%,'() is the molar amount of 4-MBA within the focal volume V./0 and thus contributing to the Raman signal, ρ '() is the molecular density of solid 4-MBA, and M '() its molar mass. V./0 can be estimated by V./0 = A $3456 z, where A $3456 is the laser illumination area and z corresponds to full width at half maximum (FWHM) of the depth-dependent Raman Intensity I(z). With ρ '() = 1.34 < <, M = 154, A 0= > =/$ $3456 = 20μm D and z = 111μm, we calculated n bulk,mba = R9 mol. 2. n SERS,MBA where n 3U4,'() n 3U4,'() = N WX ρ YX ρ 3U4,'() A YX is the molar amount of 4-MBA adsorbed on the AuNS surface available within A $3456, N WX the number of PS beads, ρ YX the AuNS density per bead, A YX the AuNS surface area and ρ 3U4,'() the bonding density of 4-MBA onto AuNS surface. With N WX = ) Z[\]^ D_6` and r = μm for the radius of the PS beads, ρ YX = 20, 80 and 120, A YX = 9752μm D (estimated from parameters measured by TEM, see main text) and ρ 3U4,'() = 0.5 h=/$ 0=`, we calculated: n ads,mba = R16 mol (high) n ads,mba = R16 mol (middle) n ads,mba = R17 mol (low) S8

9 3. Measured Intensities: I SERS = high I SERS = middle I SERS = 8047 low I Raman = 4256 bulk with we calculated EF = I X~X N "#$% N 3U4 I 3=3h EF = high EF = middle EF = low Figure S7. Bright-field photos overlaid with SERS images of the 1078 cm -1 vibration of the Raman active molecule 4-MBA using medium-loaded Au NS-PS beads incubated with MCF-7 (a) and J774 (b) cells. The white scale bars correspond to 20 µm. S9

10 Figure S8. MTT assay results of A459 cells incubated for 66 hours with different concentrations of Au NSs (black), PS (red) and Au NS-PS (blue) particles. Initial particle concentrations (Au NSs 0.5mM and PS and Au NS-PS % PS in solids) were diluted from 20 to 160 times. References (1) Kimling, J.; Maier, M.; Okenve, B.; Kotaidis, V.; Ballot, H.; Plech, A. Turkevich Method for Gold Nanoparticle Synthesis Revisited. J. Phys. Chem. B 2006, 110, (2) Yuan, H.; Khoury, C. G.; Hwang, H.; Wilson, C. M.; Grant, G. A.; Vo-Dinh, T. Gold Nanostars: Surfactant-Free Synthesis, 3D Modelling, and Two-Photon Photoluminescence Imaging. Nanotechnology 2012, 23, (3) Senthil Kumar, P.; Pastoriza-Santos, I.; Rodríguez-González, B.; Javier García de Abajo, F.; Liz-Marzán, L. M. High-Yield Synthesis and Optical Response of Gold Nanostars. Nanotechnology 2008, 19, (4) Bastús, N. G.; Merkoçi, F.; Piella, J.; Puntes, V. Synthesis of Highly Monodisperse Citrate-Stabilized Silver Nanoparticles of up to 200 Nm: Kinetic Control and Catalytic Properties. Chem. Mater. 2014, 26, S10