Multifunctional self-assembled composite colloids and their application to SERS detection† †Electronic supplementary information (ESI) available: Experimental part; magnetic separation and Raman scattering characterization of micelles; additional TEM images; and analysis of the effect of surface cha

Development of a novel hybrid colloidal system suitable for surface-enhanced Raman spectroscopy analysis.

Synthesis of gold nanostars. The surfactant-free method was used to prepare Au nanostars. 1 A solution of gold seeds (~15 nm, [Au] = 0.5 mM, 0.5 mL) prepared by the Turkevich method 2 was added to a solution (50 mL) containing HAuCl 4 (0.25 mM) and HCl (1 mM), followed by addition of AgNO 3 (10 mM, 0.15 mL) and ascorbic acid (100 mM, 0.25 mL). To increase the stability of the obtained nanostars, CTAB (100 mM, 2.5 mM) was added to the growth solution. Upon synthesis, the solution was centrifuged (4500 rpm, 30 min) to remove excess reactants, and redispersed in water.
Ligand exchange. The corresponding gold nanostar colloid (5 mM, 1 mL) was added dropwise under sonication to a THF solution (10 mL) of PS-SH (5 molecules of PS-SH per nm 2 of gold surface). The solution was kept in an ultrasonic bath for 15 min. To ensure ligand exchange, the resulting mixture was left undisturbed for 12h, and then centrifuged twice. The particles were finally dispersed in THF (final concentration 5 mM).

Electronic Supplementary Material (ESI) for Nanoscale. This journal is © The Royal Society of Chemistry 2015
Synthesis of iron oxide nanoparticles. Iron oxide nanoparticles were prepared according to the method reported by Park et al. 3 Final concentration of Fe 3 O 4 NPs in THF was 62 mg/mL.

Self-assembly of hybrid micellar clusters
In a typical assembly experiment, water (1 mL) was added dropwise to a mixture in THF containing AuNSs@PS (1.9 mL, 5.5 mM), PS 403 -b-PAA 62 (0.2 mL, 1.5 mg/mL) and Fe 3 O 4 NPs (0.01 mL, 62 mg/mL) under magnetic stirring. Subsequently, the water content was increased up to 50 wt%, followed by increasing the temperature up to 50 ºC, which was maintained for 30 min. The final solution was centrifuged twice (3500 rpm, 20 min) and the particles redispersed in pure water at a concentration of [Au] = 0.5 mM.

Self-assembly of magnetic micelles
Water (1 mL) was added to a mixture in THF containing PS 403 -b-PAA 62 (0.2 mL, 1.5 mg/mL) and Fe 3 O 4 NPs (0.01 mL, 62 mg/mL) under magnetic stirring. Subsequently, the water content was increased up to 50 wt%, followed by increasing the temperature up to 50 ºC, which was maintained for 30 min. The final solution was centrifuged twice (3500 rpm, 20 min) and the particles dispersed in pure water.

Sample preparation and SERS measurements
The hybrid colloid ([Au] = 0.5 mM) and the desired amount of analyte were incubated for 2 hours to reach thermodynamic equilibrium. A small amount (20 µL) of the sample was then used to fill a thin glass tube (~1 mm internal diameter), which was sealed at both ends by parafilm ® . A magnetic field we applied by putting a small commercial handheld magnet placed in contact with the tube. In order to avoid fluctuations in the SERS intensity due to the flow of NPs following the magnetic field, each sample was left in contact with the magnet during 15 minutes prior to recording the spectra. After this time no relevant changes in the intensities were observed.

Characterization
Optical extinction spectra were recorded using an Agilent 8453 UV/Vis diode-array spectrophotometer. SERS spectra were recorded using a Renishaw InVia Raman microscope equipped with two Peltier-cooled CCD detectors, a Leica microscope with two gratings of 1200 and 1800 lines/mm and band-pass filter optics. Excitation lasers with emission wavelengths of 633 and 532 nm were used and focused onto the sample through a 10× objective with N.A. 0.25, producing spot diameters of 3.1 and 2.6 µm for 633 and 532 nm, respectively. Except for the limit of detection experiment, all SERS spectra were collected with an integration time of 10 s, and the samples were irradiated with constant powers of 0.61 mW (633 nm) and 2.43 mW (532 nm). For the detection limit study, the samples were irradiated with 633 nm excitation at a constant power of 5.93 mW and 30s integration time. Transmission electron microscopy (TEM) images were collected with a JEOL JEM-1400PLUS instrument operating at 120 kV. HAADF-STEM images and electron tomography tilt series were acquired using a double aberration corrected cubed FEI Titan 50-80 electron microscope operated at 300kV. For the reconstruction of the series we used the SIRT algorithm, as implemented in the ASTRA toolbox. 4,5 S2. Magnetic properties of hybrid clusters Figure S1. The stable colloid of hybrid clusters phase separates upon application of an external magnetic field. Figure S2. (a-d) SERS spectra of the magnetoplasmonic assemblies without analyte. By irradiating the sample with 0.61 mW there is no signal detected without magnetic field (a) and very weak peaks once it is applied (b). On the other hand, by increasing the power at 5.93 mW there are again no peaks visible with no magnet (c) but it is possible to distinguish clearly several features after assemblies aggregation (d).  Figure S4. SERS intensity of characteristic vibrations of MG and TB (919 and 1570 cm -1 , respectively) vs. number of washing steps. Data were recorded before (black) and after (red) collecting the assemblies by applying an external magnetic field. Progressive decrease of the SERS intensity was observed for MG as compared to a steep drop for TB, suggesting the preferential adsorption of positively charged dye molecules on the surface of the negatively charged polymer micelles.