Green ” gold nanotriangles : synthesis , purification by polyelectrolyte / micelle depletion flocculation and performance in surface-enhanced Raman scattering

The aim of this study was to develop a one-step synthesis of gold nanotriangles (NTs) in the presence of mixed phospholipid vesicles followed by a separation process to isolate purified NTs. Negatively charged vesicles containing AOT and phospholipids, in the absence and presence of additional reducing agents (polyampholytes, polyanions or low molecular weight compounds), were used as a template phase to form anisotropic gold nanoparticles. Upon addition of the gold chloride solution, the nucleation process is initiated and both types of particles, i.e., isotropic spherical and anisotropic gold nanotriangles, are formed simultaneously. As it was not possible to produce monodisperse nanotriangles with such a one-step procedure, the anisotropic nanoparticles needed to be separated from the spherical ones. Therefore, a new type of separation procedure using combined polyelectrolyte/micelle depletion flocculation was successfully applied. As a result of the different purification steps, a green colored aqueous dispersion was obtained containing highly purified, well-defined negatively charged flat nanocrystals with a platelet thickness of 10 nm and an edge length of about 175 nm. The NTs produce promising results in surface-enhanced Raman scattering.


Introduction
Monodisperse gold nanoparticles (NPs) are of increasing interest due to their shape and size-dependent optical properties, 1,2 which are of enormous interest for many applications in photonics, 3 catalysis, 4 electronics 5 and biomedicine. 6It is already well established from Turkevich et al. that monodisperse spherical NPs can be synthesized by adding sodium citrate as a reducing agent to a highly diluted aqueous gold chloride solution. 7The nucleation process in the presence of sodium citrate was than further rened by Frens, as a bottom up strategy to build up monodisperse spherical NPs of different sizes. 8ecently, it was shown that asymmetric shaped NPs are formed by using special reduction agents, like lemongrass, 9 or tryptophan. 10Other strategies towards the development of anisotropic nanoparticles include templates, 11 such as dendrimers, 12 microemulsions, 13,14 or polyelectrolyte micelles. 15urrent seeding-mediated synthesis methods are very efficient in controlling the size and shape of anisotropic gold nanoparticles and, especially, those of nanorods.Over a decade ago, Jana et al. had already proposed a seed-mediated, surfactant-assisted two-step approach involving the use of the cationic surfactant, CTAB. 16The seed-mediated synthesis, in presence of CTAB, is very sensitive to halide ions, 10,17,18 meaning that nanorods are only formed in absence of iodide ions.Furthermore, the shape of gold crystals can be dramatically changed upon addition of trace amounts of iodide ions, resulting in the formation of gold nanoprisms. 19Mirkin and coworkers studied the growth of nanoparticles on CTAC-capped seeds containing iodide ions in the absence or presence of silver cations and reported that the anisotropic growth only occurs in absence of silver cations. 20Kinetically controlled reaction paths are dependent on the addition rate of ascorbic acid.Hong et al. demonstrated that nanoplates are predominantly formed in presence of iodide ions at the lowest addition rate of the mild reducing agent ascorbic acid. 21evertheless, an efficient synthetic method to produce exclusively monodisperse anisotropic gold nanoparticles of dened shape is still lacking.As a result, different attempts have been made to separate anisotropic NPs from spherical ones.However, a separation of the anisotropic particles from the spherical ones is not trivial.The most attractive separation approach was initially developed by Park et al. through the application of a method involving depletion-induced shape and size selection of gold nanoparticles. 22These authors were able to show that rod-like NPs preferential aggregate and sediment in the presence of numerous micelles.Scarabelli et al. used this method very successfully to separate and purify gold nanotriangles (NTs) in presence of CTAC micelles. 235][26] Applying a strongly alternating polyampholyte, i.e., PalPhBisCarb, and through the variation of the reaction conditions and composition of the template phase, more than 60% of the AuNPs were found to be anisotropic gold nanoplatelets.
The mechanism by which the one-step process leads to the formation of nanotriangles on the surface of mixed vesicles remains unknown.Therefore, the present work is focused, on the one hand, on the growth mechanism of triangular gold nanoplatelets on the surface of vesicles in presence of different reducing agents, i.e., polyelectrolytes and low molecular reducing agents.On the other hand, the depletion occulation process of the nanotriangles was investigated through the addition of the anionic surfactant AOT in excess.As a result of our research, puried negatively charged nanoplatelets with a yield of 99.8% aer several purication steps and a polydispersity of 10% were obtained, which can be used for SERS measurements or as a nano-platform for the construction of superstructures, for instance, with oppositely charged spherical nanoparticles.

Characterization methods
UV-vis absorption measurements were carried out with an Agilent 8453 UV-vis-NIR spectrometer covering the wavelength range 200 to 1100 nm.The shape and size of the gold nanoparticles were determined by transmission electron microscopy (TEM).Samples were dropped on carbon-coated copper grids.Upon air drying (using a suspension preparation procedure), the samples were examined using either one of two TEMs, mainly with a JEOL JEM-1011 at an acceleration voltage of 80 kV, or with a JEOL JEM-2200 FS to obtain higher resolution TEM (HRTEM) images, at an acceleration voltage of 200 kV.For the statistical evaluation (yield and polydispersity) of each sample more than 600 particles were analysed.The polydispersity was determined regarding the average edge length.The characterization of the thickness of the gold nanotriangles was performed on PicoScan-AFM from Agilent.In order to obtain the zeta potential, the Malvern Nano Zetasizer 3600, based on the principle of electrophoretic light scattering, was employed.In addition to TEM, a Hitachi S-4800 scanning electron microscope (SEM) was used to analyze the coating layer of the silica wafer for the SERS measurements.Raman spectra were recorded using a confocal Raman Microscope alpha300 from WITec equipped with laser excitation at wavelength of 785 nm.The laser beam was focused through a Nikon 20Â microscope objective with a NA of 0.4.The spectra were acquired with a thermoelectrically cooled Andor CCD detector DU401A-BV placed behind the spectrometer UHTS 300 from WITec with a spectral resolution of 3 cm À1 .The Raman band of a silicon wafer at 520 cm À1 was used to calibrate the spectrometer.

Synthesis of nanotriangles (NTs)
PL90G (0.5 wt%) and AOT (0.5 wt%) in addition to one of the applied added agents (0.01 wt%) were dispersed together in water and stirred for 24 hours at room temperature.The resulting turbid vesicle dispersion was mixed with the freshly prepared aqueous 2 mM tetrachloroaurate precursor solution.The mixtures were stirred at 45 C for 45 minutes resulting in a purple colored clear dispersion.

Purication of the NTs by depletion occulation
As a result of the diversity in shape and size, a separation method was considered necessary in order to obtain pure NTs.Such a separation procedure can be achieved through the use of depletion occulation, in the presence of polymers or micelles.The separation effect is dependent on the amount of surfactant used for the vesicle phase and the reducing agent.Once separation of triangular gold nanoparticles from the spherical ones had occurred, the dispersion was washed by centrifugation.
The depletion occulation was performed in presence of AOT micelles.The results show that only a combined polyelectrolyte/micelle depletion occulation process leads to a complete separation of the triangles.The optimum concentration of the added AOT solution was found at 0.02 M.

Preparation of SERS substrate
For the immobilization of the nanotriangles on the substrate, a silicon wafer was puried in a solution of 30 wt% H 2 O 2 and 30 wt% H 2 SO 4 for 1 h.It was rinsed in deionized water and dried before being used.The wafer was immersed in an aqueous solution of 2% 3-MPTMS ethanol for 6 h, rinsed in ethanol and dried.100 ml of the gold nanotriangles dispersion were assembled on the wafer surface and the unattached nanotriangles were removed by washing the surface with deionized water.The gold nanotriangle-coated silicon wafer was immersed in 10 mM solution of 4-NTP in ethanol overnight.The wafer was washed with ethanol and deionized water before being measured.

Template phase characterization
A turbid dispersion of negatively charged vesicles was generated under the described conditions.Dynamic light scattering (DLS) experiments of this dispersion showed that there was a very broad particle size distribution (ranging from 50 nm to 5000 nm).The intensity plot revealed a maximum at 450 nm and the number plot had a maximum at around 50 nm (Fig. 1A).The corresponding cryo-SEM micrographs (Fig. 1B) highlighted the presence small unilamellar vesicles (SUV) of about 50 nm in size, which is in good agreement with the number plot obtained from the DLS data, in addition to the signicantly larger giant unilamellar vesicles (GUV).A more detailed inspection of the interior of the GUVs by Cryo-SEM (see Fig. 1B) suggests that these are in fact multivesicular vesicles (MVV).Note, that these GUVs, with diameters up to 50 mm, can be visualized by light microscopy, as shown in Fig. 1C.
This means that the template phase is a polydisperse vesicle phase containing SUVs that are partly incorporated in GUVs.

One-step gold nanotriangle formation process
By using the mixed vesicle template phase, in the absence of any additional reducing components, an increase in the UV absorption at higher wavelengths around the peak maximum of 520 nm was observed.The plasmon resonance frequency and, thereby, the position of the localized surface plasmon resonance (LSPR) band is appreciably inuenced by the size, shape, dielectric constant of the metal particle and its surroundings.A characteristic plasmon absorption band at about 525 nm can be assigned to spherical gold nanoparticles with a particle diameter between 5 and 40 nm, 16 whereas anisotropic nanoparticles like nanorods exhibit two absorption peaks, mainly the transverse plasmon band at 520 nm and the longitudinal band between 700 and 1300 nm, depending on the aspect ratio. 28symmetric gold nanoplatelets also absorb in this region, that is between 650 and 1300 nm. 9,10,29pplying this knowledge to the UV-vis spectrum shown in Fig. 2, suggests that the formation of anisotropic gold nanoparticles, as well as spherical isotropic gold nanoparticles, has occurred.To get a more detailed understanding about the shape of the nanoparticles, TEM experiments were carried out.In addition to spherical particles of around 20 nm in size, nanotriangles with edge lengths varying between 20 nm and 200 nm were visualized (Fig. 2).
When template phases were generated in the presence of macromolecular components, quite different effects were observed.
In case of the polycation PDADMAC, only spherical isotropic gold nanoparticles were formed.This conclusion, based on the TEM micrographs (Fig. S1 †) is in good agreement with the corresponding UV-vis spectrum, which showed that there was only a broad peak maximum that was shied to lower wavelengths (Fig. 3).
By adding the polyanions PAA and AlgA, nanotriangles were formed in addition to spherical nanoparticles.The TEM micrographs as well as the UV-vis spectra indicate an enhancing effect with regard to the formation of NTs.Table 1 shows that the edge length of the NTs increases and the polydispersity decreases, in the same order of magnitude.
The use of PalPhBisCarb leads to NTs with an edge length of about 60 nm, representing 33% of all particles, with a slightly lower polydispersity of 33% and a corresponding UV-vis maximum at 950 nm.This can be explained by the fact that PalPhBisCarb behaves like a polyanion at pH 9.
Following experiments in presence of low molecular reducing components containing carboxylic moieties were performed under the same conditions as mentioned before.Table 2 shows the TEM analysis results concerning the triangular structures.The associated TEM micrographs could be This journal is © The Royal Society of Chemistry 2016 found in the ESI (Fig. S2 †).Experiments by adding ascorbic acid show that 24% of all particles are triangles permitting a shoulder at 630 nm in the UV-vis spectrum.By replacing ascorbic acid with sodium citrate, larger NTs of about 42 nm in size with a corresponding UV-vis peak at about 720 nm can be observed (Fig. 4).The use of L-lactic acid resulted in the UV-vis maximum being shied to 820 nm accompanied by the formation of triangles with an average edge length of 68 nm.Under these conditions, deformed nanoplatelets were also generated.In the presence of lemon grass oil, the individual NTs are more uniform with more dened sharp corners resulting in an absorption maximum at 900 nm.
In conclusion, the reducing agents used here show both size and shape-dependent effects.The addition of polyanions, as reducing agents, lead to an increase of nanotriangles, whereas low molecular weight compounds decrease the amount of NTs (Table 2).

Mechanism the NT formation process
To achieve a better understanding of the NT formation process, some additional time-dependent experiments were performed in presence of PalPhBisCarb.Time-dependent UV-vis absorption spectra (Fig. 5) show once a growing peak at 520 nm, which can be directly correlated to the formation of spherical gold nanoparticles, and an appearing peak at 970 nm, which can be attributed to the formation of anisotropic particles.This peak increased over time and shied towards the resulting absorption maximum at 1050 nm.As a result, the UV short wavelength at 520 nm, as well as the long wavelength absorption at about 1000 nm increased simultaneously with time.Thus, the formation of anisotropic nanotriangles starts isochronously with the formation of isotropic spherical nanoparticles.
Additional experiments performed by using light microscopy show a color change to red and green at the same time scale with the rising UV-vis absorption peak.These color effects were detected inside of the giant multivesicular vesicles, where the local concentration of AOT/phospholipon bilayer structures is at its highest.
From a mechanistic point of view one can conclude that one part of the critical nuclei started to grow up until the spherical particles reached a size of about 20 nm in a "classical" nucleation process according to the La Mer diagram.Another part of the critical nuclei grow up until anisotropic NTs were formed   due to the symmetry-breaking effects of the template phase, which was inuenced by the inner network structure inside of the multivesicular giant vesicles.Recently, we have shown that PalPhBisCarb can transform a mixed AOT vesicle phase into a tubular network structure, 30 which is a preferred weak template phase for making nanotriangles.Therefore, one can assume that the nanotriangles are predominantly formed at the junction points inside of the giant multivesicular vesicles. 25nisotropic crystals can only be formed when the cubic lattice symmetry is interrupted, e.g., by stacking faults.Crystal nuclei with stacking faults can grow to form thin plates with {111} facets on the upper and lower side, e.g., by adding ligands with the affinity for {111} faces.Such ligands can be surfactants as well as polymers.For example alkylated poly(ethyleneimines) and the cationic surfactant CTAB adsorb selective on gold or silver {111} facets. 31,32he growth process of the nanoparticles leads to a decomposition of the giant vesicles and the system becomes optically clear.Attempts to suppress the formation of spherical particles in such a one-step process failed.Therefore, a procedure to separate the anisotropic NTs from the isotropic spherical particles was required.

Gold nanotriangle separation process
Recently, Scarabelli et al. showed that the initial shape-yield of 50% nanotriangles, produced by a seed-mediated synthesis, can be elevated to 95% by a depletion induced separation process in presence of an excess of CTAC micelles. 23Related experiments with CTAC showed the principal suitability of this procedure to separate our NTs.However, a quantitative and qualitative separation with CTAC micelles was unsuccessful.A reason for that could be the opposite charge of the NTs and CTAC micelles.Therefore, we used in the following experiments an AOT-based depletion occulation process for our NTs.
To verify the quantity and quality of the shape controlled separation process in presence of AOT micelles in excess, TEM analysis and UV-Vis spectroscopy were performed.In the absence of a reducing agent (mixed vesicle template phase without additional components) only an incomplete separation process (NT yield < 50%) was observed.In presence of the low molecular reducing components, the separation procedure proved unsuccessful, too.That means the weak reducing low molecular weight compounds with carboxylic groups do not improve the AOT micelle based depletion occulation process.Much better results were obtained upon addition of the polyelectrolytes PAA, AlgA, and PalPhBisCarb.The TEM micrographs (Fig. 6) demonstrated that the efficiency of the separation process in the presence of the polyelectrolytes was related to an enforced depletion occulation.In that case negatively charged NTs with a zeta potential of about À75 mV and non-adsorbing negatively charged polyelectrolytes were combined.Consequently, the AOT micelle-based depletion occulation process is enhanced by a polymer-based depletion occulation leading to a quantitative separation of nanoplatelets (NPLs) in the presence of PalPhBisCarb (>99%) and AlgA (>99%), and an almost complete separation in the presence of PAA (96%), taking into account that the amount of well dened nanotriangles with pronounced corners is lower (Table 3).In addition to the NTs, the dispersion contains hexagonal structures, which are incompletely crystallized triangles in different growth phases.UV-vis measurements conrm the TEM analysis, meaning that the peak at around 525 nm, which is usually caused by spherical particles, disappears aer three purication (washing and centrifugation) steps (compare Fig. S3 †).Compared to the TEM analysis before the separation procedure, the average size of the NTs is increased drastically.This implies that the polyelectrolyte/micelle depletion occulation affect only the fraction of larger sized NTs.
The shape-controlled depletion occulation process showed only efficient results in presence of macromolecules, and seemed to be unaffected by AOT micelles alone.Hence, the depletion occulation of larger NTs is mainly driven by the polymer, which can act as a reducing agent and is displaced on the particle surface by AOT.By investigating the inuence of the  In summary, our results show that a combined polyelectrolyte/ micelle depletion occulation process is a sensitive and efficient procedure for the separation of larger anisotropic at nanoplatelets.The highest amount of triangles can be observed in presence of PalPhBisCarb.Surprisingly, the edge length of the isolated NTs (listed in Table 3) is signicantly larger in comparison to the initial ones (compare Table 1).Recently, Zhang et al. have shown that the micelle depletion occulation strongly depends on the type of micelles. 29Therefore, a stepwise occulation with CTAB and CTAC micelles leads to a size-dependent separation of NTs.Hence, our combined polyelectrolyte/micelle depletion occulation is even a size-selective process, yielding only the fraction of larger NTs.

Gold nanotriangle characterization
For a more comprehensive characterization, the resulting green colored dispersions were examined by means of AFM and HRTEM.
The AFM measurement in Fig. 7 shows that the nanotriangles were indeed at nanoplatelets with a thickness of 10 nm.HRTEM micrographs (compare Fig. 8) conrmed the crystalline nature of the at nanoplatelets with the top and bottom surfaces being composed of {111} facets.The triangles were single crystalline face-centered cubic (fcc) structured and as observed from the fast Fourier transform (FFT) (see inset in the HRTEM micrograph) orientated in the [111] direction.The forbidden 1/3 {422} reections indicates the presence of twin planes parallel to the (111) surface and perpendicular to the electron beam. 33Thus, the symmetry-braking effects of the AOT/ phospholipon bilayer was enhanced in presence of PalPhBis-Carb due to the adsorption of the amphoteric polymer on the  {111} facets.The special performance of PaPhBisCarb in relation to the polyanions PAA and AlgA can be related to the cationic functional groups of the polyampholyte.The quaternary N-functions may play a similar role to that of the head group of the alkylated PEI samples in the selective adsorption of gold facets. 31

SERS performance
The SERS effectiveness of the gold nanotriangles synthesized with PalPhBisCarb was studied using 4-nitrothiophenol as a probe, which can be easily chemisorbed on the gold nanoparticles by S-Au bond.First experiments with a highly diluted sample show no signicant effect.Therefore, the gold nanotriangle concentration was increased resulting in a completely covered SERS substrate (compare SEM micrograph in Fig. 9).Furthermore, Fig. 9 shows the comparison between Raman spectra of the solid 4-NTP molecules and the adsorbed 4-NTP molecules on SERS substrate.The enhancement of Raman scattering can be directly noticed from the spectra.The Raman spectrum of 4-NTP showed the characteristic symmetric stretching of the NO 2 group at 1327 cm À1 and the C]C stretching of the phenyl ring at 1568 cm À1 .
The SERS enhancement factor of the 4-NTP on the nanotriangles-fabricated SERS substrate was estimated using the equation: where I SERS and I bulk are the Raman intensity at a certain vibration for the 4-NTP molecules adsorbed on the SERS substrate and solid 4-NTP molecules, respectively.The strongest NO 2 vibration at 1327 cm À1 was chosen to calculate the SERS enhancement factor.N ads and N bulk are the number of the probed adsorbed and solid 4-NTP molecules, respectively. 34o calculate N ads , formation of a monolayer of 4-NTP on the SERS substrate was assumed. 35Knowing the area occupied by an adsorbed 4-NTP molecule and the laser spot size, the N ads was calculated to be 5.65 Â 10 6 .For N bulk , the irradiated volume was considered as a cylinder with a waist diameter of $1.2 mm and a depth of $11.5 mm.So N bulk was calculated to be 1.39 Â 10 11 .For the vibration at 1327 cm À1 , the SERS enhancement factor was estimated to be $2.76 Â 10 4 .We have repeated the measurements on three different batches of the nanotriangles and nearly the same signal enhancement was obtained.Control experiments performed at lower nanoparticle density show a lower signal enhancement.
The high SERS enhancement on the gold nanotrianglesfabricated substrate is mainly attributed to the junctions between the particles and the sharp edges of the nanotriangles, which they have been widely studied as "SERS hot spots", where the local electromagnetic eld and therefore the Raman scattering is enhanced. 36

Conclusions
A one-step synthesis of gold nanotriangles in a mixed AOT/ phospholipon vesicle phase in the presence of different reducing components with size-dependent properties was developed.
This NT formation process can be suppressed by polycations and enhanced by polyanions.The initiation of the growing process, that leads to the formation of at nanotriangles, starts at the bilayer interface facing the inside of giant multivesicular vesicles.In the case of the non-adsorbing polyanions and AOT micelles, a combined polyelectrolyte/micelle depletion occulation process was used for the complete removal of the byproducts.The green colored, highly puried and long time stable (>3 month) dispersions of 10 nm thin nanotriangles used  This article is licensed under a Creative Commons Attribution 3.0 Unported Licence.

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for SERS measurements show an enhancement factor of 2.76 Â 10 4 for the detection of 4-NTP, and will be served as a basis for building blocks for supramolecular stacking structures.

Fig. 1
Fig. 1 Analysis of the turbid vesicle template phase via DLS intensity and number plot (left), the corresponding Cryo-SEM micrograph (center), and the image of light microscopy (right).

Fig. 2
Fig. 2 TEM micrograph (left) and corresponding UV-vis spectrum (right) of the gold nanoparticles formed in the template phase without further reducing components.

Fig. 3
Fig. 3 UV-vis absorption spectra of the gold nanoparticles formed in the template phase in presence of PalPhBisCarb (A), PAA (B), AlgA (C) and PDADMAC (D).

Fig. 5
Fig. 5 Time-dependent UV-vis measurements during gold nanoparticle synthesis in the presence of PalPhBisCarb.

Fig. 4
Fig.4UV-vis absorption spectra of the gold nanoparticles formed in the template phase in presence of L-ascorbic acid (E), sodium citrate (F), L-lactic acid (G) and lemon grass oil (H).

Fig. 6
Fig. 6 TEM micrographs of the gold nanoparticles in presence of a PalPhBisCarb (A), PAA (B) and AlgA (C).

Fig. 8
Fig. 8 Analysis of the triangle crystallinity by HRTEM; the inset (right) shows the fast Fourier transform (FFT) of the single crystal with the sixfold symmetry of the fcc lattice of gold.

Fig. 9
Fig. 9 Raman spectrum of 4-nitrothiophenol in absence and presence of gold nanotriangles (left), SEM micrograph of the according substrate (right).

Table 1
TEM analysis of gold nanotriangles

Table 2
TEM analysis of gold nanotriangles

Table 3
TEM analysis of anisotropic gold nanoplatelets (NPLs) and nanotriangles (NTs) Fig.7Analysis of the triangle thickness by AFM.