We report on the improvement of the infrared optical trapping efficiency of dielectric microspheres by the controlled adhesion of gold nanorods to their surface. When trapping wavelength was equal to the surface plasmon resonance wavelength of the gold nanorods (808 nm), a 7 times improvement in the optical force acting on the microspheres was obtained. Such a gold nanorod assisted enhancement of the optical trapping efficiency enabled the intracellular manipulation of the decorated dielectric microsphere by using a low power (22 mW) infrared optical trap.
© 2014 Optical Society of America
Manipulation and trapping of nano-objects are of great interest in biology, biotechnology and medicine. The trapping and manipulation of individual cells or particles in suspension with submicrometer, or nanometer, accuracy is an essential requirement in single cell studies, high resolution single particle sensing and, also, in the emerging field of single cell proteomics. There are several methods for efficient manipulation of single cells or microparticles, including ultrasonic trapping, atomic force microscopy (AFM) and optical trapping (OT) [1–6]. Within this group of methods, OT is of particular interest since it allows both force measurement and particle manipulation in absence of physical contact thus minimizing any perturbations to the cell or particle under investigation . OT is the common term used to describe trapping of individual particles at the focus of a single tightly focused beam due to the simultaneous presence of scattering and gradient forces. OT was first demonstrated by Ashkin et al. [4, 7] who trapped water dispersed dielectric nano and micro spheres by using a single 514 nm focused laser beam. Ashkin and associates also proposed and demonstrated the potential use of optical traps for biological applications [4, 7, 8]. Since these pioneering studies, various biological objects, including viruses, bacteria, and single cells have been trapped successfully, using the single-beam configuration and a great variety of laser sources (with different trapping wavelength, λ) [9, 10]. From these studies, it was determined that near infrared (700 nm < λ < 1070nm) laser traps had the lowest detrimental effect on cell viability, compared to using lasers sources in the visible region . This feature of near-infrared optical traps has been explained in the past in terms of the reduction in the laser-induced thermal loading (water does not absorb to a significant extent in the 700-980 nm range) and also by the fact that certain wavelengths in the infrared do not promote harmful intracellular reactions [11–14]. Recent results have concluded that almost damage-free single cell manipulation can be achieved by using trapping wavelengths close to 800 nm . This opens the door for damage-free intracellular manipulation of sensing microparticles for intracellular dynamical studies. Among the different sensing microparticles, microspheres (µ-Sp) are of special relevance as they have been demonstrated to be capable of a wide range of both chemical and thermal sensing with very high accuracy [15–19]. Whilst manipulation and trapping in standard liquid environment is straightforward, intracellular manipulation of a dielectric, transparent µ-Sp by OT is far more challenging, due to the inhomogeneity and large viscosity of the intracellular medium. Intracellular optical manipulation would be only possible provided that the optical force would overcome the drag force acting on the particle due to the medium viscosity. Therefore, the larger intracellular viscosity would lead to large drag forces in such a way that the optical forces required for intracellular manipulation to be larger than those required in a low viscosity medium. The principle relationship between optical trapping force and power acting on a µ-Sp is given by ;3, 20–22]. For typical values of n1 (1.33), P (tens of mW), and Q (ranging from 10−2 to 10−3, for silica microspheres) [23, 24], F is usually of the order of few hundreds of fN. The viscosity of the intracellular medium has been estimated to be as large as 137.05 Pa·s for 1µm sized bead Murine Macrophage . Note that intracellular optical manipulation would be only possible provided that the optical force would overcome the drag force acting on the particle due to the medium viscosity. Therefore, the larger intracellular viscosity particle manipulation would make the optical forces required for intracellular manipulation to be larger than those required in a low viscosity medium at moderate/low laser powers [23, 26]. To increase trapping forces, one can potentially increase P, n1 or Q. The refractive index of the surrounding medium n1, in experiments dealing with intracellular manipulation, is that of the cytoplasm and, generally, it cannot be modified without adversely affecting the cell. Increasing the trapping power may be impractical since it may lead to absorption by the medium, causing significant heating and subsequent thermal damage . In addition, when dealing with intracellular optical manipulation the laser power should also be maintained below the membrane ablation threshold in order to avoid undesired photoporation and thermal loading . The last term, Q, has the greatest influence on the performance of the trap and determines whether the µ-Sp is trapped or not. The resultant efficiency is determined by a range of diverse parameters such as the numerical aperture of the optics, spot size, wavelength, polarization, aberrations and beam profile, which have an effect on the optical force. The efficiency parameter, Q, also takes into account the optical properties of the µ-Sp, and the size, shape, surface reflectivity and relative refractive index with respect to the surrounding medium. In the case of a silica microsphere (diameter 1 µm) the value Q is close to 0.006 . Intracellular manipulation requires the challenging task of achieving an appropriate enhancement of this parameter.
A possible approach to increase OT forces on silica microspheres is to combine them with metallic nanoparticles. Under appropriate optical excitation, metal nanoparticles display resonances, the so-termed Surface Plasmonic Resonances (SPRs). These correspond to the collective motion of surface charges. Such a collective motion is only induced for certain excitations wavelengths, denoted by λSPR, whose spectral location depends on the size and shape of the metallic nanoparticle . When a metallic nanoparticle is optically excited at λSPR, a strong field enhancement is produced, accompanied by the appearance of a large electronic polarizability and a marked variation in the refractive index of nanoparticle [28–30]. These effects make the trapping efficiency of a Rayleigh metallic particle (diameter d << λ) to be 7 fold larger than a similarly sized dielectric particle [31, 32]. Metallic nanoparticles are not only interesting because of their high optical trapping efficiency but also because they can be used to increase optical forces acting on dielectric µ-Sp when attached to their surface [33, 34]. The optical properties of such dielectric-metallic hybrid systems have been successfully described in the past by the so-called effective medium approach . This approach considers the system as a spherical silica core surrounded by an homogeneous shell with a dielectric constant that results from the combination of the dielectric constants of the medium and the metallic nanoparticles (Fig. 1(a) shows a schematic representation) . Based on this approach Burgin et al. demonstrated that the adhesion of gold nanodots to silica microspheres resulted in a relevant enhancement in the optical forces (scattering forces) acting on these hybrid particles when moving in a fluid in presence of a counter-propagating visible laser beam . Among the different metal nanoparticles, gold nanorods (GNRs) have received much attention due to their extended photonic and biological applications based on their opto-plasmonic properties [31, 36]. Currently, GNRs are considered as fundamental building blocks in modern biophotonics with applications in bioimaging and therapies . The λSPR of GNRs (associated to longitudinal electronic resonances) can be tuned into the near-infrared (700-1100 nm) by controlling their aspect ratio and length . A few publications are available which report on stable and efficient OT of GNR by using moderate/low trapping powers showing outstanding optical, chemical and mechanical stabilities . Despite these unique and noteworthy properties, the use of GNRs for the improvement of the infrared optical trapping of µ-Sp has, up to now, not being reported.
In this work, we demonstrate intracellular infrared optical manipulation of silica microspheres using GNRs as co-adjuvant nanoparticles. We have designed and synthetized a hybrid structure consisting of GNRs bonded to the modified surface of the silica µ-Sp. The optical trapping forces acting on these µ-Sp + GNR constructs were estimated by the hydrodynamic drag method at different trapping laser wavelengths. We established that if the optical trapping wavelength matches the λSPR of the GNRs, the trapping efficiency increase by approximately one order of magnitude. This plasmonic enhancement in the trapping efficiency is then used for optical intracellular manipulation of the silica µ-Sp employing moderate infrared (808 nm) laser powers (22 mW).
For the preparation of the µ-Sp + GNR constructs we used commercial silica microspheres, 1.5 µm in diameter, obtained from Microspheres-Nanospheres (Corpuscular Inc, product code C-SIO-G1.5). The Gold Nanorods (GNRs) conjugated with Polyacrylic acid (PAA) were obtained from NanopartzTM (AR12-10-808-PAA-50). We used GNR with a mean radius and length of 12 and 47 nm, respectively, showing their plasmon resonance close to 800 nm (Fig. 2(b)). The carboxylated-GNRs stock solution had a concentration of 1.21 nM; (equivalent to 7.24 x 10 11 GNP/mL).
1) Surface modification of silica microspheres with an amino functional trialkoxysilanes, the 3-aminopropyl trimethoxysilane (APTMS):
6 mg of dried SiO2 microspheres (Corpuscular, silicon oxide microspheres, Plain green Fluorescence, d = 1.5 µm) and 0.6 mL of APTMS were added in 10 mL of ethanol. After 12 h of stirring at room temperature in absence of light, the modified silica microspheres (SiO2-APTMS) were purified by washing with ethanol and isolated by centrifugation at 45,000 rpm for 3 times. The excess of APTMS was removed from the supernatants and the purified SiO2-APTMS were recuperated by centrifugation. The silanized microspheres were obtained as a pellet and redispersed in water.
2) Activation of carboxylate-groups of gold nanorods through a carbodiimide-mediated process using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), in a two-step reaction that employs the addition of N-hydroxysuccinimide (NHS) to create an intermediate ester-GNRs:
0.2 mL of the carboxylated-GNRs were suspended in 2mL of coupling buffer (50 mM MES pH 6.0). To this solution 12 mg of NHS and 12 mg of EDC were added. After 24 h of stirring, the esters-GNRs were purified by centrifugation/ redispersion in MES buffer at 45,000 rpm for 3 times. The intermediate ester-GNRs were dispersed in 1 mL MES buffer and used in the next step.
3) Reaction of the amine groups of silica microspheres with the intermediated ester-GNRs:
1mg of SiO2-APTMS microspheres was stirred with 1mL of ester-GNRs. After 2 days of mixing at room temperature in absence of light, the modified silica microspheres SiO2-microspheres@GNRs were purified by centrifugation/redispersion in water for 3 times and in acetone for 2 times at 5,000 rpm. After this process approximately 10% of the surface of silica microspheres was covered with GNRs (evaluated by fluorescence measurements).
Transmission Electron Microscopy characterization
HRTEM was performed on a JEOL JEM-2011 microscope operating at 200 kV. The sample was prepared by dropping sample solutions (1 mg/mL in water) onto a 300-mesh carbon coated copper grid (3 mm in diameter) followed by the evaporation of the solvent. From the TEM measurements the size of the SiO2 microspheres was estimated to be around 1.9 microns.
To carry out the optical trapping experiment, the microspheres were dispersed in distilled water (0.01% in weight) and sonicated for 10 min in order to break possible agglomerates between microspheres. The microspheres dispersion was placed in a 5 mm width, 100 µm height microchannel (Ibidi Inc., µ-Slide I, catalog number: 80106) and excited by using a single-mode fiber-coupler laser diode (at 808 nm and 980 nm). The laser excitation was collimated by a fiber port (Thorlabs PAF-X-7-B), expanded by using a 2X beam expander (Thorlabs BE02M), giving a beam diameter of 2 mm, which matches the back aperture of the microscope objective used. To focus the trapping beam into the microchannel, a 50x long working distance microscope objective with NA = 0.55 was used and the optical images were recorded using a CCD camera.
For thermal imaging experiments, the CdSe quantum dots(QDs) (Cat. no. Q21721MP, Invitrogen Inc) were added to the solution resulting in a final QD concentration as low as 1 × 109 cm−3. The CdSe-QDs used in this work had 14 nm length and 6 nm width leading to a broadband luminescence centered at around 655 nm. The luminescence peak wavelength shifts linearly towards longer wavelengths with temperature at a rate of 0.1 nm/K. The spatial variation of temperature can be then obtained from the spatial variation of the luminescence wavelength by using a confocal microscope that uses a 488 nm as an excitation source. Although a detailed description of the measuring procedure can be found in Reference , briefly it consists on the systematic scanning of the low power 488 nm excitation beam and point-by-point collection of CdSe-QD luminescence. The subsequent spectral analysis of the CdSe-QD luminescence is used to calculate the spatial variation of temperature and, hence, to obtain the thermal image. The aqueous solution containing both the CdSe-QDs and microspheres displayed a very stable colloidal behavior without any evidence of precipitation over several months.
The RAW 264.7 murine macrophage; Abelson murine leukemia virus transformed was grown in Dulbecco’s Modified Eagle’s Medium (DMEM, Gibco, Paisley, Scotland, UK) supplemented with fetal calf serum (FCS 10%, Gibco), and 0.5% of antibiotics (penicillin G [10000 U/mL] and streptomycin sulphate [10000 μg/mL] (Gibco)). Cells were grown in a HERAcell incubator (Heraeus, Kendro, Germany) with a 5% CO2 atmosphere, a 95% relative humidity and a constant temperature of 37 °C. For the cytotoxicity experiments cells were plated on 24-wells plates and for fluorescence experiments, cells were plated onto coverslips placed into 6-wells plates.
In vitro cell viability/cytotoxicity studies
The MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay is a simple non-radioactive colorimetric assay to measure cell cytotoxicity, proliferation or viability. MTT is a yellow, watersoluble, tetrazolium salt. Metabolically active cells are able to convert this dye into a water-insoluble dark blue formazan by reductive cleavage of the tetrazolium ring . Formazan crystals, then, can be dissolved in an organic solvent such as dimethylsulphoxide (DMSO) and quantified by measuring the absorbance of the solution at 540 nm, and the resultant value is related to the number of living cells. In this way, to determine the cytotoxicity of hybrid µ-Sp + GNR particles/viability of the cells treated with the particles, the cells were plated in a 24 well plate at 37 °C in 5% CO2 atmosphere. After 48 h of culturing, the medium in the well was replaced with the fresh medium containing the microspheres decorated with GNRs at different concentrations and cells were incubated for different periods of time. After incubation, the medium with µ-Sp + GNRs was removed and replaced by complete medium without microspheres. After 24 h, 0,5 ml of 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium-bromide (MTT) dye solution (0.05 mg/ml of MTT, Sigma) was added to each well. After 2-3 h of incubation at 37 °C and 5% CO2, the medium was removed and formazan crystals were solubilized with 0,5 ml of DMSO and the solution was vigorously mixed to dissolve the reacted dye. The absorbance of each well was read at 540 nm on a spectra fluor4 (TECAN) microplate reader. The spectrophotometer was calibrated to zero absorbance-using culture medium without cells. The relative cell viability (%) related to control wells containing cell culture in medium without nanoparticles was calculated by [A] test/[A] control x 100, where [A] test is the absorbance of the test sample and [A] control is the absorbance of control sample.
3. Results and discussion
The µ-Sp + GNR constructs used in this work were synthetized using commercially available GNRs and silica microspheres. GNRs were linked to the µ-Sp using a three step process described in detail in the experimental section and shown schematically in Fig. 1(b). Figure 1(c) shows the TEM images of the synthetized µ-Sp + GNR construct, showing the presence of the GNRs at the surface of the microsphere while preserving their original spherical morphology. The extinction cross section of the GNRs bonded to the µ-Sp surface is shown in Fig. 2(b) revealing a λSPR close to 800 nm. The synthesized µ-Sp + GNR construct was found to be highly stable, showing excellent colloidal properties. No evidence of precipitation was observed over a period of several months of the µ-Sp + GNR dispersed in a phosphate buffer solution (PBS).
Following the successful synthesis of µ-Sp + GNR construct, we studied the improvement in the trapping efficiency due to the presence of GNR at the particle surface. We firstly conducted single beam OT trapping experiments on a diluted colloidal suspension of µ-Sp + GNR constructs (0.01% in mass). Details of the simple OT trapping set-up used in this work can be found in the experimental section. We determine the lateral trapping force acting on the µ-Sp + GNR, using the so-called hydrodynamic-drag method. This consists on the creation of a controlled fluid flow around the trapped µ-Sp + GNR and then measuring the fluid speed needed to remove the µ-Sp + GNR from the optical trap. This occurs when the drag force (Fdrag) created by the flow rate exceeds the optical trapping force (Ftrap) at a specific laser power. This condition allows us to estimate the optical trapping force from the experimentally determined critical velocity using the following equation:Fig. 2(a) that shows the power dependence of the OT force using an 808 nm laser obtained for the µ-Sp + GNR construct and for the unmodified µ-Sp. As seen in Fig. 2(a) the optical trapping force is found to increase linearly with the 808 nm excitation power. These experimental data were fitted using Eq. (1) and the values of the trapping force constant, kSp + GNR808mn, and the trapping efficiency factor, QSp + GNR808mn, were found to be equal to 88.8 pN⋅µm−1⋅W−1 and 0.017, respectively for the µ-Sp + GNR construct. For µ-Sp the values of kSp808mn = 9 pN⋅µm−1⋅W−1 and QSp808mn = 0.0018 were obtained respectively. At this point it should be noted that the experimentally obtained trapping efficiency factor for non-modified µ-Sp is in good agreement with previously reported results . Thus, we may conclude that the optical excitation of the SPR of GNRs has caused a significant improvement in the optical trapping efficiency. In order to unequivocally correlate this enhancement with the plasmonic excitation of GNRs, optical trapping experiments on µ-Sp + GNR construct were also performed using a 980 nm trapping laser beam. As shown in Fig. 2(b), at 980 nm the plasmonic extinction cross section is much lower than that at 808 nm (i.e. at the surface plasmon resonance wavelength). As a consequence, if the OT force enhancement is caused by the plasmonic excitation of GNRs, then the OT forces exerted by 980 nm radiation on the µ-Sp + GNR construct should be significantly reduced in respect to those obtained at 808 nm. This has been experimentally corroborated (see Fig. 2(a)). Again a linear relation between 980 nm laser power and the OT force over µ-Sp + GNR construct has been obtained from which we have determined the 980 nm trapping force constant and efficiency factor to be kSp + GNR980mn ≅ 12 pN⋅µm−1⋅W−1 and QSp + GNR980mn ≈0.0023, respectively. These values were found to be only slightly larger than those corresponding to the µ-Sps. Thus, we conclude that when trapping wavelength does not match the plasmonic excitation peak of GNRs, the OT efficiency enhancement due to the pseudo-continuous metallic shell is negligible. This can be explained as follows: when a GNR is optically excited close to its SPR, relevant changes and singularities appear on both the real and imaginary parts of their dielectric constant . The maximum modification of the dielectric constant of the effective homogeneous shell constituted by GNRs and medium is expected to occur under optical excitation at the plasmon resonance wavelength. In previous works, optical forces were found to increase with the dielectric constant of the effective shell and the fact that optical forces were maximized under plasmonic excitation was, indeed, expected .
Plasmonic mediated heating has been also postulated to be capable of efficient thermo-optical trapping of metallic nanoparticles and of absorbing microspheres [43, 44]. The possible contribution of such thermal effects in our experiments has been evaluated by acquiring thermal images of the µ-Sp + GNR constructs in the course of the optical trapping. Figure 3(a) shows an optical image of a single µ-Sp + GNR construct optically trapped by 808 nm laser beam (22 mW power). The corresponding fluorescence thermal image (measured by Quantum Dot thermometry) is shown in Fig. 3(c) where the position of the µ-Sp + GNR construct is indicated by an arrow . As can be observed, only a small temperature increment of 1-2 °C was observed, very close to our detection limit of 1 °C. In order to provide a better estimation of the laser induced heating in µ-Sp + GNR constructs we acquired thermal images for the trapping of multiple constructs. Figure 3(b) shows an optical transmission image for 808 nm, 22 mW laser trap during simultaneous trapping of 4 µ-Sp + GNR constructs.
In this case the laser induced temperature increase was approximately 8 °C (see Figs. 3(d) and 3(e)) so we estimated the maximum laser induced heating per µ-Sp + GNR constructs to be 2 °C. The heating rate of the plasmonic constructs is then calculated to be close to 100 °C/W. This heating rate is comparable to the heating rates reported for silica microspheres decorated with metallic nanodots under visible light excitation that could be as large as 50 °C/W . Figure 3(e) shows the heating curve estimated for a single µ-Sp + GNR construct as a function of trapping power. Data were obtained by dividing the laser induced heating of multiple trapped particles by the number of optically trapped particles. For the sake of comparison Fig. 3(e) also includes the heating curve obtained for plain (non-conjugated) microspheres by following the same procedure. In this case, no heating was observed. In reference  is presented a study about the increase of the temperature as a function of the Nanorods trapped which is related to the thermal force. M. Gu et al reported that with 250 gold Nanorods (with an average length and aspect ratio of 45 nm and 4, corresponding to λSPR close to 800 nm) trapped at the focus, the net photothermal force at a place 10 mm away from the focus center can reach 0.45 fN, which is approximately the threshold to overcome the Brownian motion, which correspond to an temperature increase around 60K. So, our temperature increase of 2 °C should not produce thermal forces of the sample. Therefore, we conclude thermal effects are not playing a dominant role during the OT of single µ-Sp + GNR plasmonic constructs.
Finally, following confirmation of efficient trapping of µ-Sp + GNR construct with moderate laser powers in aqueous solution, our next challenge was to verify the possibility of achieving intracellular optical manipulation. Macrophage cells were incubated with an aqueous suspension containing µ-Sp + GNR constructs at a reduced concentration of 0.001 and 0.002 mg/mL. Figure 4(a) shows the incorporation of the µ-Sp + GNR construct into the living cell (arrows indicate their location). In absence of any external stimulus, the intracellular µ-Sp + GNR constructs were not moving inside the cell very likely due to the large viscosity of the cytoplasm. Then we focused the 808 nm, 20 mW laser beam inside the cell 5 µm away from a µ-Sp + GNR construct (red arrow in Fig. 4(b) indicates the location of the laser spot, black arrow indicates the location of the trapped Sp-GNR). The time evolution of the distance between the µ-Sp + GNR construct and the laser spot was registered and the results are included in Fig. 4(c). It is expected that the velocity of the trapped object during the trajectory increase when the object is close to the laser point. Nevertheless, the intracellular medium is highly inhomogeneous due to the presence of different intracellular components and organelles. As a consequence, the cytoplasmic viscosity will vary significantly vary from point-to-point. Such a strong local variations make the particle approach to the laser trap to be randomly accelerated and decelerated. At the same time, the possible adhesion to the pushed particle of intracellular components could also lead to the appearance of a larger drag forces. The combination of these effects could dominate the observed form of the particle velocity as it approaches the trap in such a way that the expected acceleration is not observed.
It is evident that the µ-Sp + GNR construct is pushed by optical forces into the laser focus. 130 s after laser irradiation the µ-Sp + GNR construct reached the laser focus (see Fig. 4(b)) and remained there while the laser was on. µ-Sp + GNR constructs far away from the laser spot were not subjected to any optical force and, consequently, they remained at the same position during the irradiation (compare yellow arrows in Figs. 4(a) and 4(b)). For comparison, the time evolution of the position of a non-trapped µ-Sp + GNR is shown in Fig. 4(d). In absence of optical trapping force the µ-Sp + GNR does not experienced any preferential motion. This experiment was repeated several times to ensure that the data here presented are reproducible and representative of the effect.
From the data included in Fig. 4(c) we have estimated a translational velocity for the µ-Sp + GNR construct to be close to 40 nm/s. Taking into account the medium viscosity reported by Ying-chun  for the particular cellular line used in this work, we have obtained a drag force value of 0.12 pN. The intracellular manipulation shown in Fig. 4 is here stated to be a GNR-assisted process. This is further supported by the fact that intracellular optical manipulation of bare (non-decorated) microspheres under the same experimental conditions of Fig. 4 was not observed. At this point we should note that we performed a number of different toxicity assays. We have found that incubation of macrophages with medium solutions containing µ-Sp + GNR construct does not cause any toxicity as it is shown in Fig. 5.
In conclusion, we have demonstrated how the infrared optical trapping efficiency of dielectric microspheres could be remarkably enhanced by the controlled adhesion of gold nanorods to their surface. In this work it has been found that such enhancement in the optical trapping efficiency is strongly dependent on the particular trapping wavelength. The maximum enhancement in the optical trapping efficiency was observed when the trapping laser wavelength was equal to the surface plasmon resonance wavelength of the gold nanorods decorating the microsphere surface (808 nm in our case). The gold nanorod assisted enhancement of the optical trapping efficiency enabled the intracellular manipulation of the decorated dielectric microsphere by using a low power (22 mW) infrared optical trap. This is the first demonstration of GNR assisted intracellular optical manipulation of a dielectric microsphere. Results in this work reveal a new application of gold nanorods that is in addition to their demonstrated abilities for cell imaging and therapy; they now emerge as coadjutant nanoparticles for efficient optical manipulation of dielectric micro-particles.
This work was supported by the Spanish Ministerio de Educacion y Ciencia (MAT2010-16161 and MAT2013-47395-C4-1-R). P.H.G. thanks the Spanish Ministerio de Economia y competitividad (MINECO) for Juan de la Cierva program. E.M. Rodriguez thanks ERC for Marie Curie fellowship (PIOF-GA-2010-274404 – LUNAMED). KD thanks the UK Engineering and Physical Sciences Research Council for support. J.A.C. is a Concordia University Research Chair in Nanoscience and is grateful to Concordia University for Financial support. J.A.C. is grateful for the support from (NSERC) Canada.
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