Article pubs.acs.org/JPCC
Two-Photon Fluorescence Correlation Spectroscopy of Gold Nanoparticles under Stationary and Flow Conditions Ilaria Fortunati,*,† Verena Weber,† Emilia Giorgetti,‡ and Camilla Ferrante† †
Department of Chemical Sciences and UR INSTM, University of Padova, Via Marzolo 1, 35131 Padova, Italy Istituto dei Sistemi Complessi ISCCNR, Sezione di Sesto Fiorentino, Via Madonna del Piano 10, 50019 Sesto Fiorentino, Firenze, Italy
‡
S Supporting Information *
ABSTRACT: In this work we report two-photon fluorescence correlation spectroscopy experiments run on gold nanoparticles in aqueous solution. We compare the photophysical properties of nanoparticles obtained by the Turkevich method and by laser ablation as a function of increasing laser power. Fluorescence correlation spectroscopy curves for all nanoparticles show the rotational diffusion contribution at short lag times, even if the residual rotation contrast coefficient at high excitation laser power is lower for laser ablation nanoparticles with respect to Turkevich syntheses. Moreover, in contrast to Turkevich nanoparticles, laser ablation ones do not show an increase of the number of bright tracers in the excitation volume under higher excitation power. Nanoparticles from Turkevich synthesis are also tested as possible flow tracers inside a very simple microfluidic device featuring a single straight channel. Our findings confirm that gold nanoparticles can indeed be used as extremely sensitive probes to observe local flow inside micrometric size channels, for example, such as mammalian capillaries, since it can distinguish subtle changes in flow speed, as the ones imparted by a syringe pump.
1. INTRODUCTION In the past decade great attention has been devoted to the synthesis and characterization of novel and efficient fluorescent tracers for multiphoton excitation. Pulsed lasers emitting in the near-infrared region (700−1000 nm) are largely used in biomedical applications, exploiting the intrinsic 3D spatial resolution and large optical penetration depth into human tissues of the two-photon absorption process. Among many different materials proposed for their large two-photon absorption and emission efficiencies, such as fluorescent proteins1 and semiconductor quantum dots derivatives,2,3 novel nanostructures based on noble metals have been explored. Since Boyd and colleagues measured higher emission efficiency from a rough metal surface with respect to a flat one, in particular under two-photon excitation,4 there are only few reports on applications exploiting the photoemission properties of different types of metal nanostructures.5−10 Among these, gold nanostructures (nanoparticles, nanorods, nanostars) attract particular interest because of their promising emission properties in terms of good photostability under continuous irradiation, no blinking effects, and good biocompatibility (in particular with respect to the potential leaching of toxic metal ions from semiconductor quantum dots). The possibility of tuning the absorption band by modifying the particles shape is another advantage for their application in different fields. A thorough investigation on the luminescence behavior, under one- and two-photon excitation at increasing laser power, of citrate capped gold nanoparticles (Au NP) with increasing © 2014 American Chemical Society
diameter (up to 50 nm) has been reported by Loumaigne and colleagues.11−13 From fluorescence correlation spectroscopy (FCS) analysis, they reported an apparent increase in the number of active nanoparticles (fluorescent nanoparticles) with laser intensity together with a reduced overall emission anisotropy. According to the authors, the source of these power dependent changes is the increase in local temperature on the nanoparticle surface induced by the highly focused laser beam. The heat transfer promotes the salt ligand reorganization on the surface and modifies the defect map at the interface. Since these defects are thought to be responsible for the Au NP luminescence, an increase in their number will give rise to an enhanced emission as the laser power is raised. At the same time, multiple defect sites on the same NP will make the NP emission isotropic, reducing the part of the signal sensitive to anisotropy. The fluorescence quantum yield of Au NP with dimensions larger than 5 nm in aqueous solution is extremely low (of the order of 10−5−10−6).14 It is interesting to note that it has been demonstrated that the NP emission under two-photon excitation (MAIL, multiphoton absorption induced luminescence) is more intense than the emission from one-photon pumping.9 The mechanism giving rise to the two-photon excited emission is still under debate, and a complete theory at Received: June 27, 2014 Revised: September 15, 2014 Published: September 23, 2014 24081
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surfactants. FCS experiments allow the observation of the Brownian diffusion motion, both rotational and translational, of the different nanoparticles and, as a consequence, can give an estimate of the average nanoparticle hydrodynamic radius. FCS experiments are run under increasing laser power, and the results are discussed taking into account the different structural and chemical characteristics of the investigated nanoparticles. In the second part of the paper, because of the high sensitivity and high spatial resolution of FCS, we employ this technique for the measurement of fluid speed inside a simple microfluidic device using the Au NP as tracers. The use of large fluorescent tracers, in comparison to conventional organic fluorescent molecules, improves the sensibility toward low flow speed values, which are typical of blood flow in capillary vessels, for which microfluidic devices (MFD) can constitute a viable in vitro model.
the microscopic level for these processes has not yet been formulated. Among the proposed mechanisms there are radiative recombination of excited electrons of d bands and holes of the sp conduction levels, radiative localized surface plasmons of NP, and surface plasmon enhanced sp intraband radiative transition in the NIR region.9,14−16 In addition, since the probability of two-photon absorption is proportional to the fourth power of the electric field enhancement, MAIL intensity can be amplified by many orders of magnitude when the local electric field is strong. This condition is satisfied in the presence of rough surfaces, in asymmetries in the metal nanostructures, or at the junction of two or more particles (“hot spots”).7,17,18 Because of experimental difficulties, the two-photon absorption cross-section (σTPA) has been measured only for few gold nanostructures, including gold clusters and some gold nanoparticles.19 Ramkrishna et al. reported on the increase of the two-photon absorption cross-section with nanocluster dimensions, exhibiting exceptionally giant cross sections in comparison with common organic chromophores. The authors measured σTPA of 1.5 × 106 GM for clusters of 309 Au atoms and 3.5 × 106 GM for 4 nm Au NP. Therefore, considering these data, Au NP with tens of nanometer dimensions should have comparable or even higher two-photon absorption efficiency. In addition, it is reported that each nanoparticle could activate the emission at different laser power, depending on the chemical and physical properties of the nanoparticle, in particular its surface features.13,20 Because of the development of highly sensitive detectors, such as avalanche photodiodes, and of commercial, ready to use ultrafast laser sources in the NIR, MAIL from Au NP can be now routinely exploited to track these nanoparticles in in vitro and in vivo biological experiments.21 In this respect, an emerging in vitro technique that can strongly benefit from the use of the luminescence emitted by these nanoparticles is microfluidics. Indeed, microfluidics allows the study of biological processes under controlled conditions and with an increasing degree of complexity. Apart from the obvious addition of all the physical processes connected to flow, such as laminar or turbulent flow and shear stress,22,23 microfluidics has been very successful at the realization of cocultures, which are considered the first step to generate in vitro models capable of substituting, at least in part, in vivo tests on complex tissues.24,25 As an example, epithelial and endothelial cells, capable of communicating through a porous membrane, were grown in a microfluidic device in the attempt to reconstruct the alveolar-capillary interface of human lungs.26 Au NP in microfluidic devices can be used to (i) track flow speed inside the device with micrometric resolution through FCS experiments,27 (ii) study single NP−cell interaction through time- and frequency-resolved luminescence microscopy experiments, as well as surface enhanced raman scattering microscopy, (iii) observe the cellular localization of these NP as well as their effectiveness as drug carriers or photothermal agents on different kinds of cell cultures. In the first part of this work, two-photon excited FCS is employed to characterize the photophysical behavior under high intensity laser irradiation of different batches of Au NP dispersed in water. The first two batches are obtained following Turkevich synthesis. They are used to test the reproducibility of the technique when Au NP samples with nominally equal properties are used. The third batch of Au NP is fabricated by laser ablation in pure water and in the absence of any
2. EXPERIMENTAL METHODS 2.1. Synthesis of Au NP. Two batches of gold colloidal dispersions are synthesized according to Turkevich procedure,28 and they are labeled as Au NP T1 and T2. Gold(III) chloride solution 30% w/w in dilute HCl and sodium citrate dihydrate are both purchased from Sigma-Aldrich and used without further purification. For both syntheses, 45 mL of 1 mM aqueous solution of tetrachloroauric(III) acid is reduced, under reflux conditions, with 5 mL of a 39 mM aqueous solution of sodium citrate. The third batch (Au NP LA) is prepared by laser ablation of a gold target in pure, doubly deionized water, employing a mode-locked Nd:YAG laser at 1064 nm, 15 mJ pulse energy. The resulting gold colloid is dispersed in water.29 The NP concentrations of the native solutions are 1.3 × 1012, 1.6 × 1012, 4.2 × 1013 NP/mL for Au NP T1, Au NP T2, and Au NP LA, respectively. For Turkevich syntheses, the number of NP (NNP) is calculated from the equation w(Au) = NNP × ρ(Au) × 4/3π∑nRn3An, where w(Au) is the weight of gold calculated from the precursor amount (assuming all the gold precursor is reduced by sodium citrate) and ρ(Au) is the gold bulk weight density. Rn and An are parameters extracted from the TEM dimension distribution histogram (see Figure 2): Rn is the mean radius for each n column, and An is the fraction of Au NP with the nth mean radius. This method allows for a better determination of the total number of NP because it is calculated taking into account the overall distribution of NP dimension and not only the radius mean value. Since the weight of gold is unknown for LA synthesis, the concentration of the Au NP LA can be extrapolated only from the fitting of the UV− visible spectrum, using the relations from the Mie theory.30 Fluorescence correlation spectroscopy analyses in free diffusion are carried out on NP native solutions. For flowing experiments, the Au NP T1, T2, and LA are diluted to 2.6 × 1011, 2.2 × 1011, and 7 × 1012 NP/mL, respectively, to reduce the number of spikes generated in the fluorescence signal by the larger number of aggregates flowing through the laser spot when advection is present. 2.2. Au NP Characterization. UV−vis spectra are recorded with a Varian Cary 5 spectrometer in the 350−900 nm range, and emission spectra are recorded using a Horiba-Jobin Yvon FluoroMax-3 spectrofluorimeter with excitation at 500 nm. Morphological Au NP analysis is carried out by TEM in bright-field and high-resolution modes. A field-emission gun (FEG) Tecnai F20 Super-twin (S)TEM working at 200 keV is used. Dynamic light scattering (DLS) and ζ-potential measure24082
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ments are performed with a Zetasizer Nano ZS90, Malvern Instruments. In this apparatus, the ζ-potential is calculated from the electrophoretic mobility on the basis of the Helmholtz− Smoluchowski relation. 2.3. Fabrication of MFDs. MFDs with a single microchannel (length 40 mm, width 2 mm, and height 150 μm) are realized in polydimethylsiloxane (PDMS, Sylgard 184, Dow Corning, MI) following the replica molding method.31,32 Masters for replica molding are realized by photopolymerization of a commercial photoresist (Microresist, SU-8) deposited on a silicon substrate. After thermal polymerization, the 1 mm thick PDMS layer is peeled from the master and drilled to provide the inlet and outlet for liquid flow. The PDMS is exposed to reactive oxygen plasma to permit further sticking to a coverglass (10 min for glass, 30 s for PDMS under UVO treatment). 2.4. Fluorescence Correlation Spectroscopy. For twophoton fluorescence correlation spectroscopy (TP-FCS) a Ti:sapphire laser (Coherent-Mira900, 76 MHz repetition rate, 150 fs pulse duration) is focused into the Au NP aqueous solution through a water-immersion 60× microscope objective mounted on a confocal microscope system (OlympusBX51WI/FV300). The laser wavelength is set at 820 nm, and the average power is modified through a half-wave plate coupled to a vertical polarizer. The reported laser power corresponds to the average energies on the sample. The emission signal, focused on a 100 μm core optical fiber, is split by a 40/60 beam splitter into two avalanche photodiodes (MPD-PDM100). A short-pass 750 nm filter, combined with a 572/35 bandpass one, is used for reducing the back-scattered laser light in the collection path. Time-correlated single-photon counting, supported by ultrafast electronics (PicoQuantPicoHarp300), is employed for the calculation of the crosscorrelation function in order to remove after-pulsing artifacts, mainly affecting the curve at short lag times. For simplicity, cross-correlation TP-FCS will be afterward called TP-FCS. For characterization of Au NP in flow conditions, the NP solution is injected into the MFD through a syringe, connected to the microchannel through LDPE tube, by means of a syringe pump (KD Scientific-KD200). The flow rate ranges between 5 and 70 μL min−1. The measurement of the mean fluid velocity in the channel center, as well as the velocity profiles along the channel width as a function of the flow rate, is accomplished by focusing the laser beam in different positions inside the channel. For further analysis of dynamic data, TP-FCS experiments in free diffusion conditions are recorded in the same positions within the MFD. Each data set is registered with 20 s collection time. Strong spikes, due to small aggregates, are filtered from intensity traces by post numerical treatment before calculating the crosscorrelation curve. The data hereafter reported are the average between four independent measurements, and errors are calculated as the semidifference between the maximum and the minimum values.
Figure 1. UV−visible spectra of two batches of Au NP from Turkevich method (solid black and dashed red traces, T1 and T2) and of Au NP from laser ablation (dot blue trace, LA) in water. The inset is the enlargement of the absorption maximum peak.
Au NP T2, and Au NP T1, respectively. The absence of a noticeable tail toward the NIR wavelengths is a confirmation of a negligible number of large aggregates in all solutions. The measurement of the ζ-potential indicates the presence of negative charges on the NP surface for all types of NP. The surface potential is approximately −40 mV for all NP in water solution, confirming high suspension stability also after long periods of time from the synthesis. As already reported for gold nanoparticles with diameter larger than 10 nm, which are characterized by emission quantum yields on the order of magnitude of 10−6,33 our nanoparticles are also characterized by a weak fluorescence in the visible−NIR region. The emission spectra of the three Au NP samples are similar, showing a broad band centered at 620 nm (see Supporting Information, Figure SM1). 3.2. TEM and DLS Analysis for Shape and Dimension Characterization. The dimensions of Au NP have been characterized with HR-TEM, allowing an estimation of the metal particle radius without the hydration shell. As shown in Figure 2, Turkevich synthesis gives NP dimension distributions that are symmetric around the mean value and almost comparable for the average size and distribution, while the laser ablation method bears the preparation of NP with large and asymmetric distribution.29 In Figure 2c, the histogram of Au NP LA gives evidence of the presence of a large fraction of small nanoparticles (few nanometer radius) with a tail broadened up to 20 nm. The LA distribution is fitted with a log−normal function, giving an average radius of 4.4 nm. Because of the asymmetric distribution, it is necessary to define a mean standard deviation from the central value below the average (1.3 nm) and above the average (1.8 nm). The larger average dimension of Au NP T1 and T2 triggers a slight red-shift of the absorption peak position, proportional to the NP volume as predicted from Mie theory. In addition, Au NP LA are more spherical in comparison with NP from Turkevich synthesis. TEM analysis shows that some of Turkevich NP are characterized by a more elliptical or asymmetric shape, as a consequence of asymmetric growth of gold on the seeds or of the coalescence of small nanoparticles during synthesis.34,35 The average NP radii measured with DLS are 14.7, 10.8, and 28 nm for Au NP T1, Au NP T2, and Au NP LA, respectively. These values are equal or slightly higher than those measured by TEM analysis for Au NP T1 and T2. For the sample Au NP
3. RESULTS AND DISCUSSION 3.1. UV−Visible and Fluorescence Analyses. In Figure 1 the UV−visible spectra of the three solutions are reported: solid black and dashed red traces pertain to the NP obtained following Turkevich method, while the dotted blue curve relates to the NP prepared by laser ablation. The absorption spectra are similar, showing a well-resolved plasmon band centered at 520, 524, and 525 nm for Au NP LA, 24083
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Figure 2. HR-TEM images and distribution histograms of particle radius of Au NP T1 (a), Au NP T2 (b), and Au NP LA (c).
LA, the average hydrodynamic radius from DLS is strongly overestimated with respect to TEM analysis. This can be explained by considering light scattering theory: the intensity of the scattered light is proportional to the product of the tracer molecular weight and its concentration. Larger particles, even when present at low concentrations, can strongly influence the average dimension for the distribution measured with the DLS technique. For this reason, the value obtained for Au NP LA cannot be considered reliable for the full dimensional characterization of the sample. HR-TEM and absorption experiments confirm that Turkevich synthesis, when performed under the same experimental conditions, gives Au NP batches with highly reproducible dimesions and shapes. 3.3. Analysis of Free Diffusing Au NP by Fluorescence Correlation Spectroscopy. In this work two-photon absorption is employed for optical excitation of gold nanoparticles dispersed in water. TP-FCS curves recorded at increasing laser power will be compared to analyze the behavior of different types of Au NP and to try to understand the origin of their power dependent emission properties. The TP-FCS curves of all samples display two distinct temporal features: the first at short lag times in the microsecond regime and the second one on the millisecond time scale. As already demonstrated by Loumaigne et al.,11,12 the longer time can be assigned to a Brownian translational diffusion process, while the microsecond signature is related to rotational diffusion of Au NP. Figure 3 reports the normalized TP-FCS curves for different batches of NP, freely diffusing in water, under similar laser power excitation. All of them display the Brownian rotational contribution at short times, even if the relative amplitude changes from one batch to the other. Full lines in Figure 3 are calculated from a fitting procedure using eq 1 for Gdiff(τ) describing the autocorrelation of the fluorescence fluctuations in time. Gdiff(τ) contains both rotational and translational diffusion terms.36 Gdiff (τ ) = Grot(τ )Gtransl(τ ) = [1 + C e−τ / τrot] −1/2 ⎤ ⎡ ⎛ ⎞−1⎛ τ ⎞ ⎢ 1 ⎜1 + τ ⎟ ⎜ 1 + ⎥ ⎟ 2 ⎢N⎝ ⎥ τ τ S ⎠ ⎠ ⎝ transl transl ⎣ ⎦
Figure 3. Normalized TP-FCS curves of Au NP T1 (black squares), Au NP T2 (pink dots), and Au NP LA (green triangles) at 820 nm and average laser power of 23 mW on the sample. The full lines are the result of data fitting based on eq 1 combining the rotational and translational diffusion terms.
The parameters of eq 1 are the rotation contrast C, the characteristic rotational time τrot, the particle number N in the focal volume, the time τtransl employed by the tracer to pass through the focal volume through a process of translational diffusion, and S the focal shape factor. By use of the parameters obtained from the fitting of the freediffusion TP-FCS curves, an estimate of the NP hydrodynamic radius can be done. By adoption of a classical hydrodynamic description valid for low Reynolds numbers, τrot and τtransl are proportional to the volume and the radius of the nanoparticle, respectively:37 τtransl =
τrot =
3πηR h,transl w0 2 = 8Dtransl 4kBT
4πR h,rot 3η VNPη = kBT 3kBT
(2a)
(2b)
where η is the water viscosity, kB is the Boltzmann constant, T is the temperature, w0 is the radial focal volume radius, and Rh is the NP hydrodynamic radius. For the determination of the NP hydrodynamic radius it is preferable to employ the rotational
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Figure 4. Dependence of Au NP hydrodynamic radius on laser power (bottom scale) and on laser fluence (upper scale). The values are extrapolated from the translational diffusion (full squares) and the rotational diffusion term (empty squares) of TP-FCS curves.
diffusion time instead of the translational one because τrot is independent of the radial dimension of the focal volume, and therefore, it is less affected from artifacts, including optical saturation and optical trapping occurring at high excitation energies. As mentioned in the Introduction, the fluorescence signal intensity is given by the product of the emission quantum yield and the two-photon absorption cross section. These properties can have important implications for the determination of optical saturation effects when different fluorophores are used. Indeed, for two-photon excitation and Gaussian-shaped pulses, the threshold saturation intensity is defined as ITPA sat = [(8 ln2)/ π)]1/4[1/(σTPAτp)]1/2 where τp is the laser pulse duration.38 This parameter identifies the intensity necessary for exciting half of the molecules at the center of the focal volume by each laser pulse. For organic fluorophores typically used in TP-FCS (such as rhodamines) having σTPA on the order of 100 GM, and considering the laser pulses delivered by our setup, the calculated optical saturation will correspond to an average laser power of 8 mW. For chromophores with a σTPA of 106 GM, the saturation should occur at 80 μW, which is a very low energy for two-photon excitation experiments. In addition, from numerical calculation, it has been demonstrated that the effects on the effective focal volume due to saturation are evident also at intensities lower than the saturation threshold.38 In FCS, calibration of the focal volume is commonly carried out using a free diffusing organic dye with a known Dtransl. Since organic dyes are characterized by saturation intensities markedly different from those of NP, w0 is here internally calibrated by matching the values of NP radius calculated from both relations, at lower excitation power. In this condition of minimum excitation rate, the optical saturation artifacts on the effective focal volume should be negligible. To have a confirmation of the influence that saturation can play in the analysis of the FCS curves, a comparison between the estimated NP radius from the rotational and the translational diffusion times is done at increasing laser power. In Figure 4 the estimated NP radii for all Au NP are reported as a function of laser power (bottom scale) and of average laser fluence (upper scale). Average laser fluence is calculated with the w0 value extrapolated for low laser power experiments. The errors in the graphs are calculated by the error propagation formula, considering the uncertainties on τrot or τtransl and on w0. For both Turkevich NP batches, the behavior reported in Figure 4 is similar: the average radius evaluated from rotational
diffusion is almost independent of laser power, while the radius from the translational term shows a tendency to increase with excitation power. Within the investigated power range, Rh,transl increases as τtransl because of the enlargement of the effective focal volume. Our experimental findings cannot be directly compared with the one reported by Loumaigne and colleagues for 20 nm Au NP under two-photon excitation,13 since the applied laser fluence is different. In their FCS experiments the observed τtransl did not significantly change for laser fluences up to 4 MW cm−2, while in our experiments the fluence is changed between 3 and 18 MW cm−2. Since Rh,transl is affected by large error bars, meaningful comparison between Turkevich and LA batches on this parameter cannot be carried out. On the contrary, the results for Rh,rot are affected by smaller uncertainties and therefore can be compared. As shown in Figure 4, Rh,rot for Au NP LA is constant up to 20 mW and it increases for higher power, in contrast with the data for Turkevich batches. This increase of NP average size can result from a coalescence process involving the smallest nanoparticles promoted by high applied fluences. Ultrafast laser pulses excite electrons to high energy levels, heating the nanoparticle through an electron−phonon scattering process that occurs with a characteristic time of a few picoseconds.39 Therefore, the nanoparticle undergoes a sudden temperature increase followed by thermalization with the surrounding medium on a time scale of 100 ps to 1 ns. Since electron−phonon thermalization is much faster than thermal equilibration with the surrounding bath, after a single femtosecond pulse, the temperature on the surface of the particle can be approximated by the following equation:40 ΔTNP = TNP,surf − Tbath =
σabsF VρAu cAu
(3)
where σabs is the absorption cross section, F is the laser fluence, V is the nanoparticle volume, ρAu is the gold mass density, and cAu is the gold specific heat capacity. For a NP with radius of 15 nm excited by an average irradiance in the 3−20 MW cm−2 range at 820 nm, the calculated maximum temperature rise is 30−170 °C. Considering also the heat transfer to the surround and the Au NP interface resistivity, the temperature range is reduced to approximately 10−50 °C. Following this model, the surface temperature rapidly increases with incident laser power and with the reduction of NP dimensions. Since the bulk melting temperature of Au NP dramatically decreases for particles with radius smaller than 5 nm 41 and the surface melting temperature of high surface-volume ratio particles is 24085
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Figure 5. Dependence of the number of bright nanoparticles N in the focal volume (a) and of the rotation contrast C (b) with the excitation power.
even lower than the bulk one,42 the experimental conditions used here mainly affect the Au NP LA ensemble. Moreover electrostatic stabilization of Au NP LA is accomplished only by charged oxygen bridges on “naked” NP, and the increase of surface temperature during the irradiation can more easily modify their surface charge distribution, reducing their stability. In addition, the presence of larger nanostructures can promote an increase of optical trapping processes, which, in turn, will lengthen the Brownian diffusion time. In this regard, Wang and colleagues measured the threshold power needed for trapping Au NP with different sizes, under two-photon excitation at room temperature.43 At 750 nm excitation, the trapping threshold for 15 nm Au NP is 10 MW cm−2, for 30 nm Au NP is 3 MW cm−2 and for 50 nm Au NP is
