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Cite this: DOI: 10.1039/c5cp02620b
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Quantifying the photothermal efficiency of gold nanoparticles using tryptophan as an in situ fluorescent thermometer† Ming-Jui Chiu and Li-Kang Chu* The photothermal efficiencies, denoting the efficiency of transducing incident light to heat, of gold nanoparticles of different diameters (+ = 22–86 nm) were quantified upon exposure at 532 nm. The fluorescence of tryptophan at 300–450 nm upon 280 nm excitation serves as an in situ fluorescent thermometer to illustrate the evolution of the average temperature change in the heating volume of the nanoparticle solution. The fluorescence intensity decreases as the temperature increases, having a linear gradient of 2.05% fluorescence decrease per degree Celsius increment from 20 to 45 1C. The presence of gold nanoparticles at the nM level does not perturb the temperature-dependent fluorescence of tryptophan in terms of fluorescence contour and temperature response. The heating volume was defined by overlapping the collimated 532 nm laser (+ = 0.83 mm) for exciting the nanoparticles and the 280 nm continuous-wave beam (+ = 0.81 mm) for exciting tryptophan in a 2 mm 2 mm square tube, and the fluorescence was collected perpendicularly to the collinear alignment. This method has satisfactory reproducibility and a sufficient temperature detectivity of 0.2 1C. The profiles of the average temperature evolution of the mixtures containing nanoparticles and tryptophan were derived from the
Received 6th May 2015, Accepted 27th May 2015
evolution of fluorescence and analyzed using collective energy balancing. The relative photothermal
DOI: 10.1039/c5cp02620b
those predicted using Mie theory. The employment of tryptophan as a fluorescent thermometer not
efficiencies for different sizes of gold nanoparticles with respect to the 22 nm nanoparticle agree with only provides an in situ tool to monitor the photothermal effect of nanostructures but is also applicable
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to thermal imaging in biological applications.
Introduction The photothermal effect of metallic nanoparticles upon photoexcitation, i.e., thermo-plasmonics,1–3 has been extensively employed in many aspects, such as drug delivery and release,4–6 thermotherapeutic treatment,7–10 catalysis,11 and water vapor generation.12 Metallic nanoparticles are capable of attenuating incident light via scattering and absorption, as characterized using Mie theory.13,14 The step-wise energy cascading processes, including the electron cloud thermalization (within B100 fs),15 electron–phonon thermalization (B1 ps),16,17 and external heat release to surroundings,1,18 are used to illustrate heat transfer upon ultrafast pulsed excitation.19 Thermal equilibrium inside the nanoparticles can be achieved quickly, within 0.1 ns to 1 ms, for nanoparticles of diameters of 10 nm to 1 mm.18 As the
Department of Chemistry, National Tsing Hua University, 101, Sec. 2, Kuang-Fu Rd., Hsinchu 30013, Taiwan. E-mail: [email protected]; Fax: +886-3-5711082; Tel: +886-3-5715131 ext. 33396 † Electronic supplementary information (ESI) available. See DOI: 10.1039/ c5cp02620b
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nanoparticles are irradiated with continuous illumination, the evolution of the temperature of the surroundings over a prolonged period can be depicted using the collective energy balance.20–23 In the past decade, miscellaneous experimental methods were employed to qualitatively12,24–27 and quantitatively12,20–24 monitor the photothermal effect. Hogan et al. observed steam generation upon excitation of a gold nanoparticle solution and demonstrated that light absorption creates intense localized heating, which efficiently vaporizes the surrounding liquids.24 Neumann et al. demonstrated that 80% of absorbed sunlight is converted into water vapor, and merely 20% of absorbed light energy is converted to heat in the surrounding liquid.12 The thermal imaging in the vicinity of the nanostructure arrays was recorded on the basis of the thermal-induced refractive index variation.25,26 Bendix et al. measured the heating process by using molecular partitioning and by employing the phase dependent quantum yield of DilC18 fluorophores in lipid bilayers.27 Quantitative approaches, especially those employing thermocouples, have been used to reflect heat generation upon continuous photoexcitation of gold nanoparticle suspensions by monitoring the temperature change and analyzing the energy balance.20–23
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Roper et al. recorded the heating process of a 20 nm nanoparticle suspension and derived that the efficiency of transducing incident resonant light to heat (Z) was less than 10%.20 However, Richardson et al. recorded the evolution of the temperature change of an aqueous droplet containing 20 nm nanoparticles upon photoexcitation using a thin thermocouple and demonstrated that the transduction efficiency Z is close to 100%.21 Jiang et al. have demonstrated the size-dependent Z of gold nanoparticles, but the observed values of Z were lower than those predicted by Mie theory.22 It should be noticed that the thermocouples were not placed in the heating volume;20–22 that is, these temperature detections were not the result of in situ probing. Fluorescent materials have been extensively employed in determining the temperature in materials and biomolecular applications.28,29 In this study, we have established experimental and analytical methods to illustrate the photothermal transduction efficiency of gold nanoparticles using a temperature-sensitive fluorescing molecule, tryptophan, as an in situ thermometer. Previously, organic polymers30,31 and organic molecules32,33 have been extensively employed in monitoring the temperature of a solution due to fluorescence enhancement or quenching as the temperature increases. The fluorescent molecule perylene was embedded in different film matrices to illustrate the photothermal phenomenon of varied nanostructures.34,35 The photoluminescence of quantum dots were also employed in luminescence thermometry.36,37 For instance, the fluorescence intensity of rhodamine B exhibits sufficient temperature sensitivity (3.4% 1C1).38 However, its spectral overlap with the resonant band of gold spherical nanoparticles does not allow it to function as a molecular fluorescent thermometer using a spectroscopic method. The fluorescence of tryptophan above 300 nm upon excitation at 280 nm, however, avoids spectral overlap of the plasmonic band of gold nanoparticles at 530 nm. It exhibits a sufficient temperature response as the temperature is increased,39 and it has been used in understanding protein folding dynamics.40,41 In this study, we utilized tryptophan fluorescence to monitor the average temperature change of the heating volume as the gold nanoparticle suspensions were irradiated using a continuous 532 nm laser and analyzed the evolution of the average temperature using a collective energy balance model. For gold nanoparticles of various sizes, the observed relative photothermal efficiencies were consistent with those predicted by Mie theory. Tryptophan was successfully used as an in situ fluorescent thermometer in quantifying the photothermal effects of the nanostructures. This approach could also be applicable to thermal bio-imaging.
Experimental section Materials The synthesis of gold nanoparticles of different sizes followed the protocol of the seeding growth method,42 with partial modification. The 22 nm gold seed was synthesized by adding 0.5 ml of 0.029 M chloroauric acid (HAuCl43H2O, Alfa Aesar, 99.99%) and 0.1 g sodium citrate (Na3C6H5O7, J. T. Baker, 499%) to
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50 ml DI water simultaneously, followed by vigorous stirring at 80 1C. The color of the solution rapidly turned to dark purple, and then gradually turned to ruby red over 40 minutes, indicating that the gold ions (Au3+) were completely reduced to neutral gold atoms. After the seed solution was prepared, the growth medium was used to manufacture the larger gold nanoparticles. To six conical flasks, labeled as A to F, 9 ml of 46.5 mM cetyltrimethylammonium bromide (CTAB, Sigma Aldrich, 499%) and 0.1 ml of 0.029 M HAuCl4 were added, and the mixture was stirred at 30 1C. Then 0, 1.2, 0.5, 0.25, and 0.125 ml of 22 nm gold seed were separately added to flasks A to E, and 50 ml of 0.1 M ascorbic acid (J. T. Baker, 499.5%) solution was added immediately to each flask. After 10 minutes of stirring, the solutions turned dark red, indicating the completion of the growth of nanoparticles of different sizes. In flask F, 1 ml of solution B and 50 ml of 0.1 M ascorbic acid solution were added and the mixture stirred for 10 minutes. Spherical gold nanoparticles with diameters of 22, 31, 47, 59, 74, and 86 nm were prepared. The as-prepared gold nanoparticles were concentrated to an optical density of 1.0 for 0.2 cm optical length at 532 nm using centrifugation (Model 6500, Kubota) at 25 1C for further experiments. 0.1 ml of 5 mM tryptophan solution and 0.25 ml of O.D. = 4 (for 1 cm absorption length) nanoparticle solution were mixed and diluted with DI water to a total volume of 1 ml. The samples contained 0.5 mM tryptophan, and O.D. = 1 of nanoparticles were prepared for 1 cm absorption length. The corresponding concentrations were about 0.71, 0.24, 0.06, 0.03, 0.01, and 0.008 nM for 22, 31, 47, 59, 74, and 86 nm gold nanoparticles, respectively. The characterization of the morphology of the nanoparticles is discussed in Results and discussions. Steady-state absorption and fluorescence spectra The steady-state ultraviolet-visible (UV-Vis) absorption spectra were recorded using a spectrometer (USB4000-UV-Vis, Ocean Optics), and temperature-programmable fluorescence (FL) spectra were collected using an F-7000 Hitachi spectrophotometer coupled with a thermostat (qpod TC125, Quantum Northwest) to maintain the temperature within a range of 20–45 1C, within 0.1 1C variation at the set points. The emission contours upon excitation of tryptophan at 280 nm were recorded from 300 to 450 nm with scanning steps of 0.2 nm. TEM imaging The sizes and morphologies of the gold nanoparticles were characterized using a thermal-type field emission scanning electron microscope (JSM-7000F, JEOL) and a high-resolution transmission electron microscope (JEM-2100, JEOL). Experimental setup for probing the evolution of the temperature change using fluorescence of tryptophan upon excitation at 280 nm The experimental module included a continuous-wave 532 nm laser for exciting gold nanoparticles, continuous-wave 280 nm light for exciting tryptophan, fluorescence collecting optics, and a data acquisition system, as shown in Fig. 1(a). The continuous-wave
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Fig. 1 (a) Experimental setup for monitoring the temperature change upon continuous excitation of gold nanoparticle suspensions. (b) Profiling the diameters of the 280 nm beam passing through a 100 mm pinhole at different distances from the center of the pinhole.
532 nm diode laser (DPGL-2100, Casix) provided a power of 102 mWatt in a beam diameter of 0.83 mm (full-width at half maximum) to serve as the excitation source. The incident intensity of the 532 nm laser was adjusted using the neutral density filters (FRQ-ND02, 03, and 04, Newport) and monitored using a power meter (818P-015-19, Newport). The laser was introduced to a sample cell, a 2 mm 2 mm (inner) square quartz tube (QST-2-75, Friedrick & Dimmock Inc.). The 280 nm light was provided by a solar generator (Model 70050, Newport) upon wavelength selection using a monochromator (Grating Model 77309, Newport). The 280 nm beam passed through a 100 mm pinhole (HPH-0100M, Unice E-O Service Inc.) to define the probing cross section and propagated opposite to the 532 nm beam. The diameters of the 280 nm beam at different distances from the pinhole were profiled by scanning the position of the photomultiplier (R928, Hamamatsu), which was perpendicular
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to the propagation direction of the 280 nm beam, as shown in Fig. 1(b). A 100 mm pinhole (P100S, Thorlabs Inc.) was mounted in front of the PMT to define the spatial scanning steps. At the center of the sample cell, 9 mm from the pinhole, the diameter of the 280 nm beam was 0.81 mm, slightly smaller than that of the 532 nm excitation beam (0.83 mm), to ascertain that the fluorescence intensity alteration originated from the heating volume upon excitation of the nanoparticles. The fluorescence of tryptophan was filtered using two edge filters (Model 84704, Edmund) at 450 nm to remove most of the scattering 532 nm light and a bandpass filter (BG–3–25.4, Lambda) to define the detection wavelength at 300–450 nm in front of a photomultiplier (R928, Hamamatsu). The electronic signal was recorded using an oscilloscope (WaveSurfer 24MXs-B, LeCroy). The exposure period was controlled to 500 seconds using a mechanical shutter (VS25S2T1, Uniblitz).
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Results and discussions In this work, we designed an in situ method to quantify the relative photothermal efficiencies (Zrel.) of different sizes of gold nanoparticles upon continuous excitation at 532 nm using tryptophan as a fluorescent thermometer. Steady-state extinction spectroscopy and electron microscopy were used to characterize the morphologies of the gold nanoparticles. Fluorescence spectroscopy was used to illustrate the temperature change as the mixtures of nanoparticles and tryptophan were irradiated with the modulated cw diode laser at 532 nm. By analyzing the temporal profiles of the temperature evolutions using a collective heat transport model, we derived the relative photothermal efficiencies of the gold nanoparticles, which agreed with the predictions of Mie theory. Morphology and size distribution of the gold nanoparticles When light travels through metallic nanoparticle suspensions, the intensity of the light can be attenuated due to absorption and scattering. In order to avoid the confusion of these two effects, the conventional absorption spectrum was replaced by the extinction spectrum, referring to the logarithm of the intensity ratio of the incident light before and after passing through the sample. The steady-state extinction spectra of the gold nanoparticles are shown in Fig. 2(a). The concentrations of nanoparticles were controlled to manifest an optical density (O.D.) of 1.0 for 10 mm optical length at 532 nm, equivalent to O.D. = 0.2 for further photoexcitation experiments using a 2 mm 2 mm square tube. The normalized extinction contours, shown in the inset of Fig. 2(a), exhibited a redshift as the nanoparticles increased in size. In addition to the spectroscopic characterization, the TEM and SEM patterns were collected to confirm the size distributions of these nanoparticles, as shown in Fig. 2(b). The TEM and SEM patterns indicated that the gold nanoparticles were spherical and well dispersed. The histograms of the size distributions, based on counting 100 particles for each size, are shown in the insets. The average sizes and the corresponding size deviations are listed in Table 1. A molecular fluorescent thermometer using tryptophan Before the mixtures of gold nanoparticles and tryptophan were prepared, the concentration of tryptophan had to be optimized to maximize the fluorescence intensity without saturation. Then the fluorescence of tryptophan in the presence of nanoparticles at different temperatures needed to be determined in order to correctly determine the temperature evolution during the 532 nm exposure of the nanoparticles. 1. Steady-state fluorescence. Gally and Edelman found a decrease in the fluorescence of tryptophan aqueous solution as the temperature was increased at a roughly linear rate of ca. 2% per degree Celsius in the range of 25–45 1C.43 Stevenson et al. have demonstrated that the decrease in tryptophan fluorescence is related to the non-radiative photoionization process and that the yield increases by almost five times, from 11 1C to 82 1C,39 suggesting that the fluorescence of tryptophan has sufficient temperature sensitivity to serve as a fluorescent thermometer.
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Fig. 2 (a) Steady-state extinction spectra of gold nanoparticles of different diameters, with normalized traces in the inset. (b) The electron microscopy images of the nanoparticles mentioned above. The histograms of the size distributions are shown in the insets.
The fluorescence contours of tryptophan at different concentrations and the relationship of the integrated intensity at 300–450 nm and the tryptophan concentrations are shown in Fig. 3(a) and (b), respectively. The normalized emission contours were independent of the concentration (inset of Fig. 3(a)), indicating an inconsequential aggregation of tryptophan. Before saturation, 0.5 mM of tryptophan was used to maximize the fluorescence intensity in the further experiments. Subsequently, temperature-programmable fluorescence contours for different sizes of gold nanoparticles and pure tryptophan were collected, as shown in Fig. S1 in the ESI†. The temperature was increased to within 20 1C of room temperature to prevent the non-linear response.39 The normalized contours, shown in the upper insets in Fig. S1(a)–(g) (ESI†), were almost identical in the 20–45 1C range, indicating that the integrated fluorescence
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Z (in mJ1 1C) for different nanoCM particles after fitting the traces in Fig. 6(b) using eqn (7)
Table 1
The observed values of
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Diameter of nanoparticle/nm 22 31 47 59 74 86
3 2 3 6 4 5
Z 102 CM 3.58 3.60 3.18 3.15 2.58 2.03
0.10 0.11 0.09 0.10 0.07 0.07
Zrel. (%)
Ztheory (%)
Ztheory rel. (%)
100.0 4.0 100.4 4.1 88.8 3.6 88.0 3.9 72.1 2.8 56.7 2.5
99.0 97.2 90.8 82.8 69.9 58.5
100.0 98.2 91.7 83.7 70.6 59.1
a
Z for different nanoparticles with CM respect to that of a 22 nm nanoparticle, assuming that M and C were the same in the measurements. Error propagation was taken into using consideration. The standard deviation of Zrel. can be derived 2 2 ! A DZ 2 DA DB ¼ þ the following equation: Z ¼ ; . The fracB Z A B tional error in Z is the square root of the sum of the squares of the fractional in its parts. Take Zrel. of 86 nm, for example: errors 0 1 ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi s Z 2 2 2:03 0:07 0:07 0:10 C M 86nm 86nm A ¼ 0:567 @1 ¼ þ Zrel: ¼ Z 3:58 0:10 2:03 3:58 C M 22nm a
Zrel. denotes the ratios of
¼ ð56:7 2:5Þ%:
intensity could serve as a fluorescence thermometer without spectral shift. In addition, the normalized fluorescence contours of tryptophan in different nanoparticle samples at 25 1C were almost identical, as shown in Fig. 3(c), indicating an insignificant interaction between the nanoparticles and the tryptophan. The ratios of the fluorescence intensity difference (DIFL/IFL 0 where DIFL = IFL IFL 0 ) with respect to fluorescence intensity at room temperature (IFL 0 ) integrated at 300–450 nm; plotted against the temperature difference in the lower insets in Fig. S1(a)–(g) (ESI†), they showed satisfactory linearity. The relationships of the nanoparticle sizes and the slopes are plotted in Fig. S1(h) (ESI†). We found that the slopes were independent of the nanoparticle sizes and manifested a value of ca. 2% per degree Celsius, similar to the intrinsic property of pure tryptophan solution. As a result, the ratios of the intensity differences and the temperature differences for all samples were plotted and fitted using a single linear relationship, as shown in Fig. 3(d). A fitted straight line agreed with the observed data and revealed a slope of 2.05% for the fluorescence decrease per increase in degree Celsius (% 1C1). This slope was used to convert the relative fluorescence difference to the temperature change.
Fig. 3 (a) The steady-state emission contours of tryptophan at different concentrations upon 280 nm excitation at 25 1C. The normalized contours are shown in the inset. (b) The relationship between the concentration of tryptophan and the integrated fluorescence intensities at 300–450 nm. (c) The normalized fluorescence contours of tryptophan solutions at 25 1C in the presence of gold nanoparticles of different sizes. The concentration of tryptophan was 0.5 mM. The concentrations of nanoparticles were controlled such that the optical density was 1.0 for an optical length of 1 cm at 532 nm. (d) The linear relationship FL FL FL between the relative change of the integrated fluorescence intensity at 300–450 nm (DIFL/IFL 0 , DI = I I0 ) and the temperature changes (DT = T Troom) with respect to room temperature for the samples mentioned above. Dots and solid lines represent the observed data and linear fitting, respectively.
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In addition, we also excluded the contribution of nanoparticles in altering the fluorescence properties of tryptophan upon 280 nm excitation. Because the concentration of the gold nanoparticles was at the nM level, the averaged inter-particle distance between nanoparticles was about 1 mm. Therefore, nanoparticle fluorescence quenching would play an insignificant role in perturbing the fluorescence property of the bulk tryptophan solution, since their effective distances are only several nanometers.44–46 In addition, the excitation of gold nanoparticles at 280 nm provides infinitesimal fluorescence intensity as compared to tryptophan under our experimental conditions, as shown in Fig. S2 (ESI†). As a result, the emission at 300–450 nm was solely attributed to the fluorescence of tryptophan upon excitation at 280 nm. 2. Deriving the evolution of the temperature difference from the fluorescence change. The transformation of the observed electronic signal of the photomultiplier tube to the evolution
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of temperature alteration is schematically displayed in Fig. 4. A stationary voltage attributed to the tryptophan fluorescence upon 280 nm cw illumination was recorded, as indicated by the purple segment of the waveform in Fig. 4(a). As the 532 nm laser was turned on, the fluorescence started decreasing due to the increase in temperature and leveled off, as shown by the green segment. When the 532 nm laser was turned off, the fluorescence returned to its original intensity, shown in a purple segment. However, scattering of the 532 nm laser could not be completely avoided, so a blank experiment was performed. In the absence of 280 nm illumination, the voltage from the PMT remained null, as shown in Fig. 4(b). Once the 532 nm laser was turned on, the voltage increased steeply and leveled off until the 532 nm laser was turned off. Generally, the contribution of 532 nm scattering was less than 1/100 of the voltage from tryptophan fluorescence.
Fig. 4 The schematic procedure to derive the temperature evolution from the evolution of the fluorescence intensity change of tryptophan. See details in the text.
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After temporal profiles of the fluorescence and scattering were collected, the raw electronic signal solely attributed to the fluorescence alteration, as shown in Fig. 4(c), was derived by subtracting the waveform in Fig. 4(a) from that in Fig. 4(b). The magnitude of the voltage evolution (V(t)) before introducing the 532 nm laser was averaged to serve as the fluorescence intensity at time zero (V0). By calculating (V(t) V0)/V0 and multiplying the conversion factor (x) for the fluorescence intensity-to-temperature change derived from the slope in Fig. 3(d), the evolution of the temperature was obtained (Fig. 4(d)). The detectivity of the temperature alteration was about 0.2 1C when accounting for the non-heating period. The observed evolutions of the temperature upon photoexcitation of nanoparticles of different sizes at different excitation powers are shown in Fig. S3 (ESI†). Establishing an appropriate kinetics model enabled us to estimate the heat transport process and the photothermal efficiency of the gold nanoparticles.
the power injection via the photon absorption by the nanoparticles and the heat loss to the surroundings. Qin can be evaluated by accounting for the incident excitation laser power Iex (Watts), the optical density (O.D.) of the nanoparticle suspensions, and the photothermal transduction efficiency Z, Qin = Iex(1 10O.D.)Z
O.D. was maintained at 0.2 for a 2 mm optical length in the square quartz tube. The incident power of the 532 nm laser of a circular cross section (diameter = 0.83 mm, defined by the full width at half maximum) was adjusted to 45.8–67.6 mW. Accordingly, the heating volume was about 1.08 mm3. As for the energy loss during the laser exposure, an apparent rate coefficient k, including the heat transfer coefficient of the solvent and the area of the cross section perpendicular to the heating cylinder,21,22 should be accounted for by Qout, Qout = kDT
Analysis of the temperature evolution The collective energy balance was previously employed in accounting for the temperature evolution upon photoexcitation of the gold nanoparticles,20–23 including the injection and the loss of energy, as shown in eqn (1). MC
dT ¼ Qin Qout dt
(1)
where M and C denote the mass (g) and heat capacity of the liquid (J 1C1 g1) of the heating volume; T and t represent the temperature (1C) and time (seconds); Qin and Qout represent
Fig. 5
(2)
(3)
where DT denotes the temperature gradient between the heating volume and the surroundings. In this work, the fluorescence alteration reflects the temperature change in the heating volume. However, the spatial distribution of the temperature could be slightly inhomogeneous because the solution is not homogenized mechanically. Therefore, there might have been a temperature gradient from the heating core to the outer layer, which was in contact with the bulk solution. Considering the cylindrical volume defined by the propagation of the 532 nm laser, at least two moieties, the core cylinder (m2) and the jacketed layer (m1),
The scheme of the heat release processes, including the core cylinder and jacketed layer.
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can be included to express the heat transport, as shown in Fig. 5. T2 and T1 represent the temperature evolutions of the core cylinder and jacketed layer, and T0 denotes the ambient temperature of the bulk solution before 532 nm exposure, respectively. As a result, the heat transfer processes of the core cylinder and jacketed layer can be expressed in the following form, m2 C
m1 C
m2 dðT2 T1 Þ k2 ðT2 T1 Þ ¼ Iex 1 10O:D: Z M dt (4a)
m1 dðT1 T0 Þ þ k2 ðT2 T1 Þ ¼ Iex 1 10O:D: Z M dt k1 ðT1 T0 Þ
(4b)
where M represents the total mass of the heating volume, and M = m1 + m2; mi and ki denote the masses and the apparent heat transfer coefficients for different heating volumes. Assuming that the fluorescence intensity is proportional to the masses in the core cylinder and jacketed layer, the overall temperature evolution in the heating volume can be expressed in the following form, DTðtÞ ¼ ¼
DFLðtÞ DFL1 ðtÞ DFL2 ðtÞ x¼ xþ x FLðt ¼ 0Þ FLðt ¼ 0Þ FLðt ¼ 0Þ
(5)
m1 m2 DT1 þ DT2 M M
where DT2 = (T2 T0), DT1 = (T1 T0), DT2 DT1 = (T2 T1), and x is the conversion factor for the fluorescence intensity-totemperature change. Subsequently, we obtain the evolution of the temperature in the following form, DTðtÞ
a kgðg þ kÞ bðb þ g kÞð1 ekt Þ 1 ebt k b ðb k Þ ðb þ g Þ (6) Iex 1 10O:D: Z k2 k2 ; g¼ ; and where a¼ ; b¼ m2 C m1 C MC k1 . The mathematical procedures are provided in the k¼ m1 C ESI†. The temporal profiles of the temperature alteration upon excitation of different nanoparticles with varied powers are shown in Fig. S3(a) (ESI†). According to eqn (6), the parameter a is proportional to the excitation power. Thus, DT(t) divided by the excitation power for a given nanoparticle will lead to the identical waveforms if the decay parameters b, g and k are independent of the excitation power, ¼
DTðtÞ 1 Z 1 10O:D: ¼ Iex k b ðb k Þ C M
kgðg þ kÞ kt bt 1e bðb þ g kÞð1 e Þ ðb þ gÞ (7) As shown in the frames of Fig. S3(b) (ESI†), the waveforms DTðtÞ were almost identical for the corresponding nanoparticles, Iex suggesting that this in situ fluorescent thermometer has
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DTðtÞ of gold nanoparticles (O.D. = 0.2 Iex in a 0.2 cm absorption length at 532 nm) in different sizes upon excitation. The normalized traces are shown in the inset. (b) Dots and solid lines represent the observed data and fittings using eqn (7), respectively, for the traces in (a).
Fig. 6
(a) Evolutions of averaged
DTðtÞ for a given Iex nanoparticle upon excitation at various powers are presented in Fig. 6(a). The normalized traces, shown in the inset of Fig. 6(a), did not manifest a significant difference, implying that the proposed mechanism is universally applicable to these measurements. Upon fitting the temporal profiles using eqn (7) and using O.D. = 0.2, the fitted curves agreed with the observations, as shown in Fig. 6(b). Z We thus deduced the parameters b, g, k and individual for a CM given nanoparticle, as listed in Table 1, for further derivation of the relative photothermal transduction efficiencies. sufficient reproducibility. The averaged traces of
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The apparent kinetic parameters, b, g, and k, were (1.34 0.01) 102 s1, (1.37 0.03) 103 s1, and (2.22 0.05) 101 s1, respectively. Accounting for the relationship of b/g = m1/m2, we can deduce the mass ratio of the core cylinder to the jacketed layer to be 10%, since the diameter of the core cylinder is 0.25 mm, i.e., 1/3 of the diameter of the cross-section of the heating cylinder. It is intuitively reasonable to define the two moieties of the heating volume. In addition to the apparent rate coefficients, the absolute photothermal efficiency can be derived in principle. Using the values of C (heat capacity, mJ 1C1 g1) and M (the mass of the solution in the heating volume, g), which were fixed at 4 180 and 1.08 103, respectively, the Z for a 22 nm nanoparticle was 16.2%, smaller than the value ca. 100% for the 20 nm nanoparticle reported by Richardson et al.,21 but larger than the 10% reported by Roper et al.20 This inconsistency is mainly attributed to the improper estimation of the heating volume, which was regarded as a cylinder. The diameter of its cross section was defined by the full-width-half-maximum of the 532 nm beam. If the estimated beam diameter is increased to 1.4 mm, which denotes the full width at 10% intensity, the mass of the excitation volume increases to 3.08 103 g; then the Z can be adjusted to 46.2%. If the excitation loss, ca. 5%, is taken into consideration, as the incident 532 nm was diffracted by the surface of the sample cell, Z can be modified to 48.6%. As a result, the estimated Z = 16.2% of the smallest nanoparticle (22 nm), which exhibits no significant contribution to the scattering in the extinction coefficient,47,48 can be regarded as the lowest limit of the photothermal transduction efficiency. For comparing the photothermal transduction efficiencies of different nanoparticles, the relative Z(Zrel.) with respect to a 22 nm nanoparticle will be suitable for correlating the predicted values by Mie theory. Determining the relative photothermal transduction efficiency of gold nanoparticles The extinction coefficient (eext.) of the nanoparticles includes the absorption (eabs.) and scattering (esca.)47,48 and the ratios of eabs./eext. decrease as the sizes of the nanoparticles increase.22,47 In our experimental configuration, the optical density of the nanoparticles at 532 nm was 0.2 for 2 mm optical length. The extinction coefficient (eext.) was regarded as the summation of eabs. and scattering esca., eext. = eabs. + esca.
(8)
The heat generation attributed to the absorbed photons, Qabs in , can be expressed as follows: e O:D: eabs: abs: ext: Qin ¼ Iex 1 10 (9) Thus, the photothermal transduction efficiency can be expressed as O:D:
eabs:
eext: Qabs: 1 10 Z ¼ in ¼ ext: O:D: Qin 1 10
(10)
Using eabs./eext. (ref. 22) on the basis of Mie theory and O.D. = 0.2 for the 2 mm cell at 532 nm, we are able to derive the theoretical
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Fig. 7 The comparison of observed (squares) and predicted (red line) relative photothermal transducing efficiency Zrel. for gold nanoparticles of different sizes relative to a 22 nm nanoparticle. The horizontal and vertical error bars represent the size dispersion and the standard deviation of Zrel., respectively.
Z (Ztheory), as listed in Table 1. Because we have demonstrated that the estimation of the absolute efficiency becomes impractical due to the uncertainty in the determination of the heating volume, the relative theoretical Z values, Ztheory , with respect to rel. the Ztheory of the 22 nm nanoparticle were derived for further comparison, as listed in Table 1 and schematically shown in Fig. 7. We found that the observed Zrel. satisfactorily agreed with Ztheory , implying that the in situ detection strategy using rel. tryptophan temperature-dependent fluorescence and collective heat transfer analysis could be used to detect the photothermal effects of the nanostructures. In previous studies, the thermocouple was employed to measure alterations in temperature.20–23 However, the relative positions of the thermocouple and the laser propagation are likely to have led to incorrect illustration of the heat transport process and quantification of the photothermal efficiency.22 Measuring the heating process of water droplet containing gold nanoparticles with a thermocouple did not provide sufficient signal-to-noise ratio when the temperature reached the steady state.21 Thermal microscopy is capable of mapping the temperature distribution around the source of heat, but it is difficult to obtain a well-defined temperature map if the temperature increase is less than 1 K.25 In this study, the greatest advantage of utilizing the fluorescence of tryptophan as an in situ thermometer is that it provides a satisfactory signal-tonoise ratio in probing temperature alterations during heating with high reproducibility and a sufficient temperature detectivity of 0.2 1C. If the molecular fluorescent thermometer could be attached to the nanoparticle, it would provide a niche to instantaneously reflect temperature alterations on the surfaces of nanoparticles upon illumination.
Conclusions Utilizing the intrinsic property of the temperature-dependent fluorescence of tryptophan, which decreases as temperature increases, we have successfully quantified the photothermal process of gold nanoparticles of diameters from 22 nm to
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86 nm upon continuous-wave excitation at 532 nm. The temperature-dependent fluorescence of tryptophan, in terms of fluorescence contour and temperature response, is not perturbed in the presence of gold nanoparticles. This method has satisfactory reproducibility and a sufficient temperature detectivity of 0.2 1C. The collective energy balance model successfully illustrates the temporal evolution of the heating process. Although the determination of absolute efficiencies is not easy, owing to the uncertainty in estimating the heating volume, the relative efficiencies of nanoparticles of different sizes are still predictable and satisfactorily agree with theoretical predictions, including the contributions of absorption and scatterings. Coupling spectroscopy with the aromatic amino acid tryptophan as a fluorescent thermometer not only provides an instantaneous in situ probe for monitoring the thermoplasmonic effect of nanostructures but could also be applicable to bio-nano imaging.
Acknowledgements The Ministry of Science and Technology of Taiwan (MOST 1032113-M-007-010-MY2) provided support for this research.
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Cite this: DOI: 10.1039/c5cp02620b
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Quantifying the photothermal efficiency of gold nanoparticles using tryptophan as an in situ fluorescent thermometer† Ming-Jui Chiu and Li-Kang Chu* The photothermal efficiencies, denoting the efficiency of transducing incident light to heat, of gold nanoparticles of different diameters (+ = 22–86 nm) were quantified upon exposure at 532 nm. The fluorescence of tryptophan at 300–450 nm upon 280 nm excitation serves as an in situ fluorescent thermometer to illustrate the evolution of the average temperature change in the heating volume of the nanoparticle solution. The fluorescence intensity decreases as the temperature increases, having a linear gradient of 2.05% fluorescence decrease per degree Celsius increment from 20 to 45 1C. The presence of gold nanoparticles at the nM level does not perturb the temperature-dependent fluorescence of tryptophan in terms of fluorescence contour and temperature response. The heating volume was defined by overlapping the collimated 532 nm laser (+ = 0.83 mm) for exciting the nanoparticles and the 280 nm continuous-wave beam (+ = 0.81 mm) for exciting tryptophan in a 2 mm 2 mm square tube, and the fluorescence was collected perpendicularly to the collinear alignment. This method has satisfactory reproducibility and a sufficient temperature detectivity of 0.2 1C. The profiles of the average temperature evolution of the mixtures containing nanoparticles and tryptophan were derived from the
Received 6th May 2015, Accepted 27th May 2015
evolution of fluorescence and analyzed using collective energy balancing. The relative photothermal
DOI: 10.1039/c5cp02620b
those predicted using Mie theory. The employment of tryptophan as a fluorescent thermometer not
efficiencies for different sizes of gold nanoparticles with respect to the 22 nm nanoparticle agree with only provides an in situ tool to monitor the photothermal effect of nanostructures but is also applicable
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to thermal imaging in biological applications.
Introduction The photothermal effect of metallic nanoparticles upon photoexcitation, i.e., thermo-plasmonics,1–3 has been extensively employed in many aspects, such as drug delivery and release,4–6 thermotherapeutic treatment,7–10 catalysis,11 and water vapor generation.12 Metallic nanoparticles are capable of attenuating incident light via scattering and absorption, as characterized using Mie theory.13,14 The step-wise energy cascading processes, including the electron cloud thermalization (within B100 fs),15 electron–phonon thermalization (B1 ps),16,17 and external heat release to surroundings,1,18 are used to illustrate heat transfer upon ultrafast pulsed excitation.19 Thermal equilibrium inside the nanoparticles can be achieved quickly, within 0.1 ns to 1 ms, for nanoparticles of diameters of 10 nm to 1 mm.18 As the
Department of Chemistry, National Tsing Hua University, 101, Sec. 2, Kuang-Fu Rd., Hsinchu 30013, Taiwan. E-mail: [email protected]; Fax: +886-3-5711082; Tel: +886-3-5715131 ext. 33396 † Electronic supplementary information (ESI) available. See DOI: 10.1039/ c5cp02620b
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nanoparticles are irradiated with continuous illumination, the evolution of the temperature of the surroundings over a prolonged period can be depicted using the collective energy balance.20–23 In the past decade, miscellaneous experimental methods were employed to qualitatively12,24–27 and quantitatively12,20–24 monitor the photothermal effect. Hogan et al. observed steam generation upon excitation of a gold nanoparticle solution and demonstrated that light absorption creates intense localized heating, which efficiently vaporizes the surrounding liquids.24 Neumann et al. demonstrated that 80% of absorbed sunlight is converted into water vapor, and merely 20% of absorbed light energy is converted to heat in the surrounding liquid.12 The thermal imaging in the vicinity of the nanostructure arrays was recorded on the basis of the thermal-induced refractive index variation.25,26 Bendix et al. measured the heating process by using molecular partitioning and by employing the phase dependent quantum yield of DilC18 fluorophores in lipid bilayers.27 Quantitative approaches, especially those employing thermocouples, have been used to reflect heat generation upon continuous photoexcitation of gold nanoparticle suspensions by monitoring the temperature change and analyzing the energy balance.20–23
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Roper et al. recorded the heating process of a 20 nm nanoparticle suspension and derived that the efficiency of transducing incident resonant light to heat (Z) was less than 10%.20 However, Richardson et al. recorded the evolution of the temperature change of an aqueous droplet containing 20 nm nanoparticles upon photoexcitation using a thin thermocouple and demonstrated that the transduction efficiency Z is close to 100%.21 Jiang et al. have demonstrated the size-dependent Z of gold nanoparticles, but the observed values of Z were lower than those predicted by Mie theory.22 It should be noticed that the thermocouples were not placed in the heating volume;20–22 that is, these temperature detections were not the result of in situ probing. Fluorescent materials have been extensively employed in determining the temperature in materials and biomolecular applications.28,29 In this study, we have established experimental and analytical methods to illustrate the photothermal transduction efficiency of gold nanoparticles using a temperature-sensitive fluorescing molecule, tryptophan, as an in situ thermometer. Previously, organic polymers30,31 and organic molecules32,33 have been extensively employed in monitoring the temperature of a solution due to fluorescence enhancement or quenching as the temperature increases. The fluorescent molecule perylene was embedded in different film matrices to illustrate the photothermal phenomenon of varied nanostructures.34,35 The photoluminescence of quantum dots were also employed in luminescence thermometry.36,37 For instance, the fluorescence intensity of rhodamine B exhibits sufficient temperature sensitivity (3.4% 1C1).38 However, its spectral overlap with the resonant band of gold spherical nanoparticles does not allow it to function as a molecular fluorescent thermometer using a spectroscopic method. The fluorescence of tryptophan above 300 nm upon excitation at 280 nm, however, avoids spectral overlap of the plasmonic band of gold nanoparticles at 530 nm. It exhibits a sufficient temperature response as the temperature is increased,39 and it has been used in understanding protein folding dynamics.40,41 In this study, we utilized tryptophan fluorescence to monitor the average temperature change of the heating volume as the gold nanoparticle suspensions were irradiated using a continuous 532 nm laser and analyzed the evolution of the average temperature using a collective energy balance model. For gold nanoparticles of various sizes, the observed relative photothermal efficiencies were consistent with those predicted by Mie theory. Tryptophan was successfully used as an in situ fluorescent thermometer in quantifying the photothermal effects of the nanostructures. This approach could also be applicable to thermal bio-imaging.
Experimental section Materials The synthesis of gold nanoparticles of different sizes followed the protocol of the seeding growth method,42 with partial modification. The 22 nm gold seed was synthesized by adding 0.5 ml of 0.029 M chloroauric acid (HAuCl43H2O, Alfa Aesar, 99.99%) and 0.1 g sodium citrate (Na3C6H5O7, J. T. Baker, 499%) to
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50 ml DI water simultaneously, followed by vigorous stirring at 80 1C. The color of the solution rapidly turned to dark purple, and then gradually turned to ruby red over 40 minutes, indicating that the gold ions (Au3+) were completely reduced to neutral gold atoms. After the seed solution was prepared, the growth medium was used to manufacture the larger gold nanoparticles. To six conical flasks, labeled as A to F, 9 ml of 46.5 mM cetyltrimethylammonium bromide (CTAB, Sigma Aldrich, 499%) and 0.1 ml of 0.029 M HAuCl4 were added, and the mixture was stirred at 30 1C. Then 0, 1.2, 0.5, 0.25, and 0.125 ml of 22 nm gold seed were separately added to flasks A to E, and 50 ml of 0.1 M ascorbic acid (J. T. Baker, 499.5%) solution was added immediately to each flask. After 10 minutes of stirring, the solutions turned dark red, indicating the completion of the growth of nanoparticles of different sizes. In flask F, 1 ml of solution B and 50 ml of 0.1 M ascorbic acid solution were added and the mixture stirred for 10 minutes. Spherical gold nanoparticles with diameters of 22, 31, 47, 59, 74, and 86 nm were prepared. The as-prepared gold nanoparticles were concentrated to an optical density of 1.0 for 0.2 cm optical length at 532 nm using centrifugation (Model 6500, Kubota) at 25 1C for further experiments. 0.1 ml of 5 mM tryptophan solution and 0.25 ml of O.D. = 4 (for 1 cm absorption length) nanoparticle solution were mixed and diluted with DI water to a total volume of 1 ml. The samples contained 0.5 mM tryptophan, and O.D. = 1 of nanoparticles were prepared for 1 cm absorption length. The corresponding concentrations were about 0.71, 0.24, 0.06, 0.03, 0.01, and 0.008 nM for 22, 31, 47, 59, 74, and 86 nm gold nanoparticles, respectively. The characterization of the morphology of the nanoparticles is discussed in Results and discussions. Steady-state absorption and fluorescence spectra The steady-state ultraviolet-visible (UV-Vis) absorption spectra were recorded using a spectrometer (USB4000-UV-Vis, Ocean Optics), and temperature-programmable fluorescence (FL) spectra were collected using an F-7000 Hitachi spectrophotometer coupled with a thermostat (qpod TC125, Quantum Northwest) to maintain the temperature within a range of 20–45 1C, within 0.1 1C variation at the set points. The emission contours upon excitation of tryptophan at 280 nm were recorded from 300 to 450 nm with scanning steps of 0.2 nm. TEM imaging The sizes and morphologies of the gold nanoparticles were characterized using a thermal-type field emission scanning electron microscope (JSM-7000F, JEOL) and a high-resolution transmission electron microscope (JEM-2100, JEOL). Experimental setup for probing the evolution of the temperature change using fluorescence of tryptophan upon excitation at 280 nm The experimental module included a continuous-wave 532 nm laser for exciting gold nanoparticles, continuous-wave 280 nm light for exciting tryptophan, fluorescence collecting optics, and a data acquisition system, as shown in Fig. 1(a). The continuous-wave
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Fig. 1 (a) Experimental setup for monitoring the temperature change upon continuous excitation of gold nanoparticle suspensions. (b) Profiling the diameters of the 280 nm beam passing through a 100 mm pinhole at different distances from the center of the pinhole.
532 nm diode laser (DPGL-2100, Casix) provided a power of 102 mWatt in a beam diameter of 0.83 mm (full-width at half maximum) to serve as the excitation source. The incident intensity of the 532 nm laser was adjusted using the neutral density filters (FRQ-ND02, 03, and 04, Newport) and monitored using a power meter (818P-015-19, Newport). The laser was introduced to a sample cell, a 2 mm 2 mm (inner) square quartz tube (QST-2-75, Friedrick & Dimmock Inc.). The 280 nm light was provided by a solar generator (Model 70050, Newport) upon wavelength selection using a monochromator (Grating Model 77309, Newport). The 280 nm beam passed through a 100 mm pinhole (HPH-0100M, Unice E-O Service Inc.) to define the probing cross section and propagated opposite to the 532 nm beam. The diameters of the 280 nm beam at different distances from the pinhole were profiled by scanning the position of the photomultiplier (R928, Hamamatsu), which was perpendicular
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to the propagation direction of the 280 nm beam, as shown in Fig. 1(b). A 100 mm pinhole (P100S, Thorlabs Inc.) was mounted in front of the PMT to define the spatial scanning steps. At the center of the sample cell, 9 mm from the pinhole, the diameter of the 280 nm beam was 0.81 mm, slightly smaller than that of the 532 nm excitation beam (0.83 mm), to ascertain that the fluorescence intensity alteration originated from the heating volume upon excitation of the nanoparticles. The fluorescence of tryptophan was filtered using two edge filters (Model 84704, Edmund) at 450 nm to remove most of the scattering 532 nm light and a bandpass filter (BG–3–25.4, Lambda) to define the detection wavelength at 300–450 nm in front of a photomultiplier (R928, Hamamatsu). The electronic signal was recorded using an oscilloscope (WaveSurfer 24MXs-B, LeCroy). The exposure period was controlled to 500 seconds using a mechanical shutter (VS25S2T1, Uniblitz).
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Results and discussions In this work, we designed an in situ method to quantify the relative photothermal efficiencies (Zrel.) of different sizes of gold nanoparticles upon continuous excitation at 532 nm using tryptophan as a fluorescent thermometer. Steady-state extinction spectroscopy and electron microscopy were used to characterize the morphologies of the gold nanoparticles. Fluorescence spectroscopy was used to illustrate the temperature change as the mixtures of nanoparticles and tryptophan were irradiated with the modulated cw diode laser at 532 nm. By analyzing the temporal profiles of the temperature evolutions using a collective heat transport model, we derived the relative photothermal efficiencies of the gold nanoparticles, which agreed with the predictions of Mie theory. Morphology and size distribution of the gold nanoparticles When light travels through metallic nanoparticle suspensions, the intensity of the light can be attenuated due to absorption and scattering. In order to avoid the confusion of these two effects, the conventional absorption spectrum was replaced by the extinction spectrum, referring to the logarithm of the intensity ratio of the incident light before and after passing through the sample. The steady-state extinction spectra of the gold nanoparticles are shown in Fig. 2(a). The concentrations of nanoparticles were controlled to manifest an optical density (O.D.) of 1.0 for 10 mm optical length at 532 nm, equivalent to O.D. = 0.2 for further photoexcitation experiments using a 2 mm 2 mm square tube. The normalized extinction contours, shown in the inset of Fig. 2(a), exhibited a redshift as the nanoparticles increased in size. In addition to the spectroscopic characterization, the TEM and SEM patterns were collected to confirm the size distributions of these nanoparticles, as shown in Fig. 2(b). The TEM and SEM patterns indicated that the gold nanoparticles were spherical and well dispersed. The histograms of the size distributions, based on counting 100 particles for each size, are shown in the insets. The average sizes and the corresponding size deviations are listed in Table 1. A molecular fluorescent thermometer using tryptophan Before the mixtures of gold nanoparticles and tryptophan were prepared, the concentration of tryptophan had to be optimized to maximize the fluorescence intensity without saturation. Then the fluorescence of tryptophan in the presence of nanoparticles at different temperatures needed to be determined in order to correctly determine the temperature evolution during the 532 nm exposure of the nanoparticles. 1. Steady-state fluorescence. Gally and Edelman found a decrease in the fluorescence of tryptophan aqueous solution as the temperature was increased at a roughly linear rate of ca. 2% per degree Celsius in the range of 25–45 1C.43 Stevenson et al. have demonstrated that the decrease in tryptophan fluorescence is related to the non-radiative photoionization process and that the yield increases by almost five times, from 11 1C to 82 1C,39 suggesting that the fluorescence of tryptophan has sufficient temperature sensitivity to serve as a fluorescent thermometer.
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Fig. 2 (a) Steady-state extinction spectra of gold nanoparticles of different diameters, with normalized traces in the inset. (b) The electron microscopy images of the nanoparticles mentioned above. The histograms of the size distributions are shown in the insets.
The fluorescence contours of tryptophan at different concentrations and the relationship of the integrated intensity at 300–450 nm and the tryptophan concentrations are shown in Fig. 3(a) and (b), respectively. The normalized emission contours were independent of the concentration (inset of Fig. 3(a)), indicating an inconsequential aggregation of tryptophan. Before saturation, 0.5 mM of tryptophan was used to maximize the fluorescence intensity in the further experiments. Subsequently, temperature-programmable fluorescence contours for different sizes of gold nanoparticles and pure tryptophan were collected, as shown in Fig. S1 in the ESI†. The temperature was increased to within 20 1C of room temperature to prevent the non-linear response.39 The normalized contours, shown in the upper insets in Fig. S1(a)–(g) (ESI†), were almost identical in the 20–45 1C range, indicating that the integrated fluorescence
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Z (in mJ1 1C) for different nanoCM particles after fitting the traces in Fig. 6(b) using eqn (7)
Table 1
The observed values of
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Diameter of nanoparticle/nm 22 31 47 59 74 86
3 2 3 6 4 5
Z 102 CM 3.58 3.60 3.18 3.15 2.58 2.03
0.10 0.11 0.09 0.10 0.07 0.07
Zrel. (%)
Ztheory (%)
Ztheory rel. (%)
100.0 4.0 100.4 4.1 88.8 3.6 88.0 3.9 72.1 2.8 56.7 2.5
99.0 97.2 90.8 82.8 69.9 58.5
100.0 98.2 91.7 83.7 70.6 59.1
a
Z for different nanoparticles with CM respect to that of a 22 nm nanoparticle, assuming that M and C were the same in the measurements. Error propagation was taken into using consideration. The standard deviation of Zrel. can be derived 2 2 ! A DZ 2 DA DB ¼ þ the following equation: Z ¼ ; . The fracB Z A B tional error in Z is the square root of the sum of the squares of the fractional in its parts. Take Zrel. of 86 nm, for example: errors 0 1 ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi s Z 2 2 2:03 0:07 0:07 0:10 C M 86nm 86nm A ¼ 0:567 @1 ¼ þ Zrel: ¼ Z 3:58 0:10 2:03 3:58 C M 22nm a
Zrel. denotes the ratios of
¼ ð56:7 2:5Þ%:
intensity could serve as a fluorescence thermometer without spectral shift. In addition, the normalized fluorescence contours of tryptophan in different nanoparticle samples at 25 1C were almost identical, as shown in Fig. 3(c), indicating an insignificant interaction between the nanoparticles and the tryptophan. The ratios of the fluorescence intensity difference (DIFL/IFL 0 where DIFL = IFL IFL 0 ) with respect to fluorescence intensity at room temperature (IFL 0 ) integrated at 300–450 nm; plotted against the temperature difference in the lower insets in Fig. S1(a)–(g) (ESI†), they showed satisfactory linearity. The relationships of the nanoparticle sizes and the slopes are plotted in Fig. S1(h) (ESI†). We found that the slopes were independent of the nanoparticle sizes and manifested a value of ca. 2% per degree Celsius, similar to the intrinsic property of pure tryptophan solution. As a result, the ratios of the intensity differences and the temperature differences for all samples were plotted and fitted using a single linear relationship, as shown in Fig. 3(d). A fitted straight line agreed with the observed data and revealed a slope of 2.05% for the fluorescence decrease per increase in degree Celsius (% 1C1). This slope was used to convert the relative fluorescence difference to the temperature change.
Fig. 3 (a) The steady-state emission contours of tryptophan at different concentrations upon 280 nm excitation at 25 1C. The normalized contours are shown in the inset. (b) The relationship between the concentration of tryptophan and the integrated fluorescence intensities at 300–450 nm. (c) The normalized fluorescence contours of tryptophan solutions at 25 1C in the presence of gold nanoparticles of different sizes. The concentration of tryptophan was 0.5 mM. The concentrations of nanoparticles were controlled such that the optical density was 1.0 for an optical length of 1 cm at 532 nm. (d) The linear relationship FL FL FL between the relative change of the integrated fluorescence intensity at 300–450 nm (DIFL/IFL 0 , DI = I I0 ) and the temperature changes (DT = T Troom) with respect to room temperature for the samples mentioned above. Dots and solid lines represent the observed data and linear fitting, respectively.
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In addition, we also excluded the contribution of nanoparticles in altering the fluorescence properties of tryptophan upon 280 nm excitation. Because the concentration of the gold nanoparticles was at the nM level, the averaged inter-particle distance between nanoparticles was about 1 mm. Therefore, nanoparticle fluorescence quenching would play an insignificant role in perturbing the fluorescence property of the bulk tryptophan solution, since their effective distances are only several nanometers.44–46 In addition, the excitation of gold nanoparticles at 280 nm provides infinitesimal fluorescence intensity as compared to tryptophan under our experimental conditions, as shown in Fig. S2 (ESI†). As a result, the emission at 300–450 nm was solely attributed to the fluorescence of tryptophan upon excitation at 280 nm. 2. Deriving the evolution of the temperature difference from the fluorescence change. The transformation of the observed electronic signal of the photomultiplier tube to the evolution
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of temperature alteration is schematically displayed in Fig. 4. A stationary voltage attributed to the tryptophan fluorescence upon 280 nm cw illumination was recorded, as indicated by the purple segment of the waveform in Fig. 4(a). As the 532 nm laser was turned on, the fluorescence started decreasing due to the increase in temperature and leveled off, as shown by the green segment. When the 532 nm laser was turned off, the fluorescence returned to its original intensity, shown in a purple segment. However, scattering of the 532 nm laser could not be completely avoided, so a blank experiment was performed. In the absence of 280 nm illumination, the voltage from the PMT remained null, as shown in Fig. 4(b). Once the 532 nm laser was turned on, the voltage increased steeply and leveled off until the 532 nm laser was turned off. Generally, the contribution of 532 nm scattering was less than 1/100 of the voltage from tryptophan fluorescence.
Fig. 4 The schematic procedure to derive the temperature evolution from the evolution of the fluorescence intensity change of tryptophan. See details in the text.
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After temporal profiles of the fluorescence and scattering were collected, the raw electronic signal solely attributed to the fluorescence alteration, as shown in Fig. 4(c), was derived by subtracting the waveform in Fig. 4(a) from that in Fig. 4(b). The magnitude of the voltage evolution (V(t)) before introducing the 532 nm laser was averaged to serve as the fluorescence intensity at time zero (V0). By calculating (V(t) V0)/V0 and multiplying the conversion factor (x) for the fluorescence intensity-to-temperature change derived from the slope in Fig. 3(d), the evolution of the temperature was obtained (Fig. 4(d)). The detectivity of the temperature alteration was about 0.2 1C when accounting for the non-heating period. The observed evolutions of the temperature upon photoexcitation of nanoparticles of different sizes at different excitation powers are shown in Fig. S3 (ESI†). Establishing an appropriate kinetics model enabled us to estimate the heat transport process and the photothermal efficiency of the gold nanoparticles.
the power injection via the photon absorption by the nanoparticles and the heat loss to the surroundings. Qin can be evaluated by accounting for the incident excitation laser power Iex (Watts), the optical density (O.D.) of the nanoparticle suspensions, and the photothermal transduction efficiency Z, Qin = Iex(1 10O.D.)Z
O.D. was maintained at 0.2 for a 2 mm optical length in the square quartz tube. The incident power of the 532 nm laser of a circular cross section (diameter = 0.83 mm, defined by the full width at half maximum) was adjusted to 45.8–67.6 mW. Accordingly, the heating volume was about 1.08 mm3. As for the energy loss during the laser exposure, an apparent rate coefficient k, including the heat transfer coefficient of the solvent and the area of the cross section perpendicular to the heating cylinder,21,22 should be accounted for by Qout, Qout = kDT
Analysis of the temperature evolution The collective energy balance was previously employed in accounting for the temperature evolution upon photoexcitation of the gold nanoparticles,20–23 including the injection and the loss of energy, as shown in eqn (1). MC
dT ¼ Qin Qout dt
(1)
where M and C denote the mass (g) and heat capacity of the liquid (J 1C1 g1) of the heating volume; T and t represent the temperature (1C) and time (seconds); Qin and Qout represent
Fig. 5
(2)
(3)
where DT denotes the temperature gradient between the heating volume and the surroundings. In this work, the fluorescence alteration reflects the temperature change in the heating volume. However, the spatial distribution of the temperature could be slightly inhomogeneous because the solution is not homogenized mechanically. Therefore, there might have been a temperature gradient from the heating core to the outer layer, which was in contact with the bulk solution. Considering the cylindrical volume defined by the propagation of the 532 nm laser, at least two moieties, the core cylinder (m2) and the jacketed layer (m1),
The scheme of the heat release processes, including the core cylinder and jacketed layer.
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can be included to express the heat transport, as shown in Fig. 5. T2 and T1 represent the temperature evolutions of the core cylinder and jacketed layer, and T0 denotes the ambient temperature of the bulk solution before 532 nm exposure, respectively. As a result, the heat transfer processes of the core cylinder and jacketed layer can be expressed in the following form, m2 C
m1 C
m2 dðT2 T1 Þ k2 ðT2 T1 Þ ¼ Iex 1 10O:D: Z M dt (4a)
m1 dðT1 T0 Þ þ k2 ðT2 T1 Þ ¼ Iex 1 10O:D: Z M dt k1 ðT1 T0 Þ
(4b)
where M represents the total mass of the heating volume, and M = m1 + m2; mi and ki denote the masses and the apparent heat transfer coefficients for different heating volumes. Assuming that the fluorescence intensity is proportional to the masses in the core cylinder and jacketed layer, the overall temperature evolution in the heating volume can be expressed in the following form, DTðtÞ ¼ ¼
DFLðtÞ DFL1 ðtÞ DFL2 ðtÞ x¼ xþ x FLðt ¼ 0Þ FLðt ¼ 0Þ FLðt ¼ 0Þ
(5)
m1 m2 DT1 þ DT2 M M
where DT2 = (T2 T0), DT1 = (T1 T0), DT2 DT1 = (T2 T1), and x is the conversion factor for the fluorescence intensity-totemperature change. Subsequently, we obtain the evolution of the temperature in the following form, DTðtÞ
a kgðg þ kÞ bðb þ g kÞð1 ekt Þ 1 ebt k b ðb k Þ ðb þ g Þ (6) Iex 1 10O:D: Z k2 k2 ; g¼ ; and where a¼ ; b¼ m2 C m1 C MC k1 . The mathematical procedures are provided in the k¼ m1 C ESI†. The temporal profiles of the temperature alteration upon excitation of different nanoparticles with varied powers are shown in Fig. S3(a) (ESI†). According to eqn (6), the parameter a is proportional to the excitation power. Thus, DT(t) divided by the excitation power for a given nanoparticle will lead to the identical waveforms if the decay parameters b, g and k are independent of the excitation power, ¼
DTðtÞ 1 Z 1 10O:D: ¼ Iex k b ðb k Þ C M
kgðg þ kÞ kt bt 1e bðb þ g kÞð1 e Þ ðb þ gÞ (7) As shown in the frames of Fig. S3(b) (ESI†), the waveforms DTðtÞ were almost identical for the corresponding nanoparticles, Iex suggesting that this in situ fluorescent thermometer has
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DTðtÞ of gold nanoparticles (O.D. = 0.2 Iex in a 0.2 cm absorption length at 532 nm) in different sizes upon excitation. The normalized traces are shown in the inset. (b) Dots and solid lines represent the observed data and fittings using eqn (7), respectively, for the traces in (a).
Fig. 6
(a) Evolutions of averaged
DTðtÞ for a given Iex nanoparticle upon excitation at various powers are presented in Fig. 6(a). The normalized traces, shown in the inset of Fig. 6(a), did not manifest a significant difference, implying that the proposed mechanism is universally applicable to these measurements. Upon fitting the temporal profiles using eqn (7) and using O.D. = 0.2, the fitted curves agreed with the observations, as shown in Fig. 6(b). Z We thus deduced the parameters b, g, k and individual for a CM given nanoparticle, as listed in Table 1, for further derivation of the relative photothermal transduction efficiencies. sufficient reproducibility. The averaged traces of
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The apparent kinetic parameters, b, g, and k, were (1.34 0.01) 102 s1, (1.37 0.03) 103 s1, and (2.22 0.05) 101 s1, respectively. Accounting for the relationship of b/g = m1/m2, we can deduce the mass ratio of the core cylinder to the jacketed layer to be 10%, since the diameter of the core cylinder is 0.25 mm, i.e., 1/3 of the diameter of the cross-section of the heating cylinder. It is intuitively reasonable to define the two moieties of the heating volume. In addition to the apparent rate coefficients, the absolute photothermal efficiency can be derived in principle. Using the values of C (heat capacity, mJ 1C1 g1) and M (the mass of the solution in the heating volume, g), which were fixed at 4 180 and 1.08 103, respectively, the Z for a 22 nm nanoparticle was 16.2%, smaller than the value ca. 100% for the 20 nm nanoparticle reported by Richardson et al.,21 but larger than the 10% reported by Roper et al.20 This inconsistency is mainly attributed to the improper estimation of the heating volume, which was regarded as a cylinder. The diameter of its cross section was defined by the full-width-half-maximum of the 532 nm beam. If the estimated beam diameter is increased to 1.4 mm, which denotes the full width at 10% intensity, the mass of the excitation volume increases to 3.08 103 g; then the Z can be adjusted to 46.2%. If the excitation loss, ca. 5%, is taken into consideration, as the incident 532 nm was diffracted by the surface of the sample cell, Z can be modified to 48.6%. As a result, the estimated Z = 16.2% of the smallest nanoparticle (22 nm), which exhibits no significant contribution to the scattering in the extinction coefficient,47,48 can be regarded as the lowest limit of the photothermal transduction efficiency. For comparing the photothermal transduction efficiencies of different nanoparticles, the relative Z(Zrel.) with respect to a 22 nm nanoparticle will be suitable for correlating the predicted values by Mie theory. Determining the relative photothermal transduction efficiency of gold nanoparticles The extinction coefficient (eext.) of the nanoparticles includes the absorption (eabs.) and scattering (esca.)47,48 and the ratios of eabs./eext. decrease as the sizes of the nanoparticles increase.22,47 In our experimental configuration, the optical density of the nanoparticles at 532 nm was 0.2 for 2 mm optical length. The extinction coefficient (eext.) was regarded as the summation of eabs. and scattering esca., eext. = eabs. + esca.
(8)
The heat generation attributed to the absorbed photons, Qabs in , can be expressed as follows: e O:D: eabs: abs: ext: Qin ¼ Iex 1 10 (9) Thus, the photothermal transduction efficiency can be expressed as O:D:
eabs:
eext: Qabs: 1 10 Z ¼ in ¼ ext: O:D: Qin 1 10
(10)
Using eabs./eext. (ref. 22) on the basis of Mie theory and O.D. = 0.2 for the 2 mm cell at 532 nm, we are able to derive the theoretical
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Fig. 7 The comparison of observed (squares) and predicted (red line) relative photothermal transducing efficiency Zrel. for gold nanoparticles of different sizes relative to a 22 nm nanoparticle. The horizontal and vertical error bars represent the size dispersion and the standard deviation of Zrel., respectively.
Z (Ztheory), as listed in Table 1. Because we have demonstrated that the estimation of the absolute efficiency becomes impractical due to the uncertainty in the determination of the heating volume, the relative theoretical Z values, Ztheory , with respect to rel. the Ztheory of the 22 nm nanoparticle were derived for further comparison, as listed in Table 1 and schematically shown in Fig. 7. We found that the observed Zrel. satisfactorily agreed with Ztheory , implying that the in situ detection strategy using rel. tryptophan temperature-dependent fluorescence and collective heat transfer analysis could be used to detect the photothermal effects of the nanostructures. In previous studies, the thermocouple was employed to measure alterations in temperature.20–23 However, the relative positions of the thermocouple and the laser propagation are likely to have led to incorrect illustration of the heat transport process and quantification of the photothermal efficiency.22 Measuring the heating process of water droplet containing gold nanoparticles with a thermocouple did not provide sufficient signal-to-noise ratio when the temperature reached the steady state.21 Thermal microscopy is capable of mapping the temperature distribution around the source of heat, but it is difficult to obtain a well-defined temperature map if the temperature increase is less than 1 K.25 In this study, the greatest advantage of utilizing the fluorescence of tryptophan as an in situ thermometer is that it provides a satisfactory signal-tonoise ratio in probing temperature alterations during heating with high reproducibility and a sufficient temperature detectivity of 0.2 1C. If the molecular fluorescent thermometer could be attached to the nanoparticle, it would provide a niche to instantaneously reflect temperature alterations on the surfaces of nanoparticles upon illumination.
Conclusions Utilizing the intrinsic property of the temperature-dependent fluorescence of tryptophan, which decreases as temperature increases, we have successfully quantified the photothermal process of gold nanoparticles of diameters from 22 nm to
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86 nm upon continuous-wave excitation at 532 nm. The temperature-dependent fluorescence of tryptophan, in terms of fluorescence contour and temperature response, is not perturbed in the presence of gold nanoparticles. This method has satisfactory reproducibility and a sufficient temperature detectivity of 0.2 1C. The collective energy balance model successfully illustrates the temporal evolution of the heating process. Although the determination of absolute efficiencies is not easy, owing to the uncertainty in estimating the heating volume, the relative efficiencies of nanoparticles of different sizes are still predictable and satisfactorily agree with theoretical predictions, including the contributions of absorption and scatterings. Coupling spectroscopy with the aromatic amino acid tryptophan as a fluorescent thermometer not only provides an instantaneous in situ probe for monitoring the thermoplasmonic effect of nanostructures but could also be applicable to bio-nano imaging.
Acknowledgements The Ministry of Science and Technology of Taiwan (MOST 1032113-M-007-010-MY2) provided support for this research.
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