Carlos J. Bueno
Alejo
a,
Chiara
Fasciani
a,
Michel
Grenier
a,
José Carlos
Netto-Ferreira
*ab and
Juan C.
Scaiano
*a
aCentre for Catalysis Research and Innovation, Department of Chemistry, University of Ottawa, 10 Marie Curie, Ottawa K1N 6N5, Canada. E-mail: tito@photo.chem.uottawa.ca
bDepartamento de Química, Universidade Federal Rural do Rio de Janeiro, Seropédica, 23851-970, Rio de Janeiro, Brazil. E-mail: josecarlos@photo.chem.uottawa.ca
First published on 8th September 2011
Plasmon excitation (532 nm) of gold nanoparticles in the presence of resazurin and hydroxylamine leads to their photocatalytic reduction to resorufin with great efficiency. In the case of laser excitation under laser-drop conditions the process is essentially complete following an ∼8 ns laser pulse at 532 nm. Excitation with LED sources at ∼530 nm proves to be a simple and cost efficient way to promote plasmon-assisted reactions. We propose that the catalytic reaction is thermally activated by the gold nanoparticle and takes advantage of the high temperatures achievable under plasmon excitation.
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Scheme 1 |
Nanoparticles are catalysts for many reactions; given their dimensions, these particles show some properties that differ from those of the bulk material. In the case of gold nanoparticles (AuNP), one of these properties is the surface plasmon resonance band (SPB) that provides the nanoparticle with a characteristic absorption band, located at ∼530 nm for spherical AuNP. The excitation of this band can affect molecules located near the AuNP in several ways. It can lead to transmitter/receiver antenna effects,9 induce electronic excitation10 and generate highly localized heat that can enhance the catalytic properties of the nanoparticles11–13 or initiate other chemical changes.11,14 Recent reviews show the great potential of nanoparticles for simultaneous molecular imaging and phototherapy15 as well as how the strong surface plasmon resonance band displayed by these nanoparticles can be used in a wide range of applications in catalysis, optics or chemical sensing.16
In this work, we carried out studies on the plasmon-mediated reduction of resazurin to resorufin by hydroxylamine, catalyzed by gold nanoparticles at room or higher temperatures. For this purpose we used LED and laser-drop excitation; the techniques were developed in our laboratory12,17 and are based on the irradiation of small drops of solution with 532 nm laser pulses that are capable of exciting the SPB of the AuNP.
The irradiated samples were collected in a cuvette (NaOH was added to ensure a basic pH), diluted to an absorbance of 0.1 at 532 nm (Cary-50-Bio UV-visible spectrophotometer) and their fluorescence examined in a spectrofluorimeter (Photon Technology International, PTI).
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Fig. 1 Fluorescence emission spectra for resazurin (blue) and resorufin (red). Both solutions with 0.1 absorbance at the excitation wavelength (532 nm) under basic conditions. The red arrow indicates the emission due to traces of resorufin in the resazurin sample. |
Solutions containing 1 mM resazurin and excess of NH2OH (2 mM) in water were added to an aqueous solution of AuNP, typically around ∼1 nM, and then irradiated (λ = 532 nm, 50 or 80 mJ per pulse) employing the laser-drop system. An instantaneous change in colour was observed upon firing the laser, with the initial reddish solution becoming bright orange and showing strong emission. The drops collected after the irradiation were diluted and their fluorescence spectrum was recorded, with the emission observed being consistent with the formation of the strongly fluorescent resorufin (see Scheme 1). Fig. 2 shows pictures of the drops photographed before and during laser excitation.
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Fig. 2 Pictures of the drop before (resazurin) and during laser excitation (resorufin); the bright point on the left of the drops is due to a reflection of the light source illuminating the drop, and the bright orange colour of the right drop incorporates extensive product fluorescence. |
To evaluate the effect of changes in the number of laser shots and in the laser power, similar laser-drop experiments as described above were performed employing 1 or 10 laser shots per drop, at 50 and 80 mJ per pulse. When only one shot was delivered to the drop, the laser was concentrated and focused inside the drop, leading to its explosion as shown in the ESI.† On the other hand, for experiments in which 10 shots per drop were delivered, the laser beam was concentrated but not focused into the drop in order to avoid its explosion before reaching 10 shots of irradiation.
The plot in Fig. 3 clearly shows that independent of the conditions employed in the laser-drop experiment, resorufin formation in the presence of AuNP is very efficient, as it is shown by its highly intense fluorescence emission during the first laser shot. The emission intensity after 10 shots per drop (recorded after the completion of the experiment) is practically the same compared to that after a single shot per drop, indicating that one laser pulse achieves near-complete conversion of resazurin to resorufin. In the same way, at different laser powers the fluorescence emission intensity is very similar, from which one can conclude that 50 mJ per drop is adequate for an essentially quantitative reaction following a single laser shot. Remarkably, this means that 50000 to 100
000 resazurin molecules are reduced per AuNP with a single 10 ns laser pulse!
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Fig. 3 Percent fluorescence emission increment relative to the maximum fluorescence emission of resorufin (λ = 582 nm) for three different experiments: (a) different laser energies (1 s/d); (b) different number of shots (50 mJ per pulse); (c) absence and presence of AuNP (50 mJ per pulse, 1 s/d); conditions for the experiments: resazurin 1 mM, NH2OH 2 mM and AuNP 1.4 nM, exception made for the control where AuNP were not employed. The individual error bars in each column show the errors within a single set of experiments, while the dashed error bar shows the reproducibility between different sets of experiments (‘a’, ‘b’ and ‘c’), frequently run on different days. |
To further confirm that the gold nanoparticles play a key role in the reduction of resazurin to resorufin, this reaction was performed in the absence of AuNP employing the laser-drop technique. Fig. 3 clearly shows that no conversion was obtained in the absence of the nanoparticles when this reaction is compared to that performed under the same conditions (50 mJ per pulse, 1 shot-per-drop), this time in the presence of AuNP. These results lead to the conclusion that the AuNP are required for the reaction to take place in this time scale. It is also important to note that even though a reasonable absorption at the irradiation wavelength (λ = 532 nm) is displayed by the resazurin solution due to its high concentration, no indication of resorufin formation could be observed under laser drop irradiation in the absence of AuNP.
We note that a few comparative experiments using triethylamine as a reducing agent showed only a very small fluorescence enhancement under the same laser drop exposure conditions where NH2OH causes a major fluorescence increase (see ESI†).
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Fig. 4 (Top) Kinetic traces of the experiments in the LED irradiator and the thermal bath. Solutions of resazurin (1 mM), NH2OH (2 mM) and AuNP (1.4 nM), were submitted to either the LED irradiator or a thermal bath. Samples were taken every 2 min, diluted to 0.1 absorbance and analyzed in a spectrofluorimeter to record its emission spectrum. The experiment was done at two different power settings of the LED irradiator (0.9 and 0.45 A) and at two different temperatures of the thermal bath (80 and 50 °C). (Bottom) Fluorescence emission at a given time (10 min) for the different experiments (except the LD experiment in which it corresponds to a single ∼8 ns exposure). |
Knowing that the AuNP and NH2OH19 are necessary for the reaction to occur, we decided to investigate the effect of varying the concentration of both, gold nanoparticles and NH2OH, on the reaction efficiency. For this purpose, solutions with different concentrations of NH2OH (from 0.5 to 2 mM) and AuNP (0.7 and 1.4 nM) were prepared and irradiated in the LED photoreactor. For these experiments the laser drop technique is not the best technique because the reaction is so fast that it was not possible to obtain a reasonable kinetic profile for the process. Fig. 5 shows the fluorescence intensity of the resorufin formed after LED irradiation for 2 minutes as a function of hydroxylamine and/or AuNP concentration.
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Fig. 5 Fluorescence intensity of resorufin after LED irradiation of resazurin as a function of hydroxylamine and/or AuNP concentration. Top: different concentrations of NH2OH (0.5, 1 and 2 mM) keeping constant the concentration of the other compounds (1 mM of resazurin and 1.4 nM of AuNP); inset: fluorescence intensity vs. NH2OH concentration (0.5, 1 and 2 mM). Bottom: different concentrations of AuNP (0.7 and 1.4 nM) keeping constant the concentration of the other compounds (1 mM of resazurin and 2 mM of NH2OH). |
Looking at the experiments in Fig. 5 (bottom) one notes that with 0.7 nM concentration of AuNP the reaction appears to work better than with 1.4 nM, since we observe stronger fluorescence. However, it is well known that metal nanoparticles quench the fluorescence of molecules in their vicinity;9,20 quenching of resorufin fluorescence emission was performed for different concentrations of AuNP (Fig. 6) and employing the Stern–Volmer analysis of eqn (1).21,22
![]() | (1) |
KSV = kqτ | (2) |
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Fig. 6 Top: fluorescence emission of resorufin (0.01 mM) with different concentrations of AuNP. Bottom: Stern–Volmer plots for the quenching of the emission due to the AuNP with excitation at 532 nm (red) and 560 nm (blue). |
From the data in Fig. 6 one estimates that for [AuNP] = 1.4 nM, over half of the resorufin molecules are associated to the AuNP. This translates to about 3570 molecules per nanoparticle; given an average AuNP size of 13 nm, this corresponds to about ∼15 Å2 per dye molecule; this area is just too small for molecules to lay flat on the AuNP surface and suggests that most molecules must bind ‘head on’ on the surface.
From these results we can assume that the differences in fluorescence intensities with different concentrations of AuNP are largely due to static quenching on the nanoparticle surface.
The study of the fluorescence growth dynamics has been summarized in Table 1; we note (see Fig. 4) that microwave irradiation (at 80 °C) is comparable to a water bath at 80 °C. For the LED at full power (0.9 A), four LEDs deliver 4 × 4.6 watt, or ∼18 watt, of this about 20% is actually absorbed by the sample; for the fastest reaction (row 2 in Table 1) about 500 J will actually be absorbed by 1 mL of solution during the half life of the reaction.
Energy source | [AuNP]/nM | [NH2OH]/mM | t 1/2 b/seconds | Plateau reached? |
---|---|---|---|---|
a Half-lives (t1/2) are based on the exponential growth analysis of the resorufin fluorescence monitored every 2 minutes for 20 minutes (unless the plateau level was already achieved). b Values given in brackets when the fluorescence intensity after 20 minutes was less than 80% of the plateau value, as extrapolated from the exponential growth analysis. | ||||
LED, 530 nm (nominal) | 0.7 | 2.0 | 180 | Yes |
LED, 530 nm (nominal) | 1.4 | 2.0 | 132 | Yes |
LED, 530 nm (nominal) | 1.4 | 1.0 | 186 | Yes |
LED, 530 nm (nominal) | 1.4 | 0.5 | (700) | No |
80 °C water bath | 1.4 | 2.0 | 225 | Yes |
50 °C water bath | 1.4 | 2.0 | (>1000) | No |
In spite of the fact that the reduction of resazurin to resorufin is a very well known process, the mechanism for the catalyzed reduction of the former by hydroxylamine in the presence of AuNP is still a subject of interest. Xu et al.8,19 propose the formation of nitrite (probably from nitrous acid). At the single molecule level the concentration dependence on hydroxylamine (in excess) was not examined,8 at the ensemble level and under plasmon excitation, we observe that the reaction efficiency follows a linear dependence with hydroxylamine concentration (see Fig. 5). We believe that the AuNP behave both as a heating element through photoexcitation of their surface plasmon band, as well as a true catalyst. Given the linear dependence of the fluorescence with the concentration of NH2OH (Fig. 5), hydroxylamine should be involved in a rate-determining step of the reaction; in other words, hydroxylamine needs to either reach the resazurin molecule at the reactive site on the AuNP during the brief period12 of plasmon-induced heating, or assist in the departure of the product (resorufin) following reduction. Chen et al. proposed19 that product departure occurred by two mechanisms, substitution by fresh resazurin or ‘spontaneously’; given that NH2OH dependence was not examined, it is likely that ‘spontaneous’ product release from the surface is in fact assisted by NH2OH.
In conclusion, the laser drop technique has proven to be very useful for the reduction reaction from resazurin to resorufin using the plasmon band of the AuNP. While with other techniques this reaction can take more than 10 min to be completed, in the case of laser drop it takes just some nanoseconds, that is the duration of the laser pulse. In spite of the dramatic acceleration under laser excitation, the utility of LED plasmon excitation stands out as an inexpensive, simple and relatively fast way of promoting reactions through plasmon assisted catalysis induced by photothermal effects.
Footnote |
† Electronic supplementary information (ESI) available: SEM images, drop photographs, additional fluorescence spectra and absorption spectra of dye solutions. See DOI: 10.1039/c1cy00236h |
This journal is © The Royal Society of Chemistry 2011 |