Reversible Photobleaching of Gold Nanoclusters: A Mechanistic

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Reversible Photobleaching of Gold Nanoclusters: A Mechanistic Investigation Bahram Hemmateenejad, Arezoo Shahrivar-Kevishahi, and Fatemeh Shakerizadeh-Shirazi J. Phys. Chem. C, Just Accepted Manuscript • DOI: 10.1021/acs.jpcc.6b07999 • Publication Date (Web): 18 Nov 2016 Downloaded from http://pubs.acs.org on November 22, 2016

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The Journal of Physical Chemistry

Reversible Photobleaching of Gold Nanoclusters: A Mechanistic Investigation Bahram Hemmateenejad,*a,b Arezoo Shahrivar-Kevishahi,a Fatemeh Shakerizadeh-Shirazia a

Department of Chemistry, Shiraz University, Shiraz, Iran

b

Medicinal and Natural Products Chemistry Research Center, Shiraz University of Medical

Sciences, Shiraz, Iran

* Corresponding author, Tel.: 0098-713 646 0724, Fax: 0098-713 646 0788

Email address: [email protected], [email protected]

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ACS Paragon Plus Environment


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ABSTRACT Fluorescent gold nanoclusters (AuNCs) have found widespread applications for designing of novel sensing elements for diagnostics and in vivo and in vitro imaging. Herein, we investigated the effect of ultraviolet (UV) irradiation on the fluorescence characteristics of bovine serum albumin-capped AuNCs. When the aqueous solution of these nanoclusters were exposed to UV irradiation, a rapid decrease in the emission intensity at about 620 nm in accompanying with an increase in emission at about 420 nm was observed. After turning off the UV light and saving the solution in dark, a gradual recovery of the quenched fluorescence was observed such that after about 400 min it reached to its original emission value. The mechanism of these fluorescence changes was investigated by transmission electron microscopy, x-ray photoelectron spectroscopy and monitoring of the effect of pH. The observed on-off fluorescence characteristics can be used as the basis for development of novel sensors. In this work, we used this system for assay of antioxidant activity. The antioxidant inhibited the photobleaching and thus could turn on the photoquenched fluorescence of the BSA-AuNCs.

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Introduction Fluorescent gold nanoclusters (AuNCs) or nanodots (AuNDs) are a particular kind of gold nanomaterials which do not show surface plasmon resonance (SPR) absorption in the visible region but have fluorescence in the visible to near-infrared (NIR) region.1 AuNCs have become interesting sensing, imaging and catalytical materials because of their benefits such as long lifetime, large Stokes shift, and biocompatibility.2,3,4 There are two major categories revealing the recent developed techniques for the preparation of AuNCs. The first class is through etching of larger sizes of nonfluorescent gold nanoparticles (AuNPs) by thiol compounds for example mercaptopropionic acid.5 In the second method, AuNCs are formed by reduction of Au3+ in the presence of a ligand or template (protein) such as bovine serum albumin (BSA).6 Recent advances in fluorescence imaging have made this technique indispensable not only for in vitro investigations of cells and proteins, but also for in vivo imaging.7 The efficiency of fluorescence probes generally for diagnostic purposes in vivo, though, can be limited by their low on–off contrast, irreversibility, poor delivery, and toxicity. The sensitivity of in vivo fluorescence imaging, which is usually restricted via autofluorescence, absorbance, light scattering, and penetration depth, has been enhanced considerably, for instance, by utilizing switchable probes.8Photoswitchable fluorescent probes have been utilized in recent years to allow superresolution fluorescence microscopy by single molecule imaging.9 While these properties make them excellent probes for super-resolution imaging, the mechanism by which cyanine dyes are photoconverted has yet to be determined. Such an understanding could prove useful for creating new photoswitchable probes with improved properties.10

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In an effort to make fluorescence nanocluster agents for bio applications, herein we prepared biocompatible protein-gold nanoclusters that can reversibly photobleaching and exhibit high on–off without any apparent toxicity. The photoinduced fluorescence enhancement was detected for BSAAuNCs in solution.11 however, under irradiation with UV light, photobleaching was originated; i.e., the photoconversion of AuNCs into their dark states appears upon illumination by UV light. The observed photobleaching was reversible: if the sample was kept in the dark for 100 s, the fluorescence recovered almost to the initial intensities. According to the best of our knowledge, this is the first report on the reversible fluorescent photobleaching of Au nanoclusters. Unlike the recently reported photoswitchable GFP-like proteins,12 the used AuNCs in this study are biocompatible and can be simply treated to or injected into living cells and organisms like smallmolecule fluorophores without any noticeable toxicity or immunogenic concerns. It was found that antioxidant could inhibit the photobleaching process. Therefore, these observations were used as a basis of antioxidant assay. The antioxidant activity (AOA) is described as the capability of a compound (or a mixture) to inhibit oxidative deteriorations11 and is related with compounds capable of protecting a biological system against the potentially harmful effect of processes or reactions involving reactive oxygen and nitrogen species (ROS and RNS).14-23

Experimental section

Reagents and solutions Bovine serum albumin (BSA) was purchased from Sigma Aldrich (St. Louis, MO, USA). FolinCiocalteu’s reagent, Gallic acid, ascorbic acid, α-lipoic acid, catechol, naringine, dopamine, pyrogallol, cysteine, Glutathione, Serine, tartaric acid, citric acid, methanol, sodium carbonate 4


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(Na2CO3), sodium hydroxide (NaOH), hydrochloric acid (HCl), Zinc nitrate (Zn(NO3)2), magnesium nitrate (Mg(NO3)2, phosphoric acid (H3PO4), acetic acid (CH3COOH) and sodium azide (NaN3) were obtained from Merck Chemicals Company (Darmstadt, Germany). Boric acid (H3BO3) was purchased from BDH. The AuNCs were synthesized by chemical reduction of HAuCl4 with BSA according to the green synthetic procedure reported by Xie et al.6,24 Details of the synthesis procedure are given in the supporting information section. The universal buffer solutions (UBS) over pH range of 2-11 were prepared by successive addition of different aliquots of 0.2 M NaOH to 1:1:1 aqueous mixture of boric acid (H3BO3), phosphoric acid (H3PO4) and acetic acid (CH3COOH) (all 0.04 M). All solutions were prepared with ultra-pure water. Stock solutions of antioxidants (concentration: 1.00 ×10

-2

, 1.00 ×10

-4

M)

were prepared by dissolving appropriate amount of the reagents in water. Working solutions of lower concentrations were prepared by appropriate dilution of the stock standard solutions.

Instruments All fluorescence spectra were recorded on an LS-50 Spectrofluorometer (Perkin-Elmer Co., USA) equipped with a water bath and a quartz cell (1.0×1.0 cm). The FL WinLab Software (PerkinElmer) was used to digitize the measured data. UV-Vis absorbance spectra were recorded using a Hewlett-Packard model 8452A diodearray spectrophotometer equipped with a 1.0-cm quartz cell. The TEM measurements were made on a transmission electron microscope (Zeiss, EM10C) operated at an accelerating voltage of 80 kV. Samples for TEM characterization were prepared by placing a drop of the as-prepared gold nanocluster samples on a formvar carbon coated grid Cu Mesh 300t hen drying at room temperature. 5



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X-Ray photoelectron spectroscopy (XPS) measurement was performed with an XR3E2 (VG Microtech) twin anode X-ray source using Al target (1486.6 eV) and Mg target (1252.6 eV) (www.vgmicrotech.com). The XPS curves were deconvoluted using Spectral Data Processor software (SDP v 4.1; http://www.xpsdata.com) A homemade UV exposure chamber equipped with UV light source (254 nm) was used for photochemical experiments. All measurements were made at atmospheric pressure.

Procedure of photochemical experiment A small volume of the as-prepared AuNCs was mixed with buffer solutions and then exposed to UV irradiation of 254 nm in a dark cabinet. After different lighting times, fluorescent spectra of the solution were recorded in the emission wavelength range of 390-700 nm and excitation wavelength of 370 nm. To study the effect of antioxidants on photobleaching, before UV irradiation, a fixed amount of the aqueous antioxidant solution was added to the buffered AuNCs solution and then sampling was done at different lighting time for fluorescence measurements. Antioxidant concentrations of 1.0 ×10 -2, 1.0 ×10 -3, 1.0 ×10 -4, 1.0 ×10 -5 and 1.0 ×10 -6 M were studied.

Result and discussion Here we used the direct synthesis method of AuNCs. A one –pot and “green” synthetic route of gold nanoclusters using bovine serum albumin (BSA) as template and at the physiological temperature was adapted from Xie et al.6 According to the TEM images of the solution of the prepared BSA-AuNC (Figure 1), the average size of the particle is about 2.5 nm (± 0.2). Due to magnifying limitations for transmission electron microscopy (TEM), AuNCs are not as easily imaged as Au nanoparticles.24,25 High resolution TEM can be attempted to detect AuNC size at the 6


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sub nanometer level but is not always successful. In particular, AuNC species smaller than 25 gold atoms have been difficult to observe.26 The presence of carbon, oxygen, nitrogen and sulfur elements with Au is obvious from the XPS spectra shown in Figure S2. According to the literature,6,27,28 the twin XPS peaks observed between binding energies of 81-90 eV (Figure 1E) can be attributed to Au-4f, where the peak at around 83-84 eV and 87-88 eV are attributed to Au-4f7/2 and Au-4f5/2, respectively. The Au-4f peaks could be deconvoluted into two distinct components (red and green) centered at binding energies of 83.2 and 84.3 eV (for 4f7/2 peak) and 87.0 and 87.7 eV (for 4f5/2 peak). The resolved peaks at larger binding energies (84.3 eV and 87.7 eV) could be assigned to Au(+1) and those at smaller binding energies (83.2 eV and 87.0 eV) could be assigned to Au(0).6,27,28 The area under the XPS peaks of metallic and monovalent Au suggest that at the surface of the synthesized AuNCs around 72% of the gold species are in the form of metallic gold and about 28% are in the form of Au+ ions, which are close to the reported values previously.6,29 Conventional spectroscopies, such as UV-Vis and fluorescence, are essential in proteinAuNC studies to capture their optical properties.24 However, protein-AuNCs are too small to exhibit surface plasmon resonance (SPR). As it is observed from Figure 1, no absorption features in the visible region clearly detect the presence of AuNC. On the other hand, the BSA–AuNCs exhibit a twin peak fluorescence spectrum located at 430 nm and 620 nm, when excited at 370 nm (Figure 1). The line width of the fluorescence spectrum of longest wavelength (width at half-maximum) was about 80 nm. Theoretically, photophysical properties of Au clusters should present discrete excitation-emission bands related to highest occupied molecular orbital-lowest unoccupied molecular orbital (HOMO-LUMO) electronic transitions.30,31 According to the literature,32,33,34 the observed weak emission peak centered at around 430 nm could be attributed to the blue emission of 7



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the protein (BSA) whereas the emission peak at longer wavelength (centered at around 620 nm) could be related to gold cluster core.

Photobleaching of the Fluorescence Emission Effect of UV irradiation on optical properties such as fluorescence and absorption of BSAAuNC was evaluated. It was observed that in the presence of UV light, a rapid decay in the fluorescence signal in 620 nm happened, as after 5 minute, the emission intensity decreases to 50% of original intensity before lighting (Figure 1C and Figure S1). However, it is observed that no significant changes in the fluorescence signal are observed when the solution was kept in the darkness. High stability of Au NCs in the absence of light is relevant to the electronic number fulfilling both the closed electron shell rule and geometric factors, also it is due to completed coordination of highly symmetrical Au cores by ligands.35 As shown in Figure 1C, the quenching of the red peak fluorescent is accompanied with a slight increase in the blue peak. Wei et al observed a reverse direction (decreasing of blue peak emission and increasing of red peak emission) through formation of protein-capped Au NCs.33 Thus, increasing in the blue peak emission could be attributed to partially detaching of BSA from the surface of AuNCs. Since the emission peak at 620 nm is the characteristic of Au NCs, we will only consider fluorescence changes at this peak in the next parts. In contrast to large effect of UV irradiation on the fluorescence behavior, very small changes were observed in the UV absorbance spectrum of BSA-AuNC upon lighting (Figure 1D); at wavelengths longer than 250 nm, decreasing in absorbance is observed. It is interesting that the observed photoinduced quenching is highly reproducible (see Figure S1). The relative standard

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deviation (RSD) of the fluorescent intensity after 5 min UV lighting was 1.7% for 5 repeated experiments. Understanding the origin of these observations needs an extensive knowledge about proteinnanoparticle interactions as well as photochemistry of the system. Firstly, it would be interesting to explain the mechanism of the formation of BSA-AuNC as reported by Guéve et al.27 Synthesis of protein-AuNCs starts with coordination of Au3+ with lone pairs of protein functional groups such as amine, carboxyl and thiol followed by reduction of Au3+ to Au+ through an autocatalytic process. It should be noted that the reduction capability of BSA is relating to some reducing residues like Tyr with pKa of ~10. So, adjusting the reaction pH above the pKa of Tyr can greatly improve the reduction capability of BSA.36 As the result, an Au+-protein complex is formed followed by disproportionation of Au+ to Au3+ and Au0, where Au0 accumulates to yield larger AuNC with Au0 as core and surface covered by Au+.28 This suggested synthesis mechanism agrees with the results of XPS study (Figure 1), where in the synthesized nanoclusters a ratio of 72:28 was observed for Au0:Au+. Considering the above mechanism, we did comprehensive studies to find a probable mechanism for fluorescence decay upon UV irradiation. Firstly, the oxidation states of gold nanoclusters labeled to BSA before and after UV irradiation were determined by XPS method. Examining the 4f7⁄2 and 4f5⁄2 binding energies of protein-stabilized AuNCs is an excellent method for determining the overall oxidation state of gold which could be disproporates into two distinct components assigned to Au0 and Au+.37 The obtained XPS spectra and the fitting curves are given in Figure 1 and in more detail in the supporting information Figure S2). Before UV irradiation, the ratio of Au0:Au+ in the synthesized AuNCs is about 72:28, which are consistent with XPS measurements performed by Xie et al.29 As shown in Figure 1F, after UV irradiation, the peak 9



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positions of the Au+ and Au(0) were not changed. However, a significant change in the population ratio of Au0:Au+ to 26:74 is explanatory oxidation of Au0 to Au+. Beside this, the XPS peak related to binding energies of S element (attributing to the covalent interaction of gold nanoclusters with the sulfur groups of the cysteine and oxidized sulfur)

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was disappeared after UV irradiation

(Figure S2). Transmission electron microscopy (TEM) was studied before and after UV irradiation of BSA-AuNC. Figure 1 is TEM image of BSA-AuNC before exposure to UV light that shows the particles with the average size of about 2.5 nm. An explicit alternation, indicating aggregation of AuNC after UV irradiation (but without clear evidence for changes in the size of particles) in TEM image is shown in Figure 1. It has been reported that aromatic residues on protein can capture UV light (from~250-298nm).39 Once excited by UV light they can enter photochemical pathways likely to have harmful effects on protein structures, such as disulfide bridges in proteins.39 On the other hand, UV light generates reactive oxygen species (ROS) in solution, such as 1O2 , H2O2 and HO•, which can damage different types of molecules, including proteins, nucleic acids and lipids.40 The action of radicals on proteins leads to extensive protein – protein cross-linking (Figure 1) and also cause linkages between amino acids such as disulfide linkage in Cys.41 The quenching effect of reactive oxygen species on the flourescence of AuNCs has been reported previously.11,42 The above observations and explanations suggest that the most probable mechanism of quenching is oxidation of Au0 to Au+. However, the effect of UV irradiation on this oxidation process is questionable. Two possible mechanisms can be encountered. The first is detaching of the electron-rich nitrogen, oxygen and sulfur from Au. These atoms, which can donate electron to the 5d and 6s orbitals of Au via their lone pairs,43 undergo n →π* transition when exposed to UV irradiation and thus their valence electrons emerge from Au0 and hence Au0 changes to Au+. 10


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The above mechanism was firstly evaluated by studying the effect of pH on quenching of BSA-AuNCs under UV irradiation. It is notable that the isoelectric point of a BSA is 4.9 and at pH around this value the protein was unstable and the BSA-AuNCs precipitated. So, the effect of pH on the fluorescence signal of BSA-AuNC was studied at pH lower and higher than 4.9. As it is observed in Figure 2, the rate of photoinduced fluorescence decay is faster in acidic pH. Also, the degree of photobleaching is higher in acidic medium. This observation can be attributed to the protonation of the coordinating groups of protein residues and side chain, which lowers their tendency toward coordinating of Au. Unbinding of Au with such groups would result in faster transformation of Au0 to Au+. Moreover, the reversibility of the fluorescence quenching after turning of the UV irradiation is an important and interesting issue that should be taken into account. As it is shown in Figure 3, when the photobleached AuNCs are stored in dark, the fluorescence is recovered to its original value after about 400 min. However, the emission peaks maximum shifts slightly toward shorter wavelengths (Figure S3). Interestingly, XPS analysis of the AuNCs after complete recovering of fluorescent showed a ratio of Au0/Au+ similar to that of before lighting (about 75%/25% ratio of Au0/Au+; see Figure 1 and supporting information Figure S2). This observation suggests that when the light turns off, the exited electrons of the coordinating atoms back to the ground state and then coordination of these groups to Au+ followed by reduction to Au0 will take place. This suggestion is based on the mechanism of the synthesis of BSA-AuNC which was explained before. By returning back the oxidized Au+ to the Au0, the fluorescence emission of the BSA-AuNC turns on, again. In fact, light acts as a reversible oxidizing agent.44 The second plausible mechanism is oxidation of Au0 to Au+ by the ROS produced upon UV irradiation. To explore the accuracy of this assumption, the effect of addition of sodium azide 11



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(NaN3) as a radical scavenger was examined. As it is shown in supporting information Figure S4 (2), NaN3 can effectively inhibit the BSA-AuNC photobleaching in buffer solution. This phenomenon can be attributed to rapid interaction of azide ion with free radicals and converting them to ground state before they can react with another molecule in the system. However, when the solution was bubbled by purging of Ar (to push out the dissolved oxygen), no changes in photobleaching phenomenon was observed. The production of ROS is highly dependent on the concentration of the dissolved oxygen and hence this observation declined the production of ROS.45 On the other hand, the observed effect of sodium azide can be explained based on its basic behavior. It can compete with coordinating atoms of protein to interact with H+ ion such that by increasing the concentration of azide ion, the basicity of solution is increased. As it was shown in Figure 2, in basic medium the photobleaching is declined.

Shading effect of chemical species We observed that, some chemical species represented shading effect by absorbing of UV light and hence protect BSA-AuNC from UV-irradiation.46 Therefore, they could inhibit the photobleaching of the BSA-AuNCs. Interestingly, this shading effect was concentration dependent and hence it can be used as a platform for designing of novel sensor. In this part, we represent the results of our investigation on the shading effects of antioxidants as an important class of biochemical species. To investigate the effect of experimental parameters on the reversal of fluorescence of AuNCs by addition of light absorbing species, we used ascorbic acid (AA) as a representative antioxidant. As shown in supporting information Figure S5, AA lowers down the degree of photobleaching such that the fluorescence of the photobleached Au-NCs almost backs of its original intensity in the presence of AA. To gain more details on the suggested mechanism of reversible 12


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photobleaching and to optimize the procedure of antioxidant assay, some experimental parameters including medium pH, type of buffer and concentration of Au-NCs were investigated. The results are given in Figure 4. The effect of pH on the photo-induce fluorescence decay have been studied for five different concentrations of ascorbic acid (AA). To examination of the inhibition ability for each concentration of ascorbic acid in different pH media, we traced time-dependent fluorescence intensity in absence and presence of different concentration of AA in 5 different pH media (details are given in supporting information Figure S6. As it was expected, by decreasing in concentration of AA, the percent of inhibition was also decreased. However, at acidic and neutral pH, the system represented better sensitivity and the least time-dependency for concentrations > 0.1 mM. So, pH of 7.4 as physiological pH was selected. Also, after 5 min lighting time, the fluorescence decay reach to plateau. Therefore, pH=7 and 5 min UV irradiation was selected as optimum condition. It is obvious from Figure 4B and C that universal buffer of 0.04 M concentration represented the highest sensitivity. The effect of amount of BSA-AuNC solution on fluorescence intensity is shown in Figure 4D. As observed, the effect of self-absorption is clear after 250 µL injection of the AuNCs to the system. The effect of six different antioxidants, including ascorbic acid, Gallic acid, catechol, dopamine, naringine and pyrogallol (see Figure S7 for molecular structures of these compounds) on the decay of fluorescence intensity of BSA-AuNC upon irradiation with UV source at 254 nm (UVC) was studied. The plots shown in Figure 5, explain dependency of the percent of inhibition of photobleaching on concentration of antioxidants (sigmoid shape). It is observed that at high concentrations (e.g.> 0.01 M) catechol, ascorbic acid, dopamine and naringine could reverse

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completely the photobleaching of BSA-AuNC. However, Gallic acid and pyrogallol represented lower tendency for inhibition of photobleaching. To have a measure of antioxidant activity by the suggested method, percent inhibition at the same concentration for all studied compounds was calculated. It should be note that the choice of such unique concentration for evaluation of activity is a critical point. In fact, the selected concentration should not be set too low so that the compounds are not able to inhibit the photobleaching. On the other hand, if the selected concentration is too high, most compounds will completely inhibit the photobleaching and thus, ranking of the compounds is not possible. Thus, according to plots shown in Figure 5, the concentration of 0.1 mM was selected for comparison of antioxidant activity. Accordingly, the studied compounds were ranked in the order of decreasing antioxidant ability as ascorbic acid> catechol> dopamine> Gallic acid> pyrogallol> naringine. Interestingly, this order of antioxidant activity is in close agreement with the standard method of Folin-Ciocalteus method suggested by the International Organization for Standardization (ISO) 14502-1 (Figure S8). As aforementioned, antioxidant molecules have different UV-absorbing capacity that is related to their structure, thermodynamic descriptors including O-H bond dissociation enthalpies (BDE), π-electron conjugation, H-bonds and number of OH groups.47 the capacities of deprotonation and electron transfer are also two main descriptors. The π -electron conjugation plays a crucial role to stabilize the phenoxy radical formed after hydrogen atom transfer (HAT). In fact, UV light leads to the homolytic dissociation of OH bonds of polyphenolic compounds.44 The absorbance spectra of ascorbic acid as a representative antioxidant before and upon UV light exposure were studied. As shown in supporting information Figure S9, after UV irradiation, decay in absorbance of ascorbic acid is observed, which can be attributed to the light-induced 14


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decomposition of ascorbic acid. According to this observation, and the discussions given in the previous section, it could be concluded that the most probably reason for inhibition of photobleaching by antioxidant is absorption of UV light and hence protecting the BSA-AuNC against UV irradiation. The fluorescent responses of photobleached BSA-AuNCs solutions to some common biological molecules were monitored and compared to that of antioxidants. Figure 6 shows that in addition to ascorbic acid (as a representative antioxidant) adding of biological species such as tyrosine (Tyr), cystein (Cys), phenylalanine (Phe), glutathione (GSH) and serine (Ser), individually in the same concentration of ascorbic acid, increased the fluorescence intensity of the photobleached of BSA-AuNCs. However, biological ions such as Zn+2 and Mg+2 did no effect significantly on photobleaching. The same is true for tartaric and citric acid. Conclusion In this study the reversible photobleaching of AuNCs was investigated. UV lighting of the NCs decreased significantly their red emission whereas this irradiation did not effect on the absorbance spectrum of the studied NCs. In addition, TEM images revealed aggregation of the nanoclusters after UV irradiation. Moreover, XPS study showed oxidation of the metallic Au to Au+ ions. Interestingly, the photobleached fluorescence turned on when the UV light turned off and a complete recovery of the original fluorescence was observed when the nanoclusters were stored in dark again. In this situation, the monovalent Au ions converted again to atomic gold. Such reversible photobleaching was explained excitation of the electrons of the coordinating atoms of BSA and hence detaching of these atoms from the surface of Au atoms which results in oxidation of Au atoms. By turning off the UV light this process is reversed.

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This observed on-off behavior of the fluorescence of AuNCs can be considered as a basis for designing of novel chemical sensors. As an example, we used the studied system for measurement of antioxidant activity. Antioxidant, by exerting shading effect, could turned on the fluorescence intensity of the photobleached nanoclusters. The present of inhibition of photobleaching represented a high correlation with the antioxidant activity measured by Folin-Ciocalteu method as a standard method.

Supporting Information. Procedures for synthesizing the BSA-Au NCs, measurement of antioxidant activity and extraction of polyphenols from leaves of tea; plots of time-decay curves, reproducibility of photobleaching, detailed XPS spectra, effects of sodium azide, ascorbic acid and pH on photobleaching, comparison between BSA-AuNCs method and standard method for assay of antioxidant activity; and list of the 4 types of tea brands used in this study.

Acknowledgment. Financial support of this work by Shiraz University research council is highly appreciated.

References

(1) Li-Yi Chen, Chia-Wei Wang, Zhiqin Yuan, and H.-T. C. Fluorescent Gold Nanoclusters: Recent Advances in Sensing and Imaging. Anal. Chem. 2015, 87, 216–229. (2) Zhang, L.; Wang, E. Metal Nanoclusters: New Fluorescent Probes for Sensors and Bioimaging,. Nano Today 2014, 9, 132−157.

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Legend for the Figures

Figure 1. Characterization of the prepared BSA-AuNCs in the presence of UV irradiation. (A and B) TEM image before and after UV irradiation, respectively. (C) Changes in fluorescence emission spectra during UV irradiation: excitation wavelength was 370 nm and the fluorescence spectra were recorded during 20 min UV irradiation. (D) Molecular absorption spectra before and after 20 min UV irradiation. (E and F) X-ray photoelectron spectra before and after UV irradiation, respectively. Figure 2. Effect of pH on the photobleaching of BSA-AuNCs: (A) fluorescent-time decay curves at different pHs and (B) fluorescence-pH relation after 20 min UV irradiation. F0 and F are the fluorescence intensities before and after UV irradiation, respectively. Thus, the higher value of F/F0 means the lower amount of photo-bleaching. Figure 3. Fluorescence-time decay curve for firstly 5 min UV irradiation and then turning it off and storing in dark ( λex = 370 nm and λem = 620 nm). Figure 4. Effect of experimental parameters on the inhibition of photobleaching by ascorbic acid: the lighting time was 5 min and the concentration of ascorbic acid was 0.1 mM Figure 5. Effect of the increasing in concentration of 6 representative antioxidants on the inhibition of photobleaching at optimized condition. The %inhibition was calculated using Eq. (1). Figure 6. Effect of different endogenic components on the florescence intensity of BSA-AUNCs in the presence of UV irradiation for 5 min. The fluorescence intensities are normalized relative to that of BSA-AUNCs in the presence of ascorbic acid.

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A

B

C

E

D

F

Figure 1

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A

B

Figure 2

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Figure 3

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A

B

C

D

Figure 4

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Figur e5

28 ACS Paragon Plus Environment

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Figure 6

29 ACS Paragon Plus Environment


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Reversible Photobleaching of Gold Nanoclusters: A Mechanistic

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