Microwave-Assisted Formation of Gold Nanoclusters Capped in

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Microwave-Assisted Formation of Gold Nanoclusters Capped in Bovine Serum Albumin and Exhibiting Red or Blue Emission Kuan-Ting Chuang, and Yang-Wei Lin J. Phys. Chem. C, Just Accepted Manuscript • DOI: 10.1021/acs.jpcc.7b09349 • Publication Date (Web): 08 Nov 2017 Downloaded from http://pubs.acs.org on November 11, 2017

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The Journal of Physical Chemistry

Microwave-Assisted Formation of Gold Nanoclusters Capped in Bovine Serum Albumin and Exhibiting Red or Blue Emission

Kuan-Ting Chuang, and Yang-Wei Lin*

Department of Chemistry, National Changhua University of Education, Changhua, Taiwan

E-mail: [email protected] (Y.W.L.)

Tel: 011-886-4-7232105-3553

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ACS Paragon Plus Environment


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ABSTRACT: In this study, a microwave-assisted method for preparing fluorescent gold nanoclusters capped in bovine serum albumin (AuNCs@BSA) was developed. Through fluorescence spectrometry, transmission electron microscopy, and X-ray photoelectron spectroscopy (XPS), the effect of reaction temperature on the size and oxidation state of AuNCs@BSA samples was determined. Blue-emitting AuNCs@BSA (B-AuNCs@BSA), with weak bonds between AuNCs and BSA, were prepared at 135 °C. Red-emitting AuNCs@BSA (R-AuNCs@BSA) were also formed, and their formation was followed by an aggregation of polydispersed AuNCs@BSA. Through infrared spectrometry and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, it was confirmed that the R-AuNCs@BSA, consisting of 18 Au atoms, was successfully embedded in BSA. In addition, XPS measurements revealed that Au+ ions were present only in the R-AuNCs@BSA. Steady-state and lifetime results revealed the importance of the size and oxidation state of Au+ in the stabilization of the AuNCs@BSA and in the presence of a long lifetime component.

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1. INTRODUCTION Fluorescent gold nanoclusters (AuNCs) consist of a few to several tens of Au atoms.1,2 They are considered attractive materials for several bioapplications because they display a molecule-like behavior. AuNCs exhibit highest occupied molecular orbital (HOMO) to lowest unoccupied molecular orbital (LUMO) transitions during absorption and photoluminescence.3,4 Because of the quantum confinement effect, they also exhibit size-dependent fluorescence properties.5,6 Fluorescent AuNCs protected by biomolecules are suitable for applications such as bioimaging, biosensing, cancer therapy, and antimicrobial agents7–14 because of their distinctive functionality, ease in bioconjugation, large Stoke shift, long lifetime, and high photo- and chemical stability. To make biomolecule-protected AuNCs ideal candidates for use in bioimaging and cancer therapy, their affinity toward specific cells or organs, stability during delivery, and cell penetration must be considered carefully. One class of such multifunctional AuNCs, called theranostic nanocomposites, are prepared through the conjugation of anticancer drugs with AuNCs.15 Both in vitro and in vivo results show that these multifunctional AuNCs provide positioning information and the conjugated anticancer drugs have a therapeutic effect on cancerous cells. Therefore, theranostic nanocomposites are useful for the early detection and therapy of cancerous cells. In 2009, Xie et al. were the first to demonstrate a simple method for preparing fluorescent bovine serum albumin (BSA)-protected AuNCs (BSA-AuNCs).16 These AuNCs contained 25 Au atoms that exhibited red emission. The BSA-AuNC surface was stabilized using a small number of Au+ ions. Since then, various biomolecule-based synthesis approaches for preparing fluorescent AuNCs have attracted increasing attention.17–20 In addition, AuNCs with magic numbers of Au atoms (e.g., n = 8, 11, 13, 18, 22, 25, 28, 39) have been prepared. However, such 3



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AuNCs usually only exhibit red fluorescence emission.21 Although AuNCs exhibiting blue emission can be obtained by controlling the pH conditions, they require a long preparation time and an exact pH value of 8.0.22,23 Recently, another method to synthesize alkanethiol-protected AuNCs through etching and passivation was developed.24–26 The nature of the alkanethiol used and the metal–thiol interaction are critical factors determining the structural and electronic properties of these AuNCs, as they result in different levels of fluorescence enhancement in the AuNCs. The aforementioned method can be used to prepare AuNCs with a discrete and high-quality fluorescence emission from blue to infrared, but the AuNCs still require a long etching time. Microwave (MW) heating was utilized to develop a more efficient method for preparing AuNCs. MW heating not only reduced the reaction time from tens of hours to several minutes but also suppressed side reactions, thereby improving the yield and reproducibility of a specific synthesis protocol.27–30 In addition, because the synthesis of small AuNCs is highly sensitive to reaction conditions, the MW-assisted method could provide a greater control over the growth of AuNCs and correspondingly improve their optical properties. However, AuNCs obtained using this method still exhibited red emission.31,32 In this paper, a MW-assisted method for preparing AuNCs capped with BSA (AuNCs@BSA), exhibiting red and blue emission, by using a precisely controllable MW synthesis instrument is proposed. The growth procedure and the nature of the interaction between Au atoms and BSA are described. Structural investigations and the characterization of optical properties of the AuNCs@BSA highlight the effects of reaction temperature, oxidation state of elemental Au, conformational changes in BSA, and electronic charge transfer. Finally, the reproducibility of the proposed MW-assisted method and the stability of the prepared AuNCs@BSA are also 4


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evaluated.

2. EXPERIMENTAL 2.1. Materials. All chemicals used were of analytical grade or of the highest available purity. Hydrogen tetrachloroaurate(III) trihydrate (HAuCl4·3H2O), BSA, quinine sulfate, riboflavin-5ʹ- phosphate, and sodium hydroxide (NaOH) were obtained from Sigma Aldrich (St. Louis, MO, USA). Deionized water (18.2 MΩ·cm) was used in all experiments. 2.2. Preparation of AuNCs@BSA. For the preparation of the AuNCs@BSA showing blue emission (B-AuNCs@BSA), 2.5 mL of a 65 mg/mL BSA solution was added to 2.5 mL of a 10 mM HAuCl4 solution under stirring at room temperature. Then, 0.25 mL of a 1.0 M NaOH solution was added dropwise to catalyze the formation of AuNCs because of an increase in the reduction capability of the tyrosine residue of BSA. The solution mixture was heated to 135 °C for 4 min in a 300 W single-mode microwave synthesis instrument (CEM-Discover Labmate; Kohan Instruments Co., Ltd., Taichung City, Taiwan). The solution was pale yellow. The AuNCs@BSA showing red emission (R-AuNCs@BSA) were prepared using the same method but at a lower temperature of 80 °C. The color of the solution turned from pale yellow to dark orange. A series of AuNCs@BSA were prepared at different temperatures from 80 °C to 135 °C to determine the effect of temperature on the growth of AuNCs. All samples were kept at 4 °C and exhibited no precipitation for 2 weeks. The experimental conditions used to prepare the AuNCs@BSA are listed in Table 1.

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Table 1. Microwave-Assisted Synthesis Conditions for Preparing AuNCs@BSA R-AuNCs@BSA B-AuNCs@BSA 3+ Molar ratio of Au /BSA 10.2 10.2 Reducing agent Tyrosine residue in BSA Tyrosine residue in BSA pH 11.0 11.0 Temperature 80 °C (300W) 135 °C (300W) Reaction time 4 min 4 min

2.3. Characterization of AuNCs@BSA. The UV–Vis absorption and fluorescence spectra of the solution containing the AuNCs@BSA were recorded using an UV–Vis spectrometer (Evolution 200; ThermoFisher, Waltham, MA, U.S.A.) and a fluorescence spectrometer (Cary Eclipse; Agilent, Santa Clara, CA, U.S.A.), respectively. A JEOL-1200EX II (JEOL, Tokyo, Japan) transmission electron microscopy (TEM) system was used to measure the size of the AuNCs@BSA. An energy dispersive X-ray spectroscopy (EDS) system (Oxford Instruments, Abingdon, U.K.) was used to confirm the compositions of the AuNCs@BSA. The hydrodynamic radii of the AuNCs@BSA were measured using a dynamic light scattering (DLS) spectrophotometer (SZ-100; Horiba, Kyoto, Japan). X-ray photoelectron spectroscopy (XPS) was performed to validate the oxidation state of elemental Au by using an electron spectroscopy system (VG ESCA210; VG Scientific, West Sussex, U.K.). X-ray diffraction (XRD) measurements were collected to obtain information on the crystal structure of the AuNCs@BSA by using an X-ray diffractometer (LabX XRD-6000; Shimadzu, Kyoto, Japan) with CuKα radiation (λ = 0.15418 nm). The AuNCs@BSA were freeze-dried and then characterized through infrared spectroscopy performed using a Fourier transform infrared (FTIR) spectrometer (Cary 600; Agilent, Santa Clara, CA, U.S.A.). Mass measurements of the AuNCs@BSA and BSA were performed using α-cyano-7-hydroxycinnamic acid as a matrix. The spectra were collected using a matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) system (Microflex; Bruker Daltonics, Bremen, 6


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Germany) in the positive ionization mode. The time-resolved fluorescence spectra of the AuNCs@BSA were measured using a time-correlated photon-counting spectrometer as previously reported.28 For fluorescence lifetime measurements, the polarization of the excitation light was set vertical (relative to the optical table), and the angle of the emission polarizer was set to 54.7° relative to the excitation light.

3. RESULTS AND DISCUSSION 3.1. Microwave-Assisted Synthesis of AuNCs@BSA. The concentrations of BSA, HAuCl4, and NaOH used in this study to synthesize AuNCs@BSA were the same as those used in an earlier study.27,28 However, the domestic MW oven used in the earlier study was replaced. A focused single-mode MW synthesis instrument (300 W) was selected as the MW source to avoid damage to BSA, to ensure efficient production of MW energy, and to provide rapid MW heating. This source was preferred because of the uniformity of MW magnetic fields, the stability of MW power, and the controllability and reproducibility of the MW program. No overheating was observed under MW irradiation, indicating that pauses during the heating procedure were not required. The effect of synthesis temperature on the AuNCs@BSA is discussed as follows. Figure 1a displays the fluorescence spectra of the AuNCs@BSA synthesized at different reaction temperatures under MW irradiation for 4 min. A suitable reaction temperature range yielded higher-quality AuNCs@BSA, and a clear emission intensity was observed for the AuNCs@BSA until the reaction temperature reached 80 °C. At reaction temperatures higher than 135 °C, BSA formed an insoluble gel and produced large variations (relative standard deviation, RSD% > 19.2%) in the fluorescence intensity of the AuNCs@BSA. Figure 1b presents the fluorescence 7



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intensity of the AuNCs@BSA at emission wavelengths of 436 and 676 nm under different reaction temperatures. The fluorescence intensity at 436 nm increased with increasing reaction temperature. Subsequently, the reaction temperature was increased from 80 °C to 135 °C but the concentration of the reaction species remained the same, resulting in the decay of the fluorescence intensity at 676 nm. The mechanisms of AuNC formation using BSA have been studied.17 During the preparation process, the cysteine and histidine residues in BSA act as a soft template for coordination with Au3+, and the tyrosine residues at pH > 10.0 reduce Au3+ ions to Au atoms. At 80 °C, the less the protein conformational change and secondary structure destruction occur, the less the cysteine and histidine residues are available for coordination with Au3+. Thus, the reaction rate of nucleation is lower than that of cluster growth. Therefore, the formation of larger AuNCs@BSA showing red emission was favored, followed by the aggregation of small AuNCs at the reaction temperature of 80 °C. By contrast, higher temperatures effectively induced conformational changes and the loss of the secondary BSA structure. In a short time, the tyrosine residues in BSA reduced Au3+ ions to Au atoms. These Au atoms probably acted as seeds for the subsequent growth of Au8 clusters owing to the remarkable stability of this magic cluster containing closed electronic shells.33,34 Thus, smaller AuNCs@BSA showing blue emission were observed at the reaction temperature of 135 °C. These results indicated that the proposed MW-assisted method produced a rapid and large temperature increase and homogeneous heating, favoring the reduction of Au3+ precursors and the nucleation of AuNCs and consequently producing a markedly increased fraction of fluorescent AuNCs@BSA exhibiting a different emission. To confirm this hypothesis, the AuNCs@BSA prepared at reaction temperatures of 80 °C and 135 °C were subjected to further spectroscopic investigation. 8


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Figure 1. (a) Fluorescence spectra of AuNCs@BSA prepared at different temperatures (dash-dot-dot: 80 °C, short dash: 90 °C, dot: 100 °C, dash: 120 °C, and solid: 135 °C) and (b) fluorescence intensity of AuNCs@BSA for emission at 436 and 676 nm under UV light excitation (375 nm) at different temperatures.

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3.2. Spectroscopic Investigation of AuNCs@BSA. Figure 2a displays the TEM image of R-AuNCs@BSA, which indicates that the prepared R-AuNCs@BSA were capped as aggregates in BSA. As shown in Figure 2a, the average diameter of the R-AuNCs@BSA was 4.2 ± 0.5 nm. The EDS spectrum verified the presence of elemental Au, as shown in Figure 2b. Figure 2c shows the TEM image of B-AuNCs@BSA, indicating that the prepared B-AuNCs@BSA were well dispersed with an average diameter of 3.1 ± 0.4 nm. The EDS spectrum shown in Figure 2d confirms the presence of elemental Au. The DLS results shown in Figure S1 also reveal that the hydrodynamic diameters of the R-AuNCs@BSA were clearly larger and more broadly dispersed than those of the B-AuNCs@BSA. These results showed that reaction temperature affected the formation of the AuNCs@BSA—the higher the reaction temperature, the smaller the AuNCs@BSA produced.

Figure 2. TEM images and EDS spectra of (a), (b) R-AuNCs@BSA and (c), (d) B-AuNCs@BSA. For images in (a) and (c), scale bar = 20 nm. 10


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The oxidation state of the AuNCs@BSA was evaluated using XPS. The binding energy of Au 4f7/2 for the R-AuNCs@BSA as shown in Figure 3a was deconvoluted into two distinct components centered at 83.9 and 85.8 eV assigned to Au0 (dash) and Au+ (dot), respectively. The amount of Au+ in BSA (~11%) was consistent with the findings of Xie et al.16 They demonstrated that Au+ located on the surface of the clusters could be considered an intermediate species that form to stabilize the AuNCs. A fluorescence-quenching assay to detect Hg2+ ions through the metallophilic interaction between Hg2+ and Au+ was proposed (Figure S2a).35,36 Compared with the R-AuNCs@BSA, the binding energy of Au 4f7/2 for the B-AuNCs@BSA was shifted to 84.8 eV, as shown in Figure 3b. Two possible explanations were identified for this binding energy shift to a higher value. One explanation was the high concentration of Au+ ions on the B-AuNCs@BSA surface. However, the results of the fluorescence-quenching test conducted using Hg2+ ions indicated the absence of Au+ ions on the B-AuNCs@BSA surface (Figure S2b). Another explanation was reported by Tanaka et al., who demonstrated that the binding energy of AuNCs increased with decreasing cluster size depending to the positive charge left on the AuNCs.37 Thus, small B-AuNCs@BSA contained only Au0 with a binding energy of 84.8 eV. In Figure 2, TEM images illustrate that the B-AuNCs@BSA were smaller than the R-AuNCs@BSA and that the protein had a high degree of freedom. These findings were consistent with the hypothesis regarding the temperature-dependent formation of the AuNCs@BSA. The crystal structure and phase composition of the AuNCs@BSA were further characterized through XRD (Figure S3). We observed four peaks at 2θ = 37.9°, 44.2°, 64.5°, and 77.6°, corresponding to the diffractions from the (111), (200), (220), and (311) planes, respectively, of the face-centered cubic Au lattice. These results revealed the existence 11



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of pure crystalline Au structures for both types of AuNCs@BSA.

Figure 3. XPS spectra of (a) R-AuNCs@BSA and (b) B-AuNCs@BSA. FTIR spectroscopy and MALDI-TOF MS are useful tools to characterize the protein templates of AuNCs. Figure 4 shows the FTIR spectra of BSA, R-AuNCs@BSA, and B-AuNCs@BSA. The protein amide I band from 1600 to 1700 12


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cm−1 and the amide II band at approximately 1545 cm−1 were related to the secondary structure of the protein.38 Moreover, the protein amide I band has been fully studied and can provide useful secondary structure information about α-helixes, β-sheets, turns, and disordered domains.38 Both types of AuNCs@BSA showed the characteristic vibration peaks of BSA. In addition, a clear shift in the amide I band from 1670 cm−1 to 1645–1654 cm−1 was observed for both types of AuNCs@BSA. This trend indicated a modification of the secondary structure of BSA in the AuNCs@BSA. Moreover, the tryptophan (Trp) vibration between 1400 and 1450 cm−1 for the R-AuNCs@BSA became larger than that for the B-AuNCs@BSA. This result was consistent with the XPS measurements that showed a high amount of Au+ ions bound to nitrogen.

Figure 4. FTIR spectra of BSA (solid), R-AuNCs@BSA (dot), and B-AuNCs@BSA (dash). Figure S4 exhibits the MALDI-TOF MS spectrum of the R-AuNCs@BSA, in 13



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which two peaks for BSA (m/z = 66,000 Da) and R-AuNCs@BSA (m/z = 69,669 kDa) were detected. The 3,669 Da difference between BSA and the R-AuNCs@BSA proved that each R-AuNC@BSA consisted of approximately 18 Au atoms entrapped by BSA. The number of Au atoms in the R-AuNCs@BSA was equal to the magic numbers of free Aun clusters associated with the closing of the electronic shells (e.g., n = 8, 18, 20 34).1,3,39,40 The MALDI-TOF MS spectra of BSA and the B-AuNCs@BSA are shown in Figure S5. After MW irradiation at 135 °C, the characteristic peaks of BSA were observed, as shown in Figure S5a. In addition, the B-AuNCs@BSA showed a major characteristic peak at an m/z value of 1,519 Da, which indicated that small B-AuNCs@BSA contained approximately 8 Au atoms (Figure S5b). This result is consistent with the extraordinary stability of the magic clusters. 3.3. Optical Properties of AuNCs@BSA. As shown in Figure 5a, both AuNCs@BSA solutions had absorbance bands at 220 nm and 275 nm, which was due to the absorptions of tyrosine and tryptophan residues in BSA. No surface plasmon resonance absorption band higher than 400 nm was observed for either type of AuNCs@BSA, excluding the formation of large Au nanoparticles greater than 10 nm through our synthesis method. Excitation and fluorescence spectra of both types of AuNCs@BSA are plotted in Figure 5b. The R-AuNCs@BSA emitted high-intensity red fluorescence (λem = 676 nm) and low-intensity blue fluorescence (λem = 455 nm) when excited with 375 nm light; moreover, they exhibited two excitation peaks at 310 and 500 nm. These excitation and fluorescence peaks for the R-AuNCs@BSA indicated a polydispersed distribution of AuNCs with different sizes, resulting in a broad emission spectrum. The DLS results shown in Figure S1 confirm the presence of a more polydispersed distribution of AuNCs in the R-AuNCs@BSA than in the 14


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B-AuNCs@BSA. Fluorescence measurements for BSA without the addition of Au3+ before and after MW irradiation (Figure S6) indicated that the blue emission of the AuNCs@BSA could be mainly attributed to the abundance of fluorescent AuNCs and not to the fluorescence of BSA or carbon dots. The B-AuNCs@BSA exhibited blue emission with maximum intensity observed at 436 nm and a single excitation band at 333 nm. Both B-AuNCs@BSA and R-AuNCs@BSA exhibited no significant shift in their fluorescence wavelengths upon excitation in the 300–550 nm range, confirming the stability of the AuNCs@BSA (Figure S7).

Figure 5. (a) UV–Vis absorbance and (b) excitation (dot) and fluorescence (solid) spectra

of

R-AuNCs@BSA

and

B-AuNCs@BSA.

Inset:

photographs

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AuNCs@BSA under daylight and UV light (λex = 365 nm). The quantum yields (QYs) of the R-AuNCs@BSA and B-AuNCs@BSA were determined to be 4.14% and 1.94%, respectively, through a comparative method using riboflavin-5ʹ- phosphate (QY ≈ 26%) and quinine sulfate (QY ≈ 54%) as references (Figure S8). The QY values were consistent with previous results for AuNCs in different templates with similar sizes and emission wavelengths.27,28,30,32 Figure S9 shows the lifetime measurements for the AuNCs-BSA performed at two emission wavelengths of 676 and 436 nm corresponding to the R-AuNCs@BSA and B-AuNCs@BSA, respectively. All data were fitted, and the results are listed in Table 2. Higher reaction temperatures during the synthesis increased the number of small clusters in solution and therefore led to an intense blue fluorescent emission. The lifetime values for the B-AuNCs@BSA were τ1 = 0.50 ns (64.8%), τ2 = 2.70 ns (28.8%), and τ3 = 9.02 (6.4%). These emission lifetimes may be attributed to singlet transitions between the low-lying d band (HOMO) and the excited sp band (LUMO) of the AuNCs.34 The R-AuNCs@BSA showed a very long lifetime component (τ3 = 1195.9 ns, 53.6%) due to a triplet-singlet intraband transition.33 Another explanation for this observation was that because of the presence of thiol–Au+ complexes, the R-AuNCs@BSA exhibited ligand–metal charge transfer and Au+–Au+ interactions. Indeed, XPS results confirmed the presence of Au+ (approximately 11%) in the R-AuNCs@BSA and the absence of Au+ in the B-AuNCs@BSA that did not have a long lifetime component. In 2010, the ligand’s role in fluorescent AuNCs was determined by Wu and Jin.41 They emphasized the role of the metal oxidation state and the ligand-to-metal charge transfer in enhancing the fluorescence. Therefore, our results suggested that the long lifetime component of the R-AuNCs@BSA was due to the size of the AuNCs and the oxidation state of elemental Au. However, future 16


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studies should investigate the charge transfer mechanism of the AuNCs@BSA, the triplet and singlet excited states, and the function of the oxidation state.

Table 2. Optical Properties of AuNCs@BSA A1 λex λem τ1 AuNCs@BSA (nm) (nm) (ns) (%) R-AuNCs@BSA 375 676 4.71 35.1 B-AuNCs@BSA 375 436 0.50 64.8

τ2 (ns) 69.0 2.70

A2 (%) 11.3 28.8

τ3 (ns) 1195.9 9.02

A3 (%) 53.6 6.4

QY (%) 4.14 1.94

We concluded that a high reaction temperature increased the nucleation rate to produce smaller B-AuNCs@BSA, whereas a low reaction temperature favored the growth of the larger R-AuNCs@BSA. The long lifetime measured only for the R-AuNCs@BSA was attributed to the Au–BSA electronic charge transfer and to the oxidation state of elemental Au. The fluorescence property of the B-AuNCs@BSA was due to the energy gap between their HOMO and LUMO. 3.4. Reproducibility and Stability of AuNCs@BSA. To evaluate the reliability of the proposed MW-assisted method for preparing AuNCs@BSA, the intra- and interday reproducibility of their fluorescence intensities at 436 and 676 nm were measured. Table 3 presents the results of intra- and interday precision with RSD% less than 6.5% and 8.9%, respectively. Nevertheless, the fluorescence intensities for both types of AuNCs@BSA were relatively stable, indicating that the formed AuNCs@BSA were of high quality because of the controllability of the MW procedure. To investigate the fluorescence stability of the AuNCs@BSA, both types were kept at 4 °C after each measurement. Normalized fluorescence intensity was calculated as the ratio of the fluorescence intensity of the AuNCs@BSA on the first day to that at any other time. Therefore, the differences in the scattering properties of the AuNCs@BSA need not be accounted for. Figure S10 demonstrates the slight intensity fluctuations as a function of storage time. After 16 days, the normalized 17



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intensity of the R-AuNCs@BSA was 101%. However, the normalized intensity of the B-AuNCs@BSA was reduced to 85% because of the absence of Au+ ions to stabilize the AuNCs@BSA. Nevertheless, these results show that the AuNCs@BSA can provide superior fluorescence performance in terms of stability and durability until 2 weeks. Table 3. Reproducibility Test for AuNCs@BSA Fluorescence intensity (peak height) AuNCs@BSA Intraday (RSD%)a Interday (RSD%)a R-AuNCs@BSA 277.1 (2.2%) 283.4 (4.2%) (λex/λwm = 375/676 nm) B-AuNCs@BSA 339.6 (6.5%) 334.0 (8.9%) (λex/λwm = 375/436 nm) a The average and relative standard deviation of fluorescence intensity were measured (n = 5) to assess intraday reproducibility. b The average and relative standard deviation of fluorescence intensity were measured (n = 5) to assess interday reproducibility.

4. CONCLUSIONS AuNCs@BSA with a red emission (λem = 676 nm) and a blue emission (λem = 436 nm) were prepared using a controllable MW synthesis instrument. In this study, we highlighted the effect of reaction temperature on conformational changes in the BSA protein, oxidation state of metal elements, and cluster growth. TEM and MALDI-TOF MS results demonstrated that the B-AuNCs@BSA formed at 135 °C were small and had a weak interaction with BSA. Instead, when the MW irradiation was conducted at 80 °C, large R-AuNCs@BSA with a polydispersed distribution were formed, with clusters mainly consisting of 18 Au atoms covalently bound to BSA. In addition, the AuNCs promoted the modification of BSA with a decrease in the number of helical structures. Fluorescence studies confirmed that high temperature favored the nucleation rate for the formation of B-AuNCs@BSA, the growth of R-AuNCs@BSA 18


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followed by an aggregation of blue-emitting small clusters at a low temperature, and an energy transfer between small and large clusters. Furthermore, XPS and fluorescence measurements indicated the oxidation state of elemental Au, the stability of the AuNCs@BSA, and the presence of a long lifetime component within the R-AuNCs@BSA.

ASSOCIATED CONTENT AUTHOR INFORMATION Corresponding Author Address correspondence to these authors at Department of Chemistry, National Changhua University of Education, Changhua City, PO Box 500, Taiwan; Fax: 886-4-7211190; E-mail: [email protected]

ACKNOWLEDGMENT This study was supported by the Ministry of Science and Technology under contract MOST 106-2119-M-018-001. This manuscript was edited by Wallace Academic Editing.

Supporting Information Available: Figure S1. DLS measurements for (a) R-AuNCs@BSA and (b) B-AuNCs@BSA. Figure S2. Fluorescence spectra in the presence and absence of Hg2+ for (a) R-AuNCs@BSA

and

(b)

B-AuNCs@BSA.

Figure

S3.

XRD

spectra

of

R-AuNCs@BSA and B-AuNCs@BSA. Figure S4. MALDI-TOF MS spectra of BSA (red) and R-AuNCs@BSA (black). Figure S5. MALDI-TOF MS spectra of (a) BSA and (b) B-AuNCs@BSA. Figure S6. Fluorescence spectra of BSA before and after 19



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MW irradiation. Figure S7. 2D fluorescence spectra of (a) R-AuNCs@BSA and (b) B-AuNCs@BSA for excitation between 300 and 550 nm. Figure S8. QY measurement for (a) R-AuNCs@BSA, (b) riboflavin-5ʹ-phosphate reference (QY = 26%) for R-AuNCs@BSA, (c) B-AuNCs@BSA, and (d) quinine sulfate reference (QY = 56%) for

B-AuNCs@BSA.

Figure

S9.

Fluorescence

decay

measurements

for

R-AuNCs@BSA and B-AuNCs@BSA. Figure S10. Variation of normalized fluorescence intensity of R-AuNCs@BSA and B-AuNCs@BSA over 16 days. This information is available free of charge on the Internet at http://pubs.acs.org/

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Microwave-Assisted Formation of Gold Nanoclusters Capped in

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