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Novel synthesis of gold nanoclusters templated with L-tyrosine for selective analyzing tyrosinase Xiaoming Yang 1, *, Yawen Luo 1, Yan Zhuo, Yuanjiao Feng, Shanshan Zhu College of Pharmaceutical Sciences, Southwest University, Tiansheng Road No. 2, Beibei District, Chongqing 400716, China
H I G H L I G H T S
G R A P H I C A L A B S T R A C T
A novel, one-pot strategy for synthesizing fluorescent AuNCs@Tyr was proposed. A selective and cost-effective assay for TR activity has been well established. This AuNCs@Tyr here may broaden avenues for detecting TR in clinical applications.
One-pot and novel synthesized fluorescent gold nanoclusters templated with L-tyrosine (AuNCs@Tyr) were employed for investigating tyrosinase activity on the basis of aggregations of AuNCs@Tyr on its active sites during the catalysis reactions, thus leading to the fluorescence quenching of AuNCs@Tyr.
A R T I C L E I N F O
A B S T R A C T
Article history: Received 24 March 2014 Received in revised form 11 May 2014 Accepted 31 May 2014 Available online xxx
L-Tyrosine (Tyr),
Keywords: Tyrosine Gold nanoclusters Tyrosinase Fluorescence quenching
playing roles as both a reducing reagent and a protecting ligand, has been first employed for synthesizing fluorescent gold nanoclusters (AuNCs@Tyr) via a novel one-pot strategy. The asprepared AuNCs@Tyr exhibited a fluorescence emission at 470 nm with a quantum yield of approximately 2.5%. Subsequently, the AuNCs@Tyr described here was applied for detections of tyrosinase (TR) activity, which was based on the mechanism of aggregations of AuNCs@Tyr occurring on the active sites of TR since TR was introduced, thus leading to the fluorescence quenching of AuNCs@Tyr. Accordingly, TR was analyzed in a linear range of 0.5–200 u mL1 with a detection limit of 0.08 u mL1 at a signal-to-noise ratio of 3. Significantly, TR has been considered as a critical marker for melanoma owing to its specifically expressing in melanoma cells. Therefore, this analytical method towards investigating TR activity may broaden avenues for meaningfully clinical applications. ã 2014 Elsevier B.V. All rights reserved.
1. Introduction Noble metal nanoclusters (NCs), only consisting of several to tens of atoms, existed in sizes near by Fermi wavelength, and showed properties regulated by their subnanometer dimensions [1–4]. Hence, various methods have been developed for the synthesis of fluorescent AuNCs by using kinds of reducing reagents [5–11]. Usually, AuNCs were synthesized by two major ways. One way is based on the template-assisted synthesis with polymers
* Corresponding author. Tel.: +86 23 68251225; fax: +86 23 68251225. E-mail addresses: [email protected], [email protected] (X. Yang). 1 Both authors contributed equally to this work.
[12] and biomolecules (e.g., proteins [7,13,14] and DNA [15,16] commonly as templates). Meanwhile, another way was build up on the basis of monolayer protection in the presence of molecules with thiol ligands [17–20]. As a new type of fluorescent material, AuNCs exist in ultra-small size with low toxicity compared with quantum dots. Besides, unique characteristics of AuNCs have recently attracted numerous attentions, potentiating it as a satisfactory candidate for biosensing, catalysis, and imaging [1,4,21,22]. Tyrosinase (TR), as a copper-containing enzyme widely existing in plants, animal tissues and fungi [23], mainly functions as catalyzing the hydroxylation of phenolic substrates to catechol derivatives, and further oxidizing the catechol derivatives as orthoquinone products. Additionally, these reactions have been
http://dx.doi.org/10.1016/j.aca.2014.05.050 0003-2670/ ã 2014 Elsevier B.V. All rights reserved.
Please cite this article in press as: X. Yang, et al., Novel synthesis of gold nanoclusters templated with L-tyrosine for selective analyzing tyrosinase, Anal. Chim. Acta (2014), http://dx.doi.org/10.1016/j.aca.2014.05.050
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proved as key points during the biosynthetic pathway of melanin and other natural pigments [24,25]. Importantly, TR has been recognized as a specific marker for these cells due to its specifically expressing in melanocytes and melanoma cells [26]. Thus, exploring ways for detecting TR activity showed critical value towards clinical objectives. For this purpose, diverse methods have been developed. However, low sensitivity of traditional analyses for TR has exhibited obvious limitation [27]. Interestingly, new strategies have been reported. For instance, gold nanoparticles have been applied to quantifying TR as well as improving not only the detection feasibility but also the sensitivity [28]. Again, several fluorescent probes for assaying TR have also been well built up including cyanine dyes, conjugated polymers and quantum dots [29]. Among these methods, innovative nanomaterials mainly including metal nanoparticles and semiconductor quantum dots have been generally employed for serving as probes to determine TR activity, and these ideas have indeed showed attractive prospect. Nevertheless, these promising methods are still lacking. Therefore, it is necessary to develop sensitive and selective strategies to broaden ways for more effectively monitoring TR activity. In this contribution, novel fluorescent gold nanoclusters stabilized with L-tyrosine were first successfully synthesized, while Tyr served as both a reducing reagent and a protecting ligand. Next, high resolution transmission electron microscopy (HR-TEM), UV–vis absorption and fluorescence spectroscopy were introduced to characterize the properties of AuNCs@Tyr prepared here. Moreover, the current AuNCs@Tyr was applied for sensitive and selective detections of tyrosinase. The sensing principle was based on TR induced fluorescence quenching of AuNCs@Tyr, due to the aggregations of AuNCs@Tyr on active sites of TR during the catalysis reactions (Fig. 1), which has been further confirmed by the HR-TEM. Herein, a novel, simple, selective and cost-effective fluorescent probe has been established, suggesting its potential to broaden avenues for sensing TR. 2. Experimental 2.1. Materials and reagents Hydrogen tetrachloroaurate trihydrate (HAuCl4), L-tyrosine (Tyr), tyrosinase (TR) were purchased from Sigma–Aldrich (Milwaukee, WI). Bovine serum albumin (BSA), urease, subtilisin, ExoIII, glucose oxidase and all the metal ions were obtained from Shanghai Sangon Biotechnology Co., Ltd. (Shanghai, China). Hydrochloric acid (HCl), disodium hydrogen phosphate (Na2HPO4) and sodium dihydrogen phosphate (NaH2PO4) were purchased from Dingguo Changsheng Biotechnology Co., Ltd. (Beijing, China). Ultrapure water, 18.25 MV cm, produced by an Aquapro AWL0520-P ultrapure water system (Chongqing, China), was employed for all the following experiments.
2.2. Apparatus All fluorescence measurements were performed on a Hitachi F-7000 fluorescence spectrophotometer (Tokyo, Japan) with excitation slit set at 5 nm band pass and emission at 5 nm band pass in 1 cm 1 cm quartz cells. In addition, UV–vis spectra were recorded by a Shimadzu UV-1750 spectrophotometer (Tokyo, Japan). The high resolution transmission electron microscopy (HR-TEM) images were obtained by using a TECNAI G2 F20 microscope (FEI, America) at 200 KV. Images were taken with an Olympus E-510 digital camera (Tokyo, Japan). The quantum yields were obtained by using Absolute PL quantum yield spectrometer C11347 (Hamamatsu, Japan). A Fangzhong pHS-3C digital pH meter (Chengdu, China) was used to measure pH values of the aqueous solutions and a vortex mixer QL-901 (Haimen, China) was applied to blend the solution. The thermostatic water bath (DF-101s) was purchased from Gongyi Experimental Instruments Factory (Henan, China). 2.3. Synthesis of AuNCs@Tyr All glassware were thoroughly cleaned with freshly prepared aqua regia (HNO3/HCl, 1:3) and rinsed with ultrapure water prior to use. The AuNCs@Tyr was prepared by the following procedures. L-tyrosine was dissolved in hydrochloric acid solution (0.01 M) at the very beginning. Then, an aqueous solution of HAuCl4 (1.0 mL, 10 mM) was mixed with tyrosine solution (3.0 mL, 6 mM) and stirred vigorously at 37 C for 24 h. Finally, the products were centrifuged (3000 rpm, 3 min), and the supernatant was subjected to 1000 MWCO of dialysis membrane for purification before further characterization and applications. 2.4. Detection of tyrosinase Towards the purpose of detecting TR, stock solutions of TR were prepared respectively. Then, 50 mL of phosphate buffer solution (0.2 M, pH 6.4) was mixed with 20 mL of AuNCs@Tyr initially, and 430 mL of various concentrations of TR were added. After incubation for 4 h at 37 C, the fluorescence intensity of these mixtures were measured upon being excited at 385 nm. To evaluate the interference of biological metal ions, since TR was well known as a metal-containing enzyme, a variety of metal ions (e.g., Ca2+, Ba2+, Fe3+, Ni2+, Pb2+, Mn2+, Co2+, and K+) were introduced with the identical concentration of 250 mM. Furthermore, the selectivity of this assay has been evaluated in the presence of other biological protein (BSA, 1 mg mL1) and enzymes (e.g., urease, subtilisin, ExoIII, and glucose oxidase, 50 u mL1). 2.5. Calculation of detection limit As the lowest analyte concentration, limit of detection (LOD) reflects the sensitivity of analysis methods. To obtain LOD, the
Fig. 1. Schematic illustration of the synthesis of AuNCs@Tyr and the fluorescence quenching mechanism of AuNCs@Tyr by tyrosinase.
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Fig. 2. (A) Excitation (black) and emission (red) fluorescence spectra of AuNCs@Tyr. Inset: photographs of AuNCs@Tyr under daylight (I) and UV light (II); (B) UV–vis absorption spectra of Tyr and AuNCs@Tyr; (C) stability of the as-prepared AuNCs@Tyr; (D) HR-TEM image of AuNCs@Tyr. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
IUPAC recommended methodology was employed here, which utilized an experimentally determined signal-to-noise ratio (S/N) [30]. The fluorescence intensity of AuNCs@Tyr without tyrosinase as a control was measured 20 times at a designated wavelength (470 nm). Average fluorescence intensity (averageblank) along with the associated standard deviation (SDblank) were calculated on the basis of the above data, and SDblank was considered as the noise (N). Similarly, the fluorescence intensity of AuNCs@Tyr by adding standard tyrosinase solutions with a relatively low concentration were measured for five times, and the average value (averagesample) was calculated. Finally, S/N was calculated as follow:
S=N ¼
ðaverageblank averagesample Þ SDblank
Hence, a sample concentration which meets the condition of 3 < S/N < 5 was defined as LOD here. 2.6. Analysis of actual samples Human serum samples from 5 healthy volunteers were collected from Southwest University Hospital, Chongqing. For the detection of TR, whole blood samples were frozen at 20 C prior to analysis. Acetonitrile (99.5%, w/w) was added to the blood samples to rupture red cell membranes. Insoluble material was removed by centrifugation (10,000 rpm, 10 min), then the supernatant was warmed and evaporated to remove liquid. After being cooled to 27 C, the serum samples were filtered through a 0.2 mm membrane and collected in aliquots. The serum samples were then analyzed separately with this proposed fluorescent strategy and a colorimetric method.
Fig. 3. (A) Fluorescence emission spectra of AuNCs@Tyr in the absence (a) and presence (b) of 50 u mL1 TR. Inset: photographs of fluorescent AuNCs@Tyr in the absence (I, III) and presence (II, IV) of TR under daylight (top) and UV light (bottom); (B) HR-TEM image of AuNCs@Tyr in the presence of 50 u mL1 TR. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Please cite this article in press as: X. Yang, et al., Novel synthesis of gold nanoclusters templated with L-tyrosine for selective analyzing tyrosinase, Anal. Chim. Acta (2014), http://dx.doi.org/10.1016/j.aca.2014.05.050
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Fig. 4. (A) Influence of pH values (6.0, 6.2, 6.4, 6.6, 6.8, 7.0) on the fluorescence decrease of AuNCs@Tyr in presence of TR (50 u mL1); (B) optimization of incubation time (1–8 h) for the detection procedure.
3. Results and discussion 3.1. Characterization of AuNCs@Tyr In this study, the fluorescent AuNCs@Tyr was synthesized in aqueous solution by an innovative approach, and its quantum yield was determined to be 2.5%. It was worth mentioning that these fluorescent AuNCs were prepared without additions of any other catalyst or chemical during the whole synthesis process, and the reactants were environmentally friendly, suggesting that the current synthesis procedure was a green method. To characterize the AuNCs@Tyr described here, fluorescence spectroscopy, UV–vis absorption spectroscopy and HR-TEM were successively employed. As Fig. 2A indicates, a standard fluorescence emission peak was centered at 470 nm as well as excited at 385 nm. Meanwhile, the photographs of AuNCs@Tyr were acquired under day light and UV light. Specifically, the color of AuNCs@Tyr solution was slightly yellow under visible light (photograph I), while an obvious yellow-green fluorescence appeared in view under UV light (photograph II). Subsequently, as revealed in Fig. 2B, there is no the characteristic surface plasmon resonance (SPR) peak of gold nanoparticles in UV–vis spectrum of AuNCs@Tyr [31,32]. Instead, it showed absorptions around 250–450 nm with a band edge of 450 nm, mostly due to its molecular-like properties [6,33– 35]. Additionally, it was also observed that the as-prepared AuNCs@Tyr were stable with scarce change of the fluorescence intensity even after 15 days (Fig. 2C), proving its favorable fluorescent stability. To further visualize the shape as well as measuring the diameter of AuNCs@Tyr, high resolution transmission electron microscopic images were obtained. As depicted in Fig. 2D, the majority of AuNCs@Tyr existed within the size range of 1–3 nm together with no distinct aggregations observed by HRTEM, demonstrating that the synthetic procedure allowed the sizes
of this new nanoclusters effectively controlled. Taken together, the data above suggested that AuNCs@Tyr has been well synthesized by using the novel and simple method proposed here. 3.2. Fluorescence quenching of AuNCs@Tyr by tyrosinase To address whether there exists an interaction between AuNCs@Tyr and TR or not while this nanoclusters stabilized with Tyr, TR were then introduced into AuNCs@Tyr. As shown in Fig. 3A, the fluorescence signal of AuNCs@Tyr responded to TR treatment. Briefly, the fluorescence intensity at 470 nm dramatically decreased upon addition of 50 u mL1 TR. At the same time, the color of AuNCs@Tyr solution transformed from slightly yellow (photograph I) to brown (photograph II) under daylight, and the fluorescence of the AuNCs@Tyr solution changed from yellow green (photograph III) to disappearing (photograph IV) under UV night. To further explore the quenching mechanism, HR-TEM measurements were performed. As shown in Fig. 3B, the sizes of the AuNCs@Tyr ranged from 50 nm to 70 nm in the presence of 50 u mL1 TR, indicating the sizes of AuNCs@Tyr growing up. The reasonable explanation for this phenomenon was that the substrate (Tyr) tended to aggregate on the active sites of the enzyme (TR) during the catalytic reactions, thus facilitating to induce the fluorescence quenching of AuNCs@Tyr. These results suggested that the AuNCs@Tyr prepared here could potentially be applied as a fluorescence probe to detect TR activity. 3.3. Optimization of detection conditions Towards the purpose of optimizing detection conditions, a series of experiments were performed. As reported, TR activity was influenced by different pH values, and the optimum pH value for this enzyme was identified between 6 and 7 [23]. To obtain the
Fig. 5. (A) Fluorescence emission spectra of AuNCs@Tyr in the presence of various concentrations of TR; (B) plot of fluorescence intensity decrease (F0–F) versus the logarithm of concentration of TR introduced.
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Fig. 6. (A) Interference of biological metal ions (250 mM) on the fluorescence decrease (F0–F) of AuNCs@Tyr in the presence of TR (50 u mL1); (B) F0–F of AuNCs@Tyr in the presence of tyrosinase, urease, subtilisin, ExoIII, glucose oxidase (50 u mL1) and BSA (1 mg mL1) respectively.
optimal pH condition for the detection approach, the tyrosinasepromoted aggregations of AuNCs@Tyr was performed in 0.2 M phosphate buffer at pH values of 6.0, 6.2, 6.4, 6.6, 6.8, 7.0 respectively, and the fluorescence intensities were recorded 6 h later. As shown in Fig. 4A, the fluorescent intensity decrease (F0–F) reached the maximum while pH was set at 6.4, suggesting that the phosphate buffer with the pH of 6.4 provided optimal performance for assaying TR. Considering the influence on detections by incubation time, further experiments towards reaction time (1–8 h) have been investigated respectively. Fig. 4B indicates that the tyrosinaseinduced fluorescence quenching of AuNCs@Tyr has completed within 4 h. Therefore, the phosphate buffer of pH 6.4 and incubation time of 4 h served as the optimal conditions during the following experiments. In addition, the whole procedure for testing TR was carried out at 37 C, since the body temperature of human beings were 37 C, and it was the optimum temperature for catalytic reactions regulated by tyrosinase. 3.4. Detection of tyrosinase As shown in Fig. 5A, the fluorescence intensity of AuNCs@Tyr decreased according to the amounts of TR added (0.5, 0.8, 1.0, 3.0, 5.0, 6.0, 8.0, 10.0, 30.0, 50.0, 80.0, 100.0, 200.0 u mL1). In particular, the fluorescent intensity decrease (F0–F) versus the logarithmic plots of TR various concentrations displayed a linear range from 0.5 u mL1 to 200 u mL1 (Fig. 5B). Meanwhile, the detection limit of TR was obtained as 0.08 u mL1 at a signal-to-ratio of 3. Overall, these results indicated that the fluorescence probe (AuNCs@Tyr) produced here is suitable for the measurement of TR activity. 3.5. Interference and selectivity studies As been well known, tyrosinase played a role as a metalcontaining enzyme. Thus, we asked whether foreign coexisting substance imply effect on the measurement of TR or not, further interference experiments were performed in the presence of TR (50 u mL1) together with kinds of biologically significant metal Table 1 Comparison of detection of TR in human serum samples by the colorimetric method and the proposed-fluorescent method. Samples
Colorimetric method (u mL1)
Fluorescent method (u mL1)
Serum Serum Serum Serum Serum
7.41 6.78 9.25 8.53 6.51
7.55 6.95 9.51 8.74 6.83
1 2 3 4 5
ions (Ca2+, Ba2+, Fe3+, Ni2+, Pb2+, Mn2+, Co2+, and K+). As shown in Fig. 6A, there was no obvious variation for the fluorescent signals, indicating that foreign coexisting metal ions showed scarce effect on assaying TR activity. To identify the selectivity of the current method, experiments were manipulated separately in the presence of a series of other enzymes including urease, subtilisin, ExoIII, and glucose oxidase and a typically biological protein of BSA. As shown in Fig. 6B, about a 70% decrease of the fluorescence intensity was observed upon addition of TR. In striking contrast, no obvious decrease was found by introducing other enzymes or the biological protein into AuNCs@Tyr, revealing that the satisfactory selectivity of AuNCs@Tyr for TR detection. 3.6. Detection of tyrosinase in human serum samples To evaluate its applicability, the described method was applied to assay the TR activity in five human serum samples, and the samples were also subjected to the colorimetric method at the same time. The TR activity detected in all samples was derived from standard curves and regression equations. As listed in Table 1, the results obtained by these two methods were in satisfactory agreement. Therefore, this method proposed here has potential for broadening roads of detecting TR activity in actual samples. 4. Conclusion In summary, we have successfully proposed a novel strategy to synthesize fluorescent AuNCs, while L-tyrosine serving as a reducing agent and a stabilizing ligand. Interestingly, this synthesis procedure was simple, facile and environment-friendly, and there was no any external toxic reagent and complicated instrument employed. In addition, we have utilized such AuNCs@Tyr for costeffective and selective detections of TR activity. Specifically, assaying of TR was based on the fluorescent decrease of AuNCs@Tyr, primarily due to the aggregations of AuNCs@Tyr on active sites of TR during the process of enzyme–substrate reactions. Up to date, investigating TR activity by fluorescent AuNCs@Tyr has not been reported, and there exhibited little interference originated from other biological metal ions for this method. Simultaneously, the current method was applied for testing TR activity in actual samples, confirming its practicability. Thus, the AuNCs@Tyr obtained here has potential for assaying the specific marker of TR in clinical applications. Acknowledgements We gratefully acknowledge financial support by National Natural Science Foundation of China (31100981), Research Fund
Please cite this article in press as: X. Yang, et al., Novel synthesis of gold nanoclusters templated with L-tyrosine for selective analyzing tyrosinase, Anal. Chim. Acta (2014), http://dx.doi.org/10.1016/j.aca.2014.05.050
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for the Doctoral Program of Higher Education of China (20110182120014), Natural Science Foundation Project of CQ CSTC (cstc2013jcyjA10117), Fundamental Research Funds for the Central Universities (XDJK2013B038), and Program for Innovative Research Team in University of Chongqing (2013). References [1] R. Jin, Quantum sized, thiolate-protected gold nanoclusters, Nanoscale 2 (2010) 343–362. [2] Q. Zhang, J. Xie, Y. Yu, J.Y. Lee, Monodispersity control in the synthesis of monometallic and bimetallic quasi-spherical gold and silver nanoparticles, Nanoscale 2 (2010) 1962–1975. [3] I. Díez, R.H. Ras, Fluorescent silver nanoclusters, Nanoscale 3 (2011) 1963–1970. [4] J. Zheng, P.R. Nicovich, R.M. Dickson, Highly fluorescent noble metal quantum dots, Annu. Rev. Phys. Chem. 58 (2007) 409. [5] X. Liu, C. Li, J. Xu, J. Lv, M. Zhu, Y. Guo, S. Cui, H. Liu, S. Wang, Y. Li, Surfactant-free synthesis and functionalization of highly fluorescent gold quantum dots, J. Phys. Chem. C 112 (2008) 10778–10783. [6] J. Zheng, J.T. Petty, R.M. Dickson, High quantum yield blue emission from water-soluble Au8 nanodots, J. Am. Chem. Soc. 125 (2003) 7780–7781. [7] J. Xie, Y. Zheng, J.Y. Ying, Protein-directed synthesis of highly fluorescent gold nanoclusters, J. Am. Chem. Soc. 131 (2009) 888–889. [8] W. Chen, X. Tu, X. Guo, Fluorescent gold nanoparticles-based fluorescence sensor for Cu2+ ions, Chem. Commun. (2009) 1736–1738. [9] H. Kawasaki, H. Yamamoto, H. Fujimori, R. Arakawa, Y. Iwasaki, M. Inada, Stability of the DMF-protected Au nanoclusters: photochemical, dispersion, and thermal properties, Langmuir 26 (2009) 5926–5933. [10] Y. Bao, H.-C. Yeh, C. Zhong, S.A. Ivanov, J.K. Sharma, M.L. Neidig, D.M. Vu, A.P. Shreve, R.B. Dyer, J.H. Werner, Formation and stabilization of fluorescent gold nanoclusters using small molecules, J. Phys. Chem. C 114 (2010) 15879–15882. [11] J.-a. Annie Ho, H.-C. Chang, W.-T. Su, DOPA-mediated reduction allows the facile synthesis of fluorescent gold nanoclusters for use as sensing probes for ferric ions, Anal. Chem. 84 (2012) 3246–3253. [12] J.I. Gonzalez, T.-H. Lee, M.D. Barnes, Y. Antoku, R.M. Dickson, Quantum mechanical single-gold-nanocluster electroluminescent light source at room temperature, Phys. Rev. Lett. 93 (2004) 147402. [13] C.L. Liu, H.T. Wu, Y.H. Hsiao, C.W. Lai, C.W. Shih, Y.K. Peng, K.C. Tang, H.W. Chang, Y.C. Chien, J.K. Hsiao, Insulin-directed synthesis of fluorescent gold nanoclusters: preservation of insulin bioactivity and versatility in cell imaging, Angew. Chem. Int. Ed. 50 (2011) 7056–7060. [14] H. Wei, Z. Wang, L. Yang, S. Tian, C. Hou, Y. Lu, Lysozyme-stabilized gold fluorescent cluster: synthesis and application as Hg2+ sensor, Analyst 135 (2010) 1406–1410. [15] A. Thomas, Blue emitting gold nanoclusters templated by poly-cytosine DNA at low pH and poly-adenine DNA at neutral pH, Chem. Commun. 48 (2012) 6845–6847. [16] G. Liu, Y. Shao, K. Ma, Q. Cui, F. Wu, S. Xu, Synthesis of DNA-templated fluorescent gold nanoclusters, Gold Bull. 45 (2012) 69–74.
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Analytica Chimica Acta xxx (2014) xxx–xxx
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Novel synthesis of gold nanoclusters templated with L-tyrosine for selective analyzing tyrosinase Xiaoming Yang 1, *, Yawen Luo 1, Yan Zhuo, Yuanjiao Feng, Shanshan Zhu College of Pharmaceutical Sciences, Southwest University, Tiansheng Road No. 2, Beibei District, Chongqing 400716, China
H I G H L I G H T S
G R A P H I C A L A B S T R A C T
A novel, one-pot strategy for synthesizing fluorescent AuNCs@Tyr was proposed. A selective and cost-effective assay for TR activity has been well established. This AuNCs@Tyr here may broaden avenues for detecting TR in clinical applications.
One-pot and novel synthesized fluorescent gold nanoclusters templated with L-tyrosine (AuNCs@Tyr) were employed for investigating tyrosinase activity on the basis of aggregations of AuNCs@Tyr on its active sites during the catalysis reactions, thus leading to the fluorescence quenching of AuNCs@Tyr.
A R T I C L E I N F O
A B S T R A C T
Article history: Received 24 March 2014 Received in revised form 11 May 2014 Accepted 31 May 2014 Available online xxx
L-Tyrosine (Tyr),
Keywords: Tyrosine Gold nanoclusters Tyrosinase Fluorescence quenching
playing roles as both a reducing reagent and a protecting ligand, has been first employed for synthesizing fluorescent gold nanoclusters (AuNCs@Tyr) via a novel one-pot strategy. The asprepared AuNCs@Tyr exhibited a fluorescence emission at 470 nm with a quantum yield of approximately 2.5%. Subsequently, the AuNCs@Tyr described here was applied for detections of tyrosinase (TR) activity, which was based on the mechanism of aggregations of AuNCs@Tyr occurring on the active sites of TR since TR was introduced, thus leading to the fluorescence quenching of AuNCs@Tyr. Accordingly, TR was analyzed in a linear range of 0.5–200 u mL1 with a detection limit of 0.08 u mL1 at a signal-to-noise ratio of 3. Significantly, TR has been considered as a critical marker for melanoma owing to its specifically expressing in melanoma cells. Therefore, this analytical method towards investigating TR activity may broaden avenues for meaningfully clinical applications. ã 2014 Elsevier B.V. All rights reserved.
1. Introduction Noble metal nanoclusters (NCs), only consisting of several to tens of atoms, existed in sizes near by Fermi wavelength, and showed properties regulated by their subnanometer dimensions [1–4]. Hence, various methods have been developed for the synthesis of fluorescent AuNCs by using kinds of reducing reagents [5–11]. Usually, AuNCs were synthesized by two major ways. One way is based on the template-assisted synthesis with polymers
* Corresponding author. Tel.: +86 23 68251225; fax: +86 23 68251225. E-mail addresses: [email protected], [email protected] (X. Yang). 1 Both authors contributed equally to this work.
[12] and biomolecules (e.g., proteins [7,13,14] and DNA [15,16] commonly as templates). Meanwhile, another way was build up on the basis of monolayer protection in the presence of molecules with thiol ligands [17–20]. As a new type of fluorescent material, AuNCs exist in ultra-small size with low toxicity compared with quantum dots. Besides, unique characteristics of AuNCs have recently attracted numerous attentions, potentiating it as a satisfactory candidate for biosensing, catalysis, and imaging [1,4,21,22]. Tyrosinase (TR), as a copper-containing enzyme widely existing in plants, animal tissues and fungi [23], mainly functions as catalyzing the hydroxylation of phenolic substrates to catechol derivatives, and further oxidizing the catechol derivatives as orthoquinone products. Additionally, these reactions have been
http://dx.doi.org/10.1016/j.aca.2014.05.050 0003-2670/ ã 2014 Elsevier B.V. All rights reserved.
Please cite this article in press as: X. Yang, et al., Novel synthesis of gold nanoclusters templated with L-tyrosine for selective analyzing tyrosinase, Anal. Chim. Acta (2014), http://dx.doi.org/10.1016/j.aca.2014.05.050
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proved as key points during the biosynthetic pathway of melanin and other natural pigments [24,25]. Importantly, TR has been recognized as a specific marker for these cells due to its specifically expressing in melanocytes and melanoma cells [26]. Thus, exploring ways for detecting TR activity showed critical value towards clinical objectives. For this purpose, diverse methods have been developed. However, low sensitivity of traditional analyses for TR has exhibited obvious limitation [27]. Interestingly, new strategies have been reported. For instance, gold nanoparticles have been applied to quantifying TR as well as improving not only the detection feasibility but also the sensitivity [28]. Again, several fluorescent probes for assaying TR have also been well built up including cyanine dyes, conjugated polymers and quantum dots [29]. Among these methods, innovative nanomaterials mainly including metal nanoparticles and semiconductor quantum dots have been generally employed for serving as probes to determine TR activity, and these ideas have indeed showed attractive prospect. Nevertheless, these promising methods are still lacking. Therefore, it is necessary to develop sensitive and selective strategies to broaden ways for more effectively monitoring TR activity. In this contribution, novel fluorescent gold nanoclusters stabilized with L-tyrosine were first successfully synthesized, while Tyr served as both a reducing reagent and a protecting ligand. Next, high resolution transmission electron microscopy (HR-TEM), UV–vis absorption and fluorescence spectroscopy were introduced to characterize the properties of AuNCs@Tyr prepared here. Moreover, the current AuNCs@Tyr was applied for sensitive and selective detections of tyrosinase. The sensing principle was based on TR induced fluorescence quenching of AuNCs@Tyr, due to the aggregations of AuNCs@Tyr on active sites of TR during the catalysis reactions (Fig. 1), which has been further confirmed by the HR-TEM. Herein, a novel, simple, selective and cost-effective fluorescent probe has been established, suggesting its potential to broaden avenues for sensing TR. 2. Experimental 2.1. Materials and reagents Hydrogen tetrachloroaurate trihydrate (HAuCl4), L-tyrosine (Tyr), tyrosinase (TR) were purchased from Sigma–Aldrich (Milwaukee, WI). Bovine serum albumin (BSA), urease, subtilisin, ExoIII, glucose oxidase and all the metal ions were obtained from Shanghai Sangon Biotechnology Co., Ltd. (Shanghai, China). Hydrochloric acid (HCl), disodium hydrogen phosphate (Na2HPO4) and sodium dihydrogen phosphate (NaH2PO4) were purchased from Dingguo Changsheng Biotechnology Co., Ltd. (Beijing, China). Ultrapure water, 18.25 MV cm, produced by an Aquapro AWL0520-P ultrapure water system (Chongqing, China), was employed for all the following experiments.
2.2. Apparatus All fluorescence measurements were performed on a Hitachi F-7000 fluorescence spectrophotometer (Tokyo, Japan) with excitation slit set at 5 nm band pass and emission at 5 nm band pass in 1 cm 1 cm quartz cells. In addition, UV–vis spectra were recorded by a Shimadzu UV-1750 spectrophotometer (Tokyo, Japan). The high resolution transmission electron microscopy (HR-TEM) images were obtained by using a TECNAI G2 F20 microscope (FEI, America) at 200 KV. Images were taken with an Olympus E-510 digital camera (Tokyo, Japan). The quantum yields were obtained by using Absolute PL quantum yield spectrometer C11347 (Hamamatsu, Japan). A Fangzhong pHS-3C digital pH meter (Chengdu, China) was used to measure pH values of the aqueous solutions and a vortex mixer QL-901 (Haimen, China) was applied to blend the solution. The thermostatic water bath (DF-101s) was purchased from Gongyi Experimental Instruments Factory (Henan, China). 2.3. Synthesis of AuNCs@Tyr All glassware were thoroughly cleaned with freshly prepared aqua regia (HNO3/HCl, 1:3) and rinsed with ultrapure water prior to use. The AuNCs@Tyr was prepared by the following procedures. L-tyrosine was dissolved in hydrochloric acid solution (0.01 M) at the very beginning. Then, an aqueous solution of HAuCl4 (1.0 mL, 10 mM) was mixed with tyrosine solution (3.0 mL, 6 mM) and stirred vigorously at 37 C for 24 h. Finally, the products were centrifuged (3000 rpm, 3 min), and the supernatant was subjected to 1000 MWCO of dialysis membrane for purification before further characterization and applications. 2.4. Detection of tyrosinase Towards the purpose of detecting TR, stock solutions of TR were prepared respectively. Then, 50 mL of phosphate buffer solution (0.2 M, pH 6.4) was mixed with 20 mL of AuNCs@Tyr initially, and 430 mL of various concentrations of TR were added. After incubation for 4 h at 37 C, the fluorescence intensity of these mixtures were measured upon being excited at 385 nm. To evaluate the interference of biological metal ions, since TR was well known as a metal-containing enzyme, a variety of metal ions (e.g., Ca2+, Ba2+, Fe3+, Ni2+, Pb2+, Mn2+, Co2+, and K+) were introduced with the identical concentration of 250 mM. Furthermore, the selectivity of this assay has been evaluated in the presence of other biological protein (BSA, 1 mg mL1) and enzymes (e.g., urease, subtilisin, ExoIII, and glucose oxidase, 50 u mL1). 2.5. Calculation of detection limit As the lowest analyte concentration, limit of detection (LOD) reflects the sensitivity of analysis methods. To obtain LOD, the
Fig. 1. Schematic illustration of the synthesis of AuNCs@Tyr and the fluorescence quenching mechanism of AuNCs@Tyr by tyrosinase.
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Fig. 2. (A) Excitation (black) and emission (red) fluorescence spectra of AuNCs@Tyr. Inset: photographs of AuNCs@Tyr under daylight (I) and UV light (II); (B) UV–vis absorption spectra of Tyr and AuNCs@Tyr; (C) stability of the as-prepared AuNCs@Tyr; (D) HR-TEM image of AuNCs@Tyr. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
IUPAC recommended methodology was employed here, which utilized an experimentally determined signal-to-noise ratio (S/N) [30]. The fluorescence intensity of AuNCs@Tyr without tyrosinase as a control was measured 20 times at a designated wavelength (470 nm). Average fluorescence intensity (averageblank) along with the associated standard deviation (SDblank) were calculated on the basis of the above data, and SDblank was considered as the noise (N). Similarly, the fluorescence intensity of AuNCs@Tyr by adding standard tyrosinase solutions with a relatively low concentration were measured for five times, and the average value (averagesample) was calculated. Finally, S/N was calculated as follow:
S=N ¼
ðaverageblank averagesample Þ SDblank
Hence, a sample concentration which meets the condition of 3 < S/N < 5 was defined as LOD here. 2.6. Analysis of actual samples Human serum samples from 5 healthy volunteers were collected from Southwest University Hospital, Chongqing. For the detection of TR, whole blood samples were frozen at 20 C prior to analysis. Acetonitrile (99.5%, w/w) was added to the blood samples to rupture red cell membranes. Insoluble material was removed by centrifugation (10,000 rpm, 10 min), then the supernatant was warmed and evaporated to remove liquid. After being cooled to 27 C, the serum samples were filtered through a 0.2 mm membrane and collected in aliquots. The serum samples were then analyzed separately with this proposed fluorescent strategy and a colorimetric method.
Fig. 3. (A) Fluorescence emission spectra of AuNCs@Tyr in the absence (a) and presence (b) of 50 u mL1 TR. Inset: photographs of fluorescent AuNCs@Tyr in the absence (I, III) and presence (II, IV) of TR under daylight (top) and UV light (bottom); (B) HR-TEM image of AuNCs@Tyr in the presence of 50 u mL1 TR. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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Fig. 4. (A) Influence of pH values (6.0, 6.2, 6.4, 6.6, 6.8, 7.0) on the fluorescence decrease of AuNCs@Tyr in presence of TR (50 u mL1); (B) optimization of incubation time (1–8 h) for the detection procedure.
3. Results and discussion 3.1. Characterization of AuNCs@Tyr In this study, the fluorescent AuNCs@Tyr was synthesized in aqueous solution by an innovative approach, and its quantum yield was determined to be 2.5%. It was worth mentioning that these fluorescent AuNCs were prepared without additions of any other catalyst or chemical during the whole synthesis process, and the reactants were environmentally friendly, suggesting that the current synthesis procedure was a green method. To characterize the AuNCs@Tyr described here, fluorescence spectroscopy, UV–vis absorption spectroscopy and HR-TEM were successively employed. As Fig. 2A indicates, a standard fluorescence emission peak was centered at 470 nm as well as excited at 385 nm. Meanwhile, the photographs of AuNCs@Tyr were acquired under day light and UV light. Specifically, the color of AuNCs@Tyr solution was slightly yellow under visible light (photograph I), while an obvious yellow-green fluorescence appeared in view under UV light (photograph II). Subsequently, as revealed in Fig. 2B, there is no the characteristic surface plasmon resonance (SPR) peak of gold nanoparticles in UV–vis spectrum of AuNCs@Tyr [31,32]. Instead, it showed absorptions around 250–450 nm with a band edge of 450 nm, mostly due to its molecular-like properties [6,33– 35]. Additionally, it was also observed that the as-prepared AuNCs@Tyr were stable with scarce change of the fluorescence intensity even after 15 days (Fig. 2C), proving its favorable fluorescent stability. To further visualize the shape as well as measuring the diameter of AuNCs@Tyr, high resolution transmission electron microscopic images were obtained. As depicted in Fig. 2D, the majority of AuNCs@Tyr existed within the size range of 1–3 nm together with no distinct aggregations observed by HRTEM, demonstrating that the synthetic procedure allowed the sizes
of this new nanoclusters effectively controlled. Taken together, the data above suggested that AuNCs@Tyr has been well synthesized by using the novel and simple method proposed here. 3.2. Fluorescence quenching of AuNCs@Tyr by tyrosinase To address whether there exists an interaction between AuNCs@Tyr and TR or not while this nanoclusters stabilized with Tyr, TR were then introduced into AuNCs@Tyr. As shown in Fig. 3A, the fluorescence signal of AuNCs@Tyr responded to TR treatment. Briefly, the fluorescence intensity at 470 nm dramatically decreased upon addition of 50 u mL1 TR. At the same time, the color of AuNCs@Tyr solution transformed from slightly yellow (photograph I) to brown (photograph II) under daylight, and the fluorescence of the AuNCs@Tyr solution changed from yellow green (photograph III) to disappearing (photograph IV) under UV night. To further explore the quenching mechanism, HR-TEM measurements were performed. As shown in Fig. 3B, the sizes of the AuNCs@Tyr ranged from 50 nm to 70 nm in the presence of 50 u mL1 TR, indicating the sizes of AuNCs@Tyr growing up. The reasonable explanation for this phenomenon was that the substrate (Tyr) tended to aggregate on the active sites of the enzyme (TR) during the catalytic reactions, thus facilitating to induce the fluorescence quenching of AuNCs@Tyr. These results suggested that the AuNCs@Tyr prepared here could potentially be applied as a fluorescence probe to detect TR activity. 3.3. Optimization of detection conditions Towards the purpose of optimizing detection conditions, a series of experiments were performed. As reported, TR activity was influenced by different pH values, and the optimum pH value for this enzyme was identified between 6 and 7 [23]. To obtain the
Fig. 5. (A) Fluorescence emission spectra of AuNCs@Tyr in the presence of various concentrations of TR; (B) plot of fluorescence intensity decrease (F0–F) versus the logarithm of concentration of TR introduced.
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Fig. 6. (A) Interference of biological metal ions (250 mM) on the fluorescence decrease (F0–F) of AuNCs@Tyr in the presence of TR (50 u mL1); (B) F0–F of AuNCs@Tyr in the presence of tyrosinase, urease, subtilisin, ExoIII, glucose oxidase (50 u mL1) and BSA (1 mg mL1) respectively.
optimal pH condition for the detection approach, the tyrosinasepromoted aggregations of AuNCs@Tyr was performed in 0.2 M phosphate buffer at pH values of 6.0, 6.2, 6.4, 6.6, 6.8, 7.0 respectively, and the fluorescence intensities were recorded 6 h later. As shown in Fig. 4A, the fluorescent intensity decrease (F0–F) reached the maximum while pH was set at 6.4, suggesting that the phosphate buffer with the pH of 6.4 provided optimal performance for assaying TR. Considering the influence on detections by incubation time, further experiments towards reaction time (1–8 h) have been investigated respectively. Fig. 4B indicates that the tyrosinaseinduced fluorescence quenching of AuNCs@Tyr has completed within 4 h. Therefore, the phosphate buffer of pH 6.4 and incubation time of 4 h served as the optimal conditions during the following experiments. In addition, the whole procedure for testing TR was carried out at 37 C, since the body temperature of human beings were 37 C, and it was the optimum temperature for catalytic reactions regulated by tyrosinase. 3.4. Detection of tyrosinase As shown in Fig. 5A, the fluorescence intensity of AuNCs@Tyr decreased according to the amounts of TR added (0.5, 0.8, 1.0, 3.0, 5.0, 6.0, 8.0, 10.0, 30.0, 50.0, 80.0, 100.0, 200.0 u mL1). In particular, the fluorescent intensity decrease (F0–F) versus the logarithmic plots of TR various concentrations displayed a linear range from 0.5 u mL1 to 200 u mL1 (Fig. 5B). Meanwhile, the detection limit of TR was obtained as 0.08 u mL1 at a signal-to-ratio of 3. Overall, these results indicated that the fluorescence probe (AuNCs@Tyr) produced here is suitable for the measurement of TR activity. 3.5. Interference and selectivity studies As been well known, tyrosinase played a role as a metalcontaining enzyme. Thus, we asked whether foreign coexisting substance imply effect on the measurement of TR or not, further interference experiments were performed in the presence of TR (50 u mL1) together with kinds of biologically significant metal Table 1 Comparison of detection of TR in human serum samples by the colorimetric method and the proposed-fluorescent method. Samples
Colorimetric method (u mL1)
Fluorescent method (u mL1)
Serum Serum Serum Serum Serum
7.41 6.78 9.25 8.53 6.51
7.55 6.95 9.51 8.74 6.83
1 2 3 4 5
ions (Ca2+, Ba2+, Fe3+, Ni2+, Pb2+, Mn2+, Co2+, and K+). As shown in Fig. 6A, there was no obvious variation for the fluorescent signals, indicating that foreign coexisting metal ions showed scarce effect on assaying TR activity. To identify the selectivity of the current method, experiments were manipulated separately in the presence of a series of other enzymes including urease, subtilisin, ExoIII, and glucose oxidase and a typically biological protein of BSA. As shown in Fig. 6B, about a 70% decrease of the fluorescence intensity was observed upon addition of TR. In striking contrast, no obvious decrease was found by introducing other enzymes or the biological protein into AuNCs@Tyr, revealing that the satisfactory selectivity of AuNCs@Tyr for TR detection. 3.6. Detection of tyrosinase in human serum samples To evaluate its applicability, the described method was applied to assay the TR activity in five human serum samples, and the samples were also subjected to the colorimetric method at the same time. The TR activity detected in all samples was derived from standard curves and regression equations. As listed in Table 1, the results obtained by these two methods were in satisfactory agreement. Therefore, this method proposed here has potential for broadening roads of detecting TR activity in actual samples. 4. Conclusion In summary, we have successfully proposed a novel strategy to synthesize fluorescent AuNCs, while L-tyrosine serving as a reducing agent and a stabilizing ligand. Interestingly, this synthesis procedure was simple, facile and environment-friendly, and there was no any external toxic reagent and complicated instrument employed. In addition, we have utilized such AuNCs@Tyr for costeffective and selective detections of TR activity. Specifically, assaying of TR was based on the fluorescent decrease of AuNCs@Tyr, primarily due to the aggregations of AuNCs@Tyr on active sites of TR during the process of enzyme–substrate reactions. Up to date, investigating TR activity by fluorescent AuNCs@Tyr has not been reported, and there exhibited little interference originated from other biological metal ions for this method. Simultaneously, the current method was applied for testing TR activity in actual samples, confirming its practicability. Thus, the AuNCs@Tyr obtained here has potential for assaying the specific marker of TR in clinical applications. Acknowledgements We gratefully acknowledge financial support by National Natural Science Foundation of China (31100981), Research Fund
Please cite this article in press as: X. Yang, et al., Novel synthesis of gold nanoclusters templated with L-tyrosine for selective analyzing tyrosinase, Anal. Chim. Acta (2014), http://dx.doi.org/10.1016/j.aca.2014.05.050
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for the Doctoral Program of Higher Education of China (20110182120014), Natural Science Foundation Project of CQ CSTC (cstc2013jcyjA10117), Fundamental Research Funds for the Central Universities (XDJK2013B038), and Program for Innovative Research Team in University of Chongqing (2013). References [1] R. Jin, Quantum sized, thiolate-protected gold nanoclusters, Nanoscale 2 (2010) 343–362. [2] Q. Zhang, J. Xie, Y. Yu, J.Y. Lee, Monodispersity control in the synthesis of monometallic and bimetallic quasi-spherical gold and silver nanoparticles, Nanoscale 2 (2010) 1962–1975. [3] I. Díez, R.H. Ras, Fluorescent silver nanoclusters, Nanoscale 3 (2011) 1963–1970. [4] J. Zheng, P.R. Nicovich, R.M. Dickson, Highly fluorescent noble metal quantum dots, Annu. Rev. Phys. Chem. 58 (2007) 409. [5] X. Liu, C. Li, J. Xu, J. Lv, M. Zhu, Y. Guo, S. Cui, H. Liu, S. Wang, Y. Li, Surfactant-free synthesis and functionalization of highly fluorescent gold quantum dots, J. Phys. Chem. C 112 (2008) 10778–10783. [6] J. Zheng, J.T. Petty, R.M. Dickson, High quantum yield blue emission from water-soluble Au8 nanodots, J. Am. Chem. Soc. 125 (2003) 7780–7781. [7] J. Xie, Y. Zheng, J.Y. Ying, Protein-directed synthesis of highly fluorescent gold nanoclusters, J. Am. Chem. Soc. 131 (2009) 888–889. [8] W. Chen, X. Tu, X. Guo, Fluorescent gold nanoparticles-based fluorescence sensor for Cu2+ ions, Chem. Commun. (2009) 1736–1738. [9] H. Kawasaki, H. Yamamoto, H. Fujimori, R. Arakawa, Y. Iwasaki, M. Inada, Stability of the DMF-protected Au nanoclusters: photochemical, dispersion, and thermal properties, Langmuir 26 (2009) 5926–5933. [10] Y. Bao, H.-C. Yeh, C. Zhong, S.A. Ivanov, J.K. Sharma, M.L. Neidig, D.M. Vu, A.P. Shreve, R.B. Dyer, J.H. Werner, Formation and stabilization of fluorescent gold nanoclusters using small molecules, J. Phys. Chem. C 114 (2010) 15879–15882. [11] J.-a. Annie Ho, H.-C. Chang, W.-T. Su, DOPA-mediated reduction allows the facile synthesis of fluorescent gold nanoclusters for use as sensing probes for ferric ions, Anal. Chem. 84 (2012) 3246–3253. [12] J.I. Gonzalez, T.-H. Lee, M.D. Barnes, Y. Antoku, R.M. Dickson, Quantum mechanical single-gold-nanocluster electroluminescent light source at room temperature, Phys. Rev. Lett. 93 (2004) 147402. [13] C.L. Liu, H.T. Wu, Y.H. Hsiao, C.W. Lai, C.W. Shih, Y.K. Peng, K.C. Tang, H.W. Chang, Y.C. Chien, J.K. Hsiao, Insulin-directed synthesis of fluorescent gold nanoclusters: preservation of insulin bioactivity and versatility in cell imaging, Angew. Chem. Int. Ed. 50 (2011) 7056–7060. [14] H. Wei, Z. Wang, L. Yang, S. Tian, C. Hou, Y. Lu, Lysozyme-stabilized gold fluorescent cluster: synthesis and application as Hg2+ sensor, Analyst 135 (2010) 1406–1410. [15] A. Thomas, Blue emitting gold nanoclusters templated by poly-cytosine DNA at low pH and poly-adenine DNA at neutral pH, Chem. Commun. 48 (2012) 6845–6847. [16] G. Liu, Y. Shao, K. Ma, Q. Cui, F. Wu, S. Xu, Synthesis of DNA-templated fluorescent gold nanoclusters, Gold Bull. 45 (2012) 69–74.
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