Biosensors and Bioelectronics 59 (2014) 216–220
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Biosensors and Bioelectronics journal homepage: www.elsevier.com/locate/bios
Short communication
Dithiothreitol-capped fluorescent gold nanoclusters: An efficient probe for detection of copper(II) ions in aqueous solution Han Ding a, Chunsu Liang b, Kangbo Sun a, Hui Wang c, J. Kalervo Hiltunen a,d, Zhijun Chen a,d,n, Jiacong Shen a a
State Key Laboratory of Supramolecular Structure and Materials, Jilin University, 2699 Qianjin Street, 130012 Changchun, PR China College of Life Sciences, Jilin University, 2699 Qianjin Street, 130012 Changchun, PR China c Jilin Agricultural University, 130118 Changchun, PR China d Biocenter Oulu and Faculty of Biochemistry and Molecular Medicine, University of Oulu, FI-90014 Oulu, Finland b
art ic l e i nf o
a b s t r a c t
Article history: Received 11 December 2013 Received in revised form 21 March 2014 Accepted 21 March 2014 Available online 31 March 2014
We report here a Green method for the synthesis of fluorescent gold nanoclusters using dithiothreitol (DTT) as both a capping agent and reducing agent at 22 1C and pH 8. The physical and chemical properties of the synthesized AuNCs@DTT were studied by TEM and UV–vis absorption, fluorescence, and X-ray photoelectron spectroscopy. AuNCs@DTT recognizes cupric ions with high selectivity and sensitivity, which allows this material to act as a copper(II) sensor in aqueous solution. A linear relationship was observed between the fluorescence intensity of the DTT capped gold nanoclusters and the concentration of copper(II) ions, in the range of 0–60 μM with a detection limit of 80 nM. The copper content in serum was also analyzed by using this copper sensor. It was shown that data obtained using the proposed method was comparable to values obtained by the traditional colorimetric method. This technique represents an alternative method for the determination of serum copper in clinical diagnosis especially for those laboratories which lack expensive analytical facilities. & 2014 Elsevier B.V. All rights reserved.
Keywords: Dithiothreitol-capped gold nanoclusters One-step facile synthesis Copper(II) ion detection Serum sample
1. Introduction Copper is an essential trace element in living organisms where it functions primarily as either an enzyme cofactor or free form as a catalyst for the generation of radicals (Gaggelli et al., 2006). Disturbance of copper homeostasis is associated with a number of human diseases such as Wilson's (Festa et al., 2011) and Parkinson's diseases (Barnham et al., 2008). Current serum copper detection methods mainly include atomic absorption spectrometry (Chan et al., 2000), plasma atomic emission spectrometry (Fodor et al., 1995), mass spectrometry (Wu et al., 1997), voltammetry (Sanchez et al., 1996), surface plasmon resonance (Homola et al., 1999), and colorimetric methods based on coordination chemistry (Yee et al., 1974; Makino et al., 1991; Williams et al., 1977; Zak et al., 1958; Zheng et al., 2002). Often, these traditional methods are laborious, time-consuming, and require expensive instrumentation and considerable expertise. Recently several excellent nano-based approaches were initiated for the detection of copper ions in aqueous solution by using various gold nanoclusters n Corresponding author at: State Key Laboratory of Supramolecular Structure and Materials, Jilin University, 2699 Qianjin Street, 130012 Changchun, PR China. Tel.: +86 186 8663 6807; fax: +86 431 8519 3421. E-mail address: [email protected] (Z. Chen).
http://dx.doi.org/10.1016/j.bios.2014.03.045 0956-5663/& 2014 Elsevier B.V. All rights reserved.
(see below) (Cao et al., 2013; Durgadas et al., 2011; Guo et al., 2012; Lin et al., 2012; Liu et al., 2011; Su et al., 2013). However, these nanoclusters cannot distinguish copper from some other metal ions such as mercury ions. Therefore, we have prepared a nanomaterial that has high selectivity for cupric ions and can be applied to environmental samples. Recently fluorescent gold nanoclusters (AuNCs) have stimulated extensive interest due to their remarkable water-soluble, non-toxic and optical properties. AuNCs can act as sensors for the detection of mercury (Xie et al., 2010), iron (Mu et al., 2013), CN (Liu et al., 2010), and H2O2 (Wen et al., 2011). AuNCs are photostable and show a wide detection range (Huang et al., 2009; Shang et al., 2009; Shiang et al., 2009); thus they are also used as a labeling tag (Lin et al., 2009; Sperling et al., 2008), and an imaging tool (Huang et al., 2011; Retnakumari et al., 2010; Yu et al., 2007). The classical methods for the synthesis of AuNCs use polyamidoamine or cetyltrimethylammonium bromide as a stabilizer and NaBH4 as the reducing agent (Wu et al., 2012; Zheng et al., 2004). Recently many laboratories have developed alternative approaches to the synthesis of AuNCs by using biomolecules such as DNA (Lin et al., 2009; Liu et al., 2012), proteins (Kawasaki et al., 2011; Wei et al., 2010; Xavier et al., 2010; Xie et al., 2009) and dihydrolipoic acid (Shang et al., 2011) as a stabilizer and/or reducing agents. The reactions are commonly carried out at
H. Ding et al. / Biosensors and Bioelectronics 59 (2014) 216–220
37 1C under the strong alkaline conditions or using a microwave (Shang et al., 2012; Yue et al., 2012) or high temperature (Chen et al., 2013). Cost-effective, non-toxic and Green methods for the preparation of AuNCs are urgently needed. Here we report a simple environment-friendly synthesis of fluorescent AuNCs by using a small molecule dithiothreitol (DTT), as a stabilizer and a reducing agent at 22 1C and pH 8 (Fig. 1a). Furthermore, we show that these AuNCs@DTT selectively detect copper(II) in aqueous solutions and in serum.
2. Experimental
217
Bedford, MA, USA). The copper colorimetric assay kit was purchased from Chuangye biological Ltd.. (Shangyu, China). 2.2. Instrumentation Fluorescence and UV–visible spectra were obtained using a Fluorescence Max-P fluorometer (HORIBA JobinYvon Inc., USA) and a Cary 5000 spectrophotometer (Varian, USA), respectively. X-ray photoelectron spectroscopy (XPS) data were collected on a Kratos AXIS X-ray photoelectrons spectrometer. Transmission electron microscope (TEM) images were collected on a JEM-2100F Transmission Electron Microscope (Japan).
2.1. Chemicals and materials 2.3. Synthesis of AuNCs@DTT Chloroauric acid and dithiothreitol (DTT) were purchased from Sinopharm Chemical Reagent and Beijing DingGuoChangsheng Biotechnology Co., Ltd., respectively. Sodium hydroxide and all other chemicals were purchased from Beijing Chemical Reagent Company. The double-distilled water (ddH2O) used throughout the experiments was produced by a Milli-Q system (Millipore,
All glasswares were cleaned in aqua regia bath (HCl:HNO3 ¼ 3:1) and rinsed thoroughly with ddH2O before use. For the synthesis, 4.4 mg of DTT was dissolved in 4 ml ddH2O. Then 320 μL of 1.25% chloroauric acid was added to the solution. It was stirred for 5 min until the color of the mixture changed
Fig. 1. Synthesis and spectroscopic characterization of AuNCs@DTT. (a) Scheme shows the synthesis of fluorescent AuNCs@DTT and quenching of the fluorescence by Cu2 þ . (b) The excitation (black) and emission (red) spectra of AuNCs@DTT. (c) Time course of the fluorescence signal during the synthesis with excitation and emission at 330 nm and 604 nm, respectively. (d) AuNCs@DTT under visible (left) or UV light (right). (e) TEM micrograph of the gold clusters (bar: 50 nm); inset: the magnified image (bar: 2 nm) and the size distribution of the nanoclusters. (f) XPS spectra show that Au(0) and Au(I) were formed at 84 eV and 87 eV, respectively. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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H. Ding et al. / Biosensors and Bioelectronics 59 (2014) 216–220
from yellow to pale yellow. The pH of the solution was then adjusted to 8.0 using sodium hydroxide, and the solution became colorless. The reaction was carried out overnight with continuous stirring at 22 1C. The reaction mixture was dialyzed against ddH2O or suitable buffers such as 20 mM HEPES buffer (pH ¼7.4). The synthesized AuNCs@DTT was lyophilized and stored at 4 1C for later use. The pH (Figs. S2 and S3), ratio of substrates (Figs. S4 and S5), and buffer systems (Figs. S6 and S7) that might affect the synthetic process were evaluated (see below). Some organic molecules having structural or functional similarity to DTT, namely isopropyl-β-D-1-thiogalactopyranoside (IPTG), glutathione (GSH), thioctic acid (DHLA), citric acid (CA), and cysteine (Cys), were also tested for the synthesis of gold nanoclusters (Figs. S8 and S9). The calculation of the concentration of AuNCs@DTT was based on an assumption that all the Au3 þ ions during the synthesis of nanoclusters were reduced to form AuNCs.
2.4. Preparations of samples for TEM and XPS characterization AuNCs@DTT at a concentration of 0.01 μM was sonicated for 15 min. 60 μL of the dispersed samples was then dropped onto Cu200 mesh (Dajikeyi, Beijing, China) for TEM analysis. AuNCs@DTT at a concentration of 0.15 mM was dialyzed against ddH2O, and lyophilized before XPS analysis.
2.5. Sensing copper ions in aqueous solution and serum AuNCs@DTT was diluted with 20 mM HEPES buffer (pH ¼7.4) to a final concentration of 0.15 mM. For copper(II) ion detection, aliquots of a stock solution (50 mM) of Cu2 þ were added to AuNCs@DTT solutions, reaching a final concentration of 0.05– 500 μM. The mixture was immediately examined using fluorescence spectroscopy. For the detection of copper in serum, blood samples were centrifuged at 1000g for 10 min at 4 1C. The supernatant was mixed with an equal volume of ice cold 20% trichloroacetic acid and incubated on ice for 1 h. After centrifugation at 14,000g for 10 min at 4 1C, an aliquot of 100 μL from the supernatant was mixed with 900 μL AuNCs@DTT solution (in 20 mM HEPES buffer, pH 7.4) for fluorescence intensity analysis.
2.6. Statistical analysis All data were expressed as mean result7standard deviation (SD). All figures shown were obtained from three independent experiments with similar results. Statistical analyses were performed by using Origin 8.5 software.
3. Results and discussion 3.1. Fluorescence properties of AuNCs@DTT The excitation and emission wavelengths of the DTT-capped gold nanoclusters were 330 and 604 nm, respectively (Fig. 1b). During the synthesis, the fluorescence intensity approached a maximum at 200 min (Fig. 1c) and the samples were collected at this point for further studies. AuNCs@DTT emitted red fluorescence under UV light irradiation (Fig. 1d). This material is stabile over a wide range of pH (Fig. S10) and resistant to bleaching (Fig. S11), which is comparable to reported quantum dots (Guevel et al., 2012). The quantum yield of the nanoclusters in 20 mM HEPES buffer, pH 7.4 was 2.09%, calculated using Rhodamine B as a reference.
3.2. TEM and XPS characterization of AuNCs@DTT The morphology of AuNCs@DTT was characterized by transmission electron microscopy (TEM). It was shown that the nanoclusters were spherical in shape with a mean diameter of 2.25 nm (Fig. 1e). X-ray photoelectron spectroscopy (XPS) was used to characterize the valence and composition of the clusters. The Au 4f XPS spectrum showed a binding energy of AuNCs@DTT at 84.17 (4f 7/2) and 87.97 eV (Au 4f 5/2) (Fig. 1f), which confirms the presence of both Au0 and Au þ (Anthony et al., 1984; Kawasaki et al., 2011; Thomas et al., 1986; Wei et al., 2010; Xavier et al., 2010; Xie et al., 2009). 3.3. Copper(II) ion detection To investigate the ability of AuNCs@DTT to detect metal ions in aqueous solutions, divalent metal ions were incubated with AuNCs@DTT and the fluorescence responses were carefully recorded and analyzed. Interestingly, AuNCs@DTT showed a high selective quenching effect toward copper ions against all the other metal ions tested (Fig. 2a), which suggests its potential as a Cu2 þ sensor. It is worth noting that most of currently available fluorescent gold nanoclusters can be quenched by Hg2 þ (Cao et al., 2013; Durgadas et al., 2011; Guo et al., 2012; Kawasaki et al., 2011; Lin et al., 2012; Liu et al., 2011; Su et al., 2013; Wei et al., 2010; Xavier et al., 2010; Xie et al., 2009); masking agents are required in these cases for the detection of metal ions other than mercury (Cao et al., 2013; Durgadas et al., 2011; Guo et al., 2012; Lin et al., 2012; Liu et al., 2011; Su et al., 2013). The selectivity of AuNCs@DTT for Cu2 þ is similar to that of L-proline-stabilized nanoclusters, which are selective for iron (Mu et al., 2013). The intensity of the fluorescence decreased with increasing concentrations of Cu2 þ and the emission was quenched completely by cupric ion at 200 μM (Fig. 2b). The limit of detection for Cu2 þ was 80 nM. The relationship between the fluorescence intensity and the concentration of cupric ions was linear in the 0–60 μM range (Fig. 2c), which corresponds to the range needed for the detection of serum copper. The sizes of the nanoclusters did not increase when they were incubated with 100 μM Cu2 þ as shown in TEM images (Fig. S13), which suggests that the fluorescence quenching was not caused by the aggregation of AuNCs. As a paramagnetic ion, Cu2 þ might quench the fluorescence of AuNCs@DTT by coordinating with the thiol or hydroxyl groups of DTT through promotion of intersystem crossing (Durgadas et al., 2011; Guo et al., 2012). 3.4. Determination of serum copper ion level Detection of serum copper content is significant for certain clinical diagnoses, as an abnormal level of Cu2 þ is associated with a number of human diseases (Barnham et al., 2008; Festa et al., 2011; Gaggelli et al., 2006). We have shown that the DTT coated fluorescent gold nanoclusters responded to Cu2 þ in a highly sensitive and selective manner. To determine the actual applicability of the putative Cu2 þ sensor, the sera from eight volunteers were tested by using both traditional colorimetric analyses and AuNCs@DTT probes. The sera were pre-treated with 20% trichloroacetic acid before the experiment to precipitate proteins (Kyaw et al., 1976). The copper content in the serum was analyzed based on the change of the fluorescence intensity when samples and probes were mixed. The calculation was based on the relationship between the fluorescence intensity and the concentration of Cu2 þ (Fig. 2c). Importantly, the concentration of cupric ions determined by the fluorescence of AuNCs@DTT was consistent with that obtained by traditional colorimetric methods (Yee et al., 1974; Makino et al., 1991; Williams et al., 1977; Zak et al., 1958; Zheng et al., 2002) (Table 1). Human serum also contains such as
H. Ding et al. / Biosensors and Bioelectronics 59 (2014) 216–220
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Table 1 Copper concentration of serum samples determined by a colorimetric method or by AuNCs@DTT probe (n¼5). The concentration of AuNCs@DTT used in the experiment was 0.15 mM. Sample
1 2 3 4 5 6 7 8
Colorimetric method
AuNCs@DTT probe
Copper (μM)
RSD
Copper (μM)
RSD
7.6 16.7 10.1 10.7 8.4 17.6 17.8 12.2
0.08 0.02 0.09 0.04 0.06 0.03 0.01 0.05
8.2 15.4 9.0 9.5 8.7 17.6 17.8 11.9
0.04 0.03 0.04 0.02 0.05 0.03 0.03 0.02
Table S1). Thus, our data indicate that AuNCs@DTT probes can serve as an alternative method to detect serum copper.
4. Conclusion Fluorescent AuNCs@DTT was synthesized by using a simple, rapid, cost-effective and eco-friendly method. The reaction was carried out at near neutral pH and room temperature. Sophisticated equipment and skills are not required for the synthesis of the nanoclusters. DTT is a commercially available reagent and is widely used as a reducing agent to prevent disulfide bond formation in proteins. To the best of our knowledge, this is the first time that fluorescent gold nanoclusters were synthesized under such simple and mild condition. Moreover, the nanomaterial showed special properties with high selectivity toward copper ions and was not quenched by mercury ions. Further, AuNCs@DTT can be used for the detection of serum copper content. Given the importance of serum copper analysis in clinical diagnosis, we expect that our approach can be a useful alternative especially for those laboratories lacking expensive instrumentation.
Acknowledgment This work was supported by the National Natural Science Foundation of China (NSFC) (No. 21372097), Open Fund of the National Laboratory of Protein and Plant Gene Research at Peking University, and a CIMO grant from Finland.
Appendix A. Supporting information Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.bios.2014.03.045. Fig. 2. AuNCs@DTT recognizes cupric ions with high selectivity and sensitivity. (a) The fluorescence of AuNCs@DTT was quenched by Cu2 þ (50 μM) but not by other metal ions (100 μM). (b) The concentration dependent quenching effect of Cu2 þ toward AuNCs@DTT. (c) Relative fluorescence intensity of AuNCs@DTT in contrast to the logarithm of the Cu2 þ concentration (I0/I: the ratio of the fluorescence intensity at 604 nm in the absence and presence of Cu2 þ ), the linear fitting could be expressed as I0/I ¼1.04þ 0.011[Cu2 þ ]. The concentration of AuNCs@DTT used in these experiments was 0.15 mM. For all of these measurements, 20 mM HEPES buffer, pH 7.4 was used.
glutathione (GSH) and cysteine (Cys) and possibility that these molecules interfere with the detection of Cu2 þ is carefully investigated. The fluorescence quenching of samples that contained a given concentration of glutathione (50 μM) and cysteine 30 μM did not differ significantly from the control samples (Fig. S14 and
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Contents lists available at ScienceDirect
Biosensors and Bioelectronics journal homepage: www.elsevier.com/locate/bios
Short communication
Dithiothreitol-capped fluorescent gold nanoclusters: An efficient probe for detection of copper(II) ions in aqueous solution Han Ding a, Chunsu Liang b, Kangbo Sun a, Hui Wang c, J. Kalervo Hiltunen a,d, Zhijun Chen a,d,n, Jiacong Shen a a
State Key Laboratory of Supramolecular Structure and Materials, Jilin University, 2699 Qianjin Street, 130012 Changchun, PR China College of Life Sciences, Jilin University, 2699 Qianjin Street, 130012 Changchun, PR China c Jilin Agricultural University, 130118 Changchun, PR China d Biocenter Oulu and Faculty of Biochemistry and Molecular Medicine, University of Oulu, FI-90014 Oulu, Finland b
art ic l e i nf o
a b s t r a c t
Article history: Received 11 December 2013 Received in revised form 21 March 2014 Accepted 21 March 2014 Available online 31 March 2014
We report here a Green method for the synthesis of fluorescent gold nanoclusters using dithiothreitol (DTT) as both a capping agent and reducing agent at 22 1C and pH 8. The physical and chemical properties of the synthesized AuNCs@DTT were studied by TEM and UV–vis absorption, fluorescence, and X-ray photoelectron spectroscopy. AuNCs@DTT recognizes cupric ions with high selectivity and sensitivity, which allows this material to act as a copper(II) sensor in aqueous solution. A linear relationship was observed between the fluorescence intensity of the DTT capped gold nanoclusters and the concentration of copper(II) ions, in the range of 0–60 μM with a detection limit of 80 nM. The copper content in serum was also analyzed by using this copper sensor. It was shown that data obtained using the proposed method was comparable to values obtained by the traditional colorimetric method. This technique represents an alternative method for the determination of serum copper in clinical diagnosis especially for those laboratories which lack expensive analytical facilities. & 2014 Elsevier B.V. All rights reserved.
Keywords: Dithiothreitol-capped gold nanoclusters One-step facile synthesis Copper(II) ion detection Serum sample
1. Introduction Copper is an essential trace element in living organisms where it functions primarily as either an enzyme cofactor or free form as a catalyst for the generation of radicals (Gaggelli et al., 2006). Disturbance of copper homeostasis is associated with a number of human diseases such as Wilson's (Festa et al., 2011) and Parkinson's diseases (Barnham et al., 2008). Current serum copper detection methods mainly include atomic absorption spectrometry (Chan et al., 2000), plasma atomic emission spectrometry (Fodor et al., 1995), mass spectrometry (Wu et al., 1997), voltammetry (Sanchez et al., 1996), surface plasmon resonance (Homola et al., 1999), and colorimetric methods based on coordination chemistry (Yee et al., 1974; Makino et al., 1991; Williams et al., 1977; Zak et al., 1958; Zheng et al., 2002). Often, these traditional methods are laborious, time-consuming, and require expensive instrumentation and considerable expertise. Recently several excellent nano-based approaches were initiated for the detection of copper ions in aqueous solution by using various gold nanoclusters n Corresponding author at: State Key Laboratory of Supramolecular Structure and Materials, Jilin University, 2699 Qianjin Street, 130012 Changchun, PR China. Tel.: +86 186 8663 6807; fax: +86 431 8519 3421. E-mail address: [email protected] (Z. Chen).
http://dx.doi.org/10.1016/j.bios.2014.03.045 0956-5663/& 2014 Elsevier B.V. All rights reserved.
(see below) (Cao et al., 2013; Durgadas et al., 2011; Guo et al., 2012; Lin et al., 2012; Liu et al., 2011; Su et al., 2013). However, these nanoclusters cannot distinguish copper from some other metal ions such as mercury ions. Therefore, we have prepared a nanomaterial that has high selectivity for cupric ions and can be applied to environmental samples. Recently fluorescent gold nanoclusters (AuNCs) have stimulated extensive interest due to their remarkable water-soluble, non-toxic and optical properties. AuNCs can act as sensors for the detection of mercury (Xie et al., 2010), iron (Mu et al., 2013), CN (Liu et al., 2010), and H2O2 (Wen et al., 2011). AuNCs are photostable and show a wide detection range (Huang et al., 2009; Shang et al., 2009; Shiang et al., 2009); thus they are also used as a labeling tag (Lin et al., 2009; Sperling et al., 2008), and an imaging tool (Huang et al., 2011; Retnakumari et al., 2010; Yu et al., 2007). The classical methods for the synthesis of AuNCs use polyamidoamine or cetyltrimethylammonium bromide as a stabilizer and NaBH4 as the reducing agent (Wu et al., 2012; Zheng et al., 2004). Recently many laboratories have developed alternative approaches to the synthesis of AuNCs by using biomolecules such as DNA (Lin et al., 2009; Liu et al., 2012), proteins (Kawasaki et al., 2011; Wei et al., 2010; Xavier et al., 2010; Xie et al., 2009) and dihydrolipoic acid (Shang et al., 2011) as a stabilizer and/or reducing agents. The reactions are commonly carried out at
H. Ding et al. / Biosensors and Bioelectronics 59 (2014) 216–220
37 1C under the strong alkaline conditions or using a microwave (Shang et al., 2012; Yue et al., 2012) or high temperature (Chen et al., 2013). Cost-effective, non-toxic and Green methods for the preparation of AuNCs are urgently needed. Here we report a simple environment-friendly synthesis of fluorescent AuNCs by using a small molecule dithiothreitol (DTT), as a stabilizer and a reducing agent at 22 1C and pH 8 (Fig. 1a). Furthermore, we show that these AuNCs@DTT selectively detect copper(II) in aqueous solutions and in serum.
2. Experimental
217
Bedford, MA, USA). The copper colorimetric assay kit was purchased from Chuangye biological Ltd.. (Shangyu, China). 2.2. Instrumentation Fluorescence and UV–visible spectra were obtained using a Fluorescence Max-P fluorometer (HORIBA JobinYvon Inc., USA) and a Cary 5000 spectrophotometer (Varian, USA), respectively. X-ray photoelectron spectroscopy (XPS) data were collected on a Kratos AXIS X-ray photoelectrons spectrometer. Transmission electron microscope (TEM) images were collected on a JEM-2100F Transmission Electron Microscope (Japan).
2.1. Chemicals and materials 2.3. Synthesis of AuNCs@DTT Chloroauric acid and dithiothreitol (DTT) were purchased from Sinopharm Chemical Reagent and Beijing DingGuoChangsheng Biotechnology Co., Ltd., respectively. Sodium hydroxide and all other chemicals were purchased from Beijing Chemical Reagent Company. The double-distilled water (ddH2O) used throughout the experiments was produced by a Milli-Q system (Millipore,
All glasswares were cleaned in aqua regia bath (HCl:HNO3 ¼ 3:1) and rinsed thoroughly with ddH2O before use. For the synthesis, 4.4 mg of DTT was dissolved in 4 ml ddH2O. Then 320 μL of 1.25% chloroauric acid was added to the solution. It was stirred for 5 min until the color of the mixture changed
Fig. 1. Synthesis and spectroscopic characterization of AuNCs@DTT. (a) Scheme shows the synthesis of fluorescent AuNCs@DTT and quenching of the fluorescence by Cu2 þ . (b) The excitation (black) and emission (red) spectra of AuNCs@DTT. (c) Time course of the fluorescence signal during the synthesis with excitation and emission at 330 nm and 604 nm, respectively. (d) AuNCs@DTT under visible (left) or UV light (right). (e) TEM micrograph of the gold clusters (bar: 50 nm); inset: the magnified image (bar: 2 nm) and the size distribution of the nanoclusters. (f) XPS spectra show that Au(0) and Au(I) were formed at 84 eV and 87 eV, respectively. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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from yellow to pale yellow. The pH of the solution was then adjusted to 8.0 using sodium hydroxide, and the solution became colorless. The reaction was carried out overnight with continuous stirring at 22 1C. The reaction mixture was dialyzed against ddH2O or suitable buffers such as 20 mM HEPES buffer (pH ¼7.4). The synthesized AuNCs@DTT was lyophilized and stored at 4 1C for later use. The pH (Figs. S2 and S3), ratio of substrates (Figs. S4 and S5), and buffer systems (Figs. S6 and S7) that might affect the synthetic process were evaluated (see below). Some organic molecules having structural or functional similarity to DTT, namely isopropyl-β-D-1-thiogalactopyranoside (IPTG), glutathione (GSH), thioctic acid (DHLA), citric acid (CA), and cysteine (Cys), were also tested for the synthesis of gold nanoclusters (Figs. S8 and S9). The calculation of the concentration of AuNCs@DTT was based on an assumption that all the Au3 þ ions during the synthesis of nanoclusters were reduced to form AuNCs.
2.4. Preparations of samples for TEM and XPS characterization AuNCs@DTT at a concentration of 0.01 μM was sonicated for 15 min. 60 μL of the dispersed samples was then dropped onto Cu200 mesh (Dajikeyi, Beijing, China) for TEM analysis. AuNCs@DTT at a concentration of 0.15 mM was dialyzed against ddH2O, and lyophilized before XPS analysis.
2.5. Sensing copper ions in aqueous solution and serum AuNCs@DTT was diluted with 20 mM HEPES buffer (pH ¼7.4) to a final concentration of 0.15 mM. For copper(II) ion detection, aliquots of a stock solution (50 mM) of Cu2 þ were added to AuNCs@DTT solutions, reaching a final concentration of 0.05– 500 μM. The mixture was immediately examined using fluorescence spectroscopy. For the detection of copper in serum, blood samples were centrifuged at 1000g for 10 min at 4 1C. The supernatant was mixed with an equal volume of ice cold 20% trichloroacetic acid and incubated on ice for 1 h. After centrifugation at 14,000g for 10 min at 4 1C, an aliquot of 100 μL from the supernatant was mixed with 900 μL AuNCs@DTT solution (in 20 mM HEPES buffer, pH 7.4) for fluorescence intensity analysis.
2.6. Statistical analysis All data were expressed as mean result7standard deviation (SD). All figures shown were obtained from three independent experiments with similar results. Statistical analyses were performed by using Origin 8.5 software.
3. Results and discussion 3.1. Fluorescence properties of AuNCs@DTT The excitation and emission wavelengths of the DTT-capped gold nanoclusters were 330 and 604 nm, respectively (Fig. 1b). During the synthesis, the fluorescence intensity approached a maximum at 200 min (Fig. 1c) and the samples were collected at this point for further studies. AuNCs@DTT emitted red fluorescence under UV light irradiation (Fig. 1d). This material is stabile over a wide range of pH (Fig. S10) and resistant to bleaching (Fig. S11), which is comparable to reported quantum dots (Guevel et al., 2012). The quantum yield of the nanoclusters in 20 mM HEPES buffer, pH 7.4 was 2.09%, calculated using Rhodamine B as a reference.
3.2. TEM and XPS characterization of AuNCs@DTT The morphology of AuNCs@DTT was characterized by transmission electron microscopy (TEM). It was shown that the nanoclusters were spherical in shape with a mean diameter of 2.25 nm (Fig. 1e). X-ray photoelectron spectroscopy (XPS) was used to characterize the valence and composition of the clusters. The Au 4f XPS spectrum showed a binding energy of AuNCs@DTT at 84.17 (4f 7/2) and 87.97 eV (Au 4f 5/2) (Fig. 1f), which confirms the presence of both Au0 and Au þ (Anthony et al., 1984; Kawasaki et al., 2011; Thomas et al., 1986; Wei et al., 2010; Xavier et al., 2010; Xie et al., 2009). 3.3. Copper(II) ion detection To investigate the ability of AuNCs@DTT to detect metal ions in aqueous solutions, divalent metal ions were incubated with AuNCs@DTT and the fluorescence responses were carefully recorded and analyzed. Interestingly, AuNCs@DTT showed a high selective quenching effect toward copper ions against all the other metal ions tested (Fig. 2a), which suggests its potential as a Cu2 þ sensor. It is worth noting that most of currently available fluorescent gold nanoclusters can be quenched by Hg2 þ (Cao et al., 2013; Durgadas et al., 2011; Guo et al., 2012; Kawasaki et al., 2011; Lin et al., 2012; Liu et al., 2011; Su et al., 2013; Wei et al., 2010; Xavier et al., 2010; Xie et al., 2009); masking agents are required in these cases for the detection of metal ions other than mercury (Cao et al., 2013; Durgadas et al., 2011; Guo et al., 2012; Lin et al., 2012; Liu et al., 2011; Su et al., 2013). The selectivity of AuNCs@DTT for Cu2 þ is similar to that of L-proline-stabilized nanoclusters, which are selective for iron (Mu et al., 2013). The intensity of the fluorescence decreased with increasing concentrations of Cu2 þ and the emission was quenched completely by cupric ion at 200 μM (Fig. 2b). The limit of detection for Cu2 þ was 80 nM. The relationship between the fluorescence intensity and the concentration of cupric ions was linear in the 0–60 μM range (Fig. 2c), which corresponds to the range needed for the detection of serum copper. The sizes of the nanoclusters did not increase when they were incubated with 100 μM Cu2 þ as shown in TEM images (Fig. S13), which suggests that the fluorescence quenching was not caused by the aggregation of AuNCs. As a paramagnetic ion, Cu2 þ might quench the fluorescence of AuNCs@DTT by coordinating with the thiol or hydroxyl groups of DTT through promotion of intersystem crossing (Durgadas et al., 2011; Guo et al., 2012). 3.4. Determination of serum copper ion level Detection of serum copper content is significant for certain clinical diagnoses, as an abnormal level of Cu2 þ is associated with a number of human diseases (Barnham et al., 2008; Festa et al., 2011; Gaggelli et al., 2006). We have shown that the DTT coated fluorescent gold nanoclusters responded to Cu2 þ in a highly sensitive and selective manner. To determine the actual applicability of the putative Cu2 þ sensor, the sera from eight volunteers were tested by using both traditional colorimetric analyses and AuNCs@DTT probes. The sera were pre-treated with 20% trichloroacetic acid before the experiment to precipitate proteins (Kyaw et al., 1976). The copper content in the serum was analyzed based on the change of the fluorescence intensity when samples and probes were mixed. The calculation was based on the relationship between the fluorescence intensity and the concentration of Cu2 þ (Fig. 2c). Importantly, the concentration of cupric ions determined by the fluorescence of AuNCs@DTT was consistent with that obtained by traditional colorimetric methods (Yee et al., 1974; Makino et al., 1991; Williams et al., 1977; Zak et al., 1958; Zheng et al., 2002) (Table 1). Human serum also contains such as
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Table 1 Copper concentration of serum samples determined by a colorimetric method or by AuNCs@DTT probe (n¼5). The concentration of AuNCs@DTT used in the experiment was 0.15 mM. Sample
1 2 3 4 5 6 7 8
Colorimetric method
AuNCs@DTT probe
Copper (μM)
RSD
Copper (μM)
RSD
7.6 16.7 10.1 10.7 8.4 17.6 17.8 12.2
0.08 0.02 0.09 0.04 0.06 0.03 0.01 0.05
8.2 15.4 9.0 9.5 8.7 17.6 17.8 11.9
0.04 0.03 0.04 0.02 0.05 0.03 0.03 0.02
Table S1). Thus, our data indicate that AuNCs@DTT probes can serve as an alternative method to detect serum copper.
4. Conclusion Fluorescent AuNCs@DTT was synthesized by using a simple, rapid, cost-effective and eco-friendly method. The reaction was carried out at near neutral pH and room temperature. Sophisticated equipment and skills are not required for the synthesis of the nanoclusters. DTT is a commercially available reagent and is widely used as a reducing agent to prevent disulfide bond formation in proteins. To the best of our knowledge, this is the first time that fluorescent gold nanoclusters were synthesized under such simple and mild condition. Moreover, the nanomaterial showed special properties with high selectivity toward copper ions and was not quenched by mercury ions. Further, AuNCs@DTT can be used for the detection of serum copper content. Given the importance of serum copper analysis in clinical diagnosis, we expect that our approach can be a useful alternative especially for those laboratories lacking expensive instrumentation.
Acknowledgment This work was supported by the National Natural Science Foundation of China (NSFC) (No. 21372097), Open Fund of the National Laboratory of Protein and Plant Gene Research at Peking University, and a CIMO grant from Finland.
Appendix A. Supporting information Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.bios.2014.03.045. Fig. 2. AuNCs@DTT recognizes cupric ions with high selectivity and sensitivity. (a) The fluorescence of AuNCs@DTT was quenched by Cu2 þ (50 μM) but not by other metal ions (100 μM). (b) The concentration dependent quenching effect of Cu2 þ toward AuNCs@DTT. (c) Relative fluorescence intensity of AuNCs@DTT in contrast to the logarithm of the Cu2 þ concentration (I0/I: the ratio of the fluorescence intensity at 604 nm in the absence and presence of Cu2 þ ), the linear fitting could be expressed as I0/I ¼1.04þ 0.011[Cu2 þ ]. The concentration of AuNCs@DTT used in these experiments was 0.15 mM. For all of these measurements, 20 mM HEPES buffer, pH 7.4 was used.
glutathione (GSH) and cysteine (Cys) and possibility that these molecules interfere with the detection of Cu2 þ is carefully investigated. The fluorescence quenching of samples that contained a given concentration of glutathione (50 μM) and cysteine 30 μM did not differ significantly from the control samples (Fig. S14 and
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