Citation

Zhiqun Bai, Xiangling Ren, Zhen Gong, Chenxi Hao, Yongmei Chen, Pingyu Wan, Xianwei Meng. 11-Mercaptoundecanoic acid functionalized gold nanoclusters as fluorescent probes for the sensitive detection of Cu²⁺ and Fe³⁺ ions[J]. Chin. Chem. Lett., 2017, 28(9): 1901-1904  

Zhiqun Bai^a,b, Xiangling Ren^b, Zhen Gong^a,b, Chenxi Hao^a,b, Yongmei Chen^a, Pingyu Wan^a, Xianwei Meng^b    

Metal ions are widespread in the biology as the necessary nutrients and trace elements for living bodies. Their deficiency can lead to the growth and metabolic disorders and their excess can be highly damaging and even fatal. For example, Cu²⁺ is involved in life activities as an enzyme cofactor, and also participates in the formation of red blood cells [1]. However excessive Cu²⁺ may cause nephropathy, Ischemic heart disease, anemia, bone diseases, etc. [2-5]. Fe³⁺ is an essential trace element for both plants and animals, which plays an important role in cellular metabolism, enzyme catalysis, as well as a cofactor in many enzymatic reactions. The intracellular iron levels need to be carefully controlled, because excessive Fe³⁺ can cause some disease such as tissue damaging, liver and spleen dysfunction, skin pigmentation, etc. [6-8]. Hence, the detection of metal ions in the blood has a very necessary practical significance.

The traditional metal ions detection methods are atomic absorption spectrometry (AAS), inductively coupled plasmaatomic emission spectrometry (ICP-AES), voltammetry, extraction spectrophotometry (ES), resonance scattering spectrometry and so on [9-11]. ICP-AES has the advantages of high sensitivity, low interference and wide linear range, but the instrument is expensive and high cost [12, 13]. AAS has the advantages of high analytical precision, fast analysis speed and can analyze many kinds of compounds, but it cannot be used for multi-element simultaneous determination [14]. In general, although the above methods have certain advantages in metal ions selectivity and sensitivity, they need expensive and elaborate instruments with complicated detection procedures. They are not suitable for the detections of large quantities of samples and real-time detections. Therefore, it is significant for biological research and medical diagnosis to design a facile method with low cost and high sensitivity, which can realize real-time detections of metal ions.

In recent years, Au NCs with ultra-small size become the research focus in materials science and biology. Au NCs are attractive for not only their small size, good photostability, large Stokes shift, facile preparation and innoxious, but also their ability to monitor the metal ions in real-time by fluorescence imaging technology. Au NCs have a promising application in the fields of analytical detection, biomarker, fluorescence imaging and so on. For example, Huang and his partners designed the competitive homologous fluorescence quenching method, which is applied in the analysis of protein where biologically modificated Au NCs serves as the energy donor and the spherical Au nanoparticles as the receptor [15]. Retnakumari has synthesized gold nanoclusters protected by bovine serum albumin (BSA) binding folic acid for specifical mark of the oral cancer cells and the breast cancer cells [16]. Jin and his partners completed the synthesis of 11-mercaptoundecanoic acid (11-MUA)-Au NCs and the selective detection of Cr³⁺ [17]. But the main studies focused on the metal ions detection in the environment. The detection of metal ions in serum using Au NCs is still rare.

In this paper, we designed 11-MUA-Au NCs with good fluorescence properties to detect metal ions. There were obvious linear relationships between the concentration of metal ions (Cu²⁺ and Fe³⁺) and the fluorescence intensity of Au NCs. After added ethylenediaminetetraacetic acid (EDTA), the quenching effect of Cu²⁺ and Fe³⁺ on Au NCs showed different phenomenon. In this way, a rapid and quantitative method for the determination of Cu²⁺ was established by masking the Fe³⁺ with EDTA. At the same time, the quantitative determination of Fe³⁺ and Cu²⁺ in the serum has been achieved.

11-MUA-Au NCs were obtained via a method described in the literature with minor modification [18]. First, 0.061mmol 11-MUA was dissolved in the NaOH solution (10mL, 15mmol/L), then HAuCl₄·4H₂O solution (5mL, 3.25mmol/L) was added in the solution. Then NaOH solution (40 μL, 0.15mmol/L) and fresh NaBH₄ solution (30 μL, 26.4 mmol/L) were added in the solution for 24h with stirring.

The transmission electron microscopy(TEM) of 11-MUA-AuNCs was showed respectively in Fig. 1A. The obtained 11-MUA-Au NCs were spherical with uniform distribution of particle size and good monodispersity. The statistics found that average size of Au NCs was 1.7nm. The HRTEM image demonstrated that Au NCs have excellent crystallinity [19].

Figure 1

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Fig. 1. (A) Typical TEM image (1), HRTEM image (2) and the size distribution histogram of the 11-MUA-Au NCs (3). (B) Emission spectra (1) and fluorescence excitation (2) of 11-MUA-Au NCs. Insets: the corresponding photos of 11-MUA-Au NCs under the daylight (a) and ultraviolet light (b), respectively. (C) Emission spectra of 11-MUA-Au NCs at different excitation wavelengths from 230 nm to 370nm. (D) Fluorescence changes of 11-MUA-Au NCs in the presence of different metal ions. The concentrations of all metal ions are 2 μmol/L. I0 and I correspond to the fluorescence intensity of Au NCs in the absence and presence of metal ions.

The prepared 11-MUA-Au NCs dispersion was colorless under daylight, which showed a clear red light under the UV lamp (365nm), in Fig. 1B insets. The ultraviolet absorption spectrum and fluorescence emission spectrum of the 11-MUA-Au NCs were showed in Fig. 1B. The curve 1 showed the ultraviolet absorption spectrum of Au NCs, which have wide and strong absorption near 330nm. Near 240nm absorption was generally attributed to 11-MUA molecules on the surface of the Au NCs [10]. Curve 2 represented the fluorescence emission spectrum of 11-MUA-Au NCs, the emission peak was 610nm when the excitation wavelength was 330nm.

The emission spectra of 11-MUA-Au NCs under different excitation wavelength have been studied. As shown in Fig. 1C, from top to bottom the excitation wavelength were 250nm, 230nm, 270nm, 290nm, 310nm, 330nm, 350nm, 370nm respectively. It can be seen the fluorescence intensity was the highest under 250nm excitation wavelength, and second one was under 230nm. From 270 nm to 370nm, the fluorescence intensity was reduced accordingly, in agree with the ultraviolet absorption spectrum. But 250nm wavelength belongs to the far ultraviolet light, we chose frequently used 330nm wavelengths for the following tests.

The effect of metal ions on the fluorescence intensity of asprepared 11-MUA-Au NCs has been investigated as shown in Fig. 1D. After 2 μmol/L metal ions added, most of ions did not show significant change on the fluorescence intensity of Au NCs. However, the fluorescence emission of the Au NCs was greatly quenched in the presence of Cu²⁺ and Fe³⁺. The quenching percentage ((I₀ -I)/I₀, I₀ and I correspond to the fluorescence intensityof Au NCsin the absence and in the presenceof metal ions respectively) of Cu²⁺ and Fe³⁺ were 51.4% and 27% respectively. The results indicated that Cu²⁺ has a stronger quenching effect on the fluorescence of Au NCs.

The degree of the fluorescence quenching of 11-MUA-Au NCs with different concentrations of Fe³⁺ was evaluated. According to Fig. 2A, the fluorescence intensity of Au NCs decreases with the increasing concentration of Fe³⁺. The degree of the fluorescence quenching of Au NCs was 55% in the presence of Fe³⁺ (10 μmol/L), which showed that the as-prepared fluorescent Au NCs probe responds to Fe³⁺ sensitively. There were two linear relationships between the quenching degree and the concentration of Fe³⁺ based on Fig. 2B. The limit of detection (LOD) of Fe³⁺ was 0.5 μmol/L. When the concentration of Fe³⁺ ranged from 0.8 μmol/L to 4.5 μmol/L, the linear regression equation was I/I₀=1.0281-0.0730C (R²=0.992). While the concentration of Fe³⁺ ranged from 4.5 μmol/L to 11.0 μmol/L, the linear relationship was I/I₀=0.9005-0.0440C (R²=0.997). The table of detection of Fe³⁺ by other nanomaterial was shown in Table S1 in Supporting information.

Figure 2

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Fig. 2. (A) Fluorescence emission spectra of the 11-MUA-Au NCs with the addition of different Fe3+ concentrations increasing from 0.1 μmol/L to 11 μmol/L (top to bottom, excitation at 330 nm). (B) Fluorescence intensity ratios (I/I0, at 610 nm) for 11-MUA-Au NCs versus the Fe3+ concentrations. (C) Fluorescence emission spectra of the 11-MUAAu NCs with the addition of different Cu2+ concentrations increasing from 0.01 μmol/L to 7.5 μmol/L (top to bottom, excitation at 330 nm). (D) Fluorescence intensity ratios (I/I0, at 610 nm) for 11-MUA-Au NCs versus the Cu2+ concentrations.

In addition, we also evaluated the degree of the fluorescence quenching of 11-MUA-Au NCs under different concentrations of Cu²⁺. Fig. 2C suggested the fluorescence intensity of Au NCs decreased along with the concentration of Cu²⁺ increasing. When the presence of Cu²⁺ was 7.5 μmol/L, the degree of the fluorescence quenching of Au NCs was up to 55%, indicating the as-prepared fluorescent Au NCs probe responded to Cu²⁺ sensitively. It can be seen from Fig. 2D that the fluorescence intensity went down as the concentration of Cu²⁺ increased. Meanwhile, in a certain range of concentration, there was a linear relationship between the quenching degree and the concentration of Cu²⁺. The detection limit of Cu²⁺ was 0.05 μmol/L. When the concentration of Cu²⁺ ranged from 0.1 μmol/L to 1.0 μmol/L, the linear regression equation was I/I₀=0.9175-0.3407C (R²=0.993). The table of detection of Cu²⁺ by other nanomaterial was shown in Table S2 in Supporting information.

Based on previous reports, the quenching mechanism should be referred to the coordination of metal ions with the carboxyl group of 11-MUA and then constructed the charge transfer from Au NCs to metal ions [18, 19]. After added EDTA to the system, the detection of Cu²⁺ or Fe³⁺ obtained different consequences as showed in Fig. 3A and B. The fluorescence quenching of Fe³⁺ or Cu²⁺ towards 11-MUA-Au NCs both became weakened when the EDTA added prior to the addition of metal ions. This may be because Fe³⁺ and Cu²⁺ preferentially selects to complex with EDTA so that the fluorescence quenching of 11-MUA-Au NCs decreased. Therefore, It can be speculated that complexation between Fe³⁺ or Cu²⁺ and EDTA is stronger than that of Fe³⁺ or Cu²⁺ and 11-MUA-Au NCs. However, the fluorescence of 11-MUA-Au NCs with Fe³⁺ was obvious recovery, while the Cu²⁺ quenching fluorescence was still remarkable when the EDTA added after the addition of metal ions. This means that the binding of Cu²⁺ and carboxylic groups of 11-MUA directly lead to the destruction of ligands' protection of Au NCs unlike the reversible fluorescence quenching behavior caused by Fe³⁺. The EDTA could not eliminate the quenching effect of Cu²⁺ on the fluorescence of 11-MUA-Au NCs. So EDTA was capable of being masking agent to Fe³⁺.

Figure 3

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Fig. 3. Effects of Cu2+, Fe3+ and EDTA on the fluorescence intensity of 11-MUA-Au NCs (A, B). (1), (2), (3) and (4) correspond to the 11-MUA-Au NCs, Cu2+, Fe3+ and EDTA, respectively. (C) Fluorescence emission spectra of the 11-MUA-Au NCs with the addition of different Cu2+ concentrations increasing from 1.5 μmol/L to 7.0 μmol/L (top to bottom, excitation at 330 nm) in the presence of Fe3+ and EDTA. (D) Fluorescence intensity ratios (I/I0, at 610 nm) for 11-MUA-Au NCs versus the Cu2+ concentrations.

We used 11-MUA-Au NCs to detect Cu²⁺ in the presence of Fe³⁺ and EDTA. As shown in Fig. 3C, when the concentration of Fe³⁺ and EDTA were fixed, the fluorescence intensity of Au NCs showed a decrease with the increasing concentration of Cu²⁺. As shown in Fig. 3D, there was a linear relationship between Cu²⁺ and the quenching fluorescence of Au NCs. When the concentration of Cu²⁺ ranged from 1.5 μmol/L to 3.5 μmol/L, the linear regression equation was I/I₀ = 1.4180-0.2900C (R² = 0.995).

For application in real samples, this method to detect the concentrations of Fe³⁺ and Cu²⁺ was used in serum solution. As shown in Fig. 4A, the fluorescence intensity of the 11-MUA-Au NCs decreased as the increasing concentrations of Fe³⁺ and Cu²⁺ in serum solution. According to Fig. 4B, two good linear correlations were obtained between concentration and the extent of quenching fluorescence. The equation of linear regression was I/I₀ = 0.9999-0.1813C (R² = 0.993) as the Fe³⁺ concentration ranging from 0.1 μmol/L to 1.0 μmol/L. For Fe³⁺ ranging from 1.0 μmol/L to 9.0 μmol/L, the equation of linear regression was I/I₀ = 0.8434-0.0262C (R² = 0.991). As shown in Fig. 4C, Cu²⁺ has a good linear relationship with the extent of quenching fluorescence over a specific concentration range. When the concentration of Cu²⁺ ranges from 0.1 μmol/L to 1.0 μmol/L, a linear regression equation can be obtained (I/I₀ = 1.0076-0.1249C (R² = 0.996)) (Fig. 4D). The known content of Cu²⁺ in human body from an adult male is 11.0-22.0 μmol/L (70-140 μg/dL), so the limit of detection is far below normal content in blood [20].

Figure 4

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Fig. 4. (A) Fluorescence emission spectra of the 11-MUA-Au NCs with the addition of different Fe3+ concentrations increasing from 0 to 9 μmol/L in serum solution (top to bottom, excitation at 330 nm). (B) Fluorescence intensity ratios (I/I0, at 610 nm) for 11-MUA-Au NCs versus the Fe3+ concentrations in serum solution. (A) Fluorescence emission spectra of the 11-MUA-Au NCs with the addition of different Cu2+ concentrations increasing from 0.1 μmol/L to 5.0 μmol/L in serum solution (top to bottom, excitation at 330 nm). (B) Fluorescence intensity ratios (I/I0, at 610 nm) for 11-MUA-Au NCs versus the Cu2+ concentrations in serum solution.

In conclusion, we obtained Au NCs with good fluorescent performance by one step in aqueous solution using 11-MUA as the protecting agent. 11-MUA-Au NCs show the ability to detect Cu²⁺ and Fe³⁺ based on the quenching effects of the metal ions towards its fluorescence. The linear relationships between the fluorescence intensity of 11-MUA-Au NCs and the concentration of Cu²⁺ or Fe³⁺ were obtained. The different effects of Cu²⁺ and Fe³⁺ on the fluorescence of 11-MUA-Au NCs were studied by adding EDTA. We build a fast, convenient, sensitive and selective method for quantitative detection of Cu²⁺. In addition, the Cu²⁺ and Fe³⁺ detection based on the 11-MUA-Au NCs was achieved satisfactorily in serum samples. Our studies indicated that 11-MUA-Au NCs can be a kind of facile and effective fluorescent probe for metal ions detection.

We acknowledge the financial support from the National Natural Science Foundation of China (Nos. 61571426, 61671435), the National Key Technology R & D Program (No. 2015BAI23H00), Beijing Natural Science Foundation (No. 4161003) and Beijing Key Laboratory of Environmentally Harmful Chemical Analysis.

Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.cclet.2017.05.005.