A “turn-off” fluorescent sensor for the selective and sensitive detection of copper(II) ions using lysozyme stabilized gold nanoclusters

Abstract

In this contribution, we have developed a simple, environmentally friendly fluorescent turn-off sensor for the detection of copper (Cu²⁺) ions in aqueous solution by using lysozyme stabilized gold nanoclusters (Lys-AuNCs) as a fluorescent probe. Since lysozyme acted as both a reducing and stabilizing agent, the use of other external reducing or stabilizing agents was averted in the preparation of Lys-AuNCs. High resolution transmission electron microscopy (HR-TEM) results revealed that Lys-AuNCs are mono-dispersed spherical particles with an average particle size of 2.5 ± 0.3 nm. Lys-AuNCs exhibit strong fluorescence and emit red photoluminescence under illumination with ultraviolet light. The emission intensity of Lys-AuNCs decreased linearly upon the addition of increasing concentrations of Cu²⁺ ions in a wide range of 0.40 × 10⁻⁷ mol dm⁻³ to 4.00 × 10⁻⁷ mol dm⁻³ and the corresponding linear plot had a correlation coefficient of 0.9976. The limit of detection for this method was calculated as 9.00 × 10⁻⁹ mol dm⁻³ (S/N = 3). Lys-AuNCs exhibit good selectivity towards the selective determination of Cu²⁺ ions even in the presence of 100-fold higher concentration of common interfering cations. To further evaluate the analytical performance of this sensing system, the concentrations of Cu²⁺ ions were determined in real water samples using tap water, drinking water and sea water.

---

1 Introduction

In recent years, noble metal nanoclusters (NCs) have gained great attention because of their unique physical, chemical, electrical and optical properties. These NCs have potential applications in catalysis, chemical sensors, electronic devices and biological imaging.^1–4 Owing to their excellent photostability, photoluminescence and large Stokes shifts, these NCs possess very low toxicity compared to quantum dots.^5,6 Several NCs of noble metals such as gold and silver have been synthesized by using various templates such as peptides, carboxylic acids, dendrimers, proteins, polymers and DNA.^7,8 Among them, the synthesis of NCs using protein templates has more advantages in biological applications.⁹ The protecting ligands or templates are considered not only essential for the stability of the colloid, but are also related to the origin of fluorescence from most of the resulting NCs.¹⁰ As the size of NCs approach the Fermi wavelength of an electron, they exhibit strong luminescence due to quantum confinement.^11,12 Gold nanoclusters (AuNCs) have emerged as a class of promising optical probes for the construction of excellent chemical sensors due to their ultra small size, strong luminescence, good photostability and low toxicity.¹³ In recent past, lysozyme (Lys) has also been applied as template for the preparation of fluorescent silver nanoclusters (AgNCs).¹⁴ Lys is a small monomeric low molecular weight (∼14 kDa) globular protein consisting of 129 amino acid residues with free carboxylic groups, amino groups and four disulfide bonds.¹⁴ Lys is highly stable and its bioactivity is well retained even when treated at 90 °C for 15 minutes, which is highly desirable for fluorescent AuNCs with specific bio-recognition ability.¹⁵ Owing to their high photoluminescence and stability, Lys-AuNCs have been applied in the detection of various analytes, including cations, anions and bacteria.^14–16

Copper (Cu²⁺) ions play an important role in a number of physiological process occurring in living organisms and are also an important environmental pollutant.^17–19 Cu²⁺ ions are extensively utilized in many industrial processes such as chemical, electronic, environment and biological field, resulting in extensive contamination of soil, water and food. However, at elevated concentrations, Cu²⁺ ions are highly toxic to organisms such as certain algae,²⁰ fungi,²¹ bacteria and viruses.^22,23 Aberrant levels of Cu²⁺ ions can result in oxidative stress and has been linked to the development of Indian childhood cirrhosis, prion disease, Menkes disease, Parkinson's disease and Wilson disease.²⁴ However, an excessive uptake of Cu²⁺ can cause serious health problems including ischemic heart disease, kidney disease, neurodegenerative disease, anaemia and bone disorders.²⁵ The tolerable amount of Cu²⁺ in drinking water approved by the United States Environment Protection Agency (USEPA) is 1.30 ppm (20.00 × 10⁻⁶ mol dm⁻³).²⁶ Therefore, it is significant to develop highly sensitive and selective analytical methods for detection of Cu²⁺ ions in real samples. In recent years, many classical techniques have been developed for detection of cations, anions, biomolecules and organic molecules.^27–32 Several methods have been developed for detection of Cu²⁺ ions including atomic adsorption spectrometry (AAS), electron paramagnetic resonance (EPR), inductively coupled plasma mass spectrometry (ICP-MS) and synchrotron radiation X-ray spectrometry (SRXRS).^33–36 However, most of these methods are relatively time-consuming, expensive and require complex sample treatment and sophisticated instruments. On the other hand, spectrofluorometric determination has received much attention because of its high selectivity and sensitivity, reproducibility, less time consumption and ease of handling. However, it still remains imperative that a simple, cost-effective, ultrasensitive and selective method is developed for the determination of Cu²⁺ ions in biological, toxicological and environmental samples to overcome most of these difficulties.

Herein, we report water soluble Lys-AuNCs as the fluorophore for selective determination of Cu²⁺ ions in water samples (Scheme 1). The morphologies and diameter distribution of Lys-AuNCs were characterized by HR-TEM measurements. The emission intensity of Lys-AuNCs diminished gradually upon the addition of Cu²⁺ ions. Based on the quenching of Lys-AuNCs, the concentration of Cu²⁺ ions were determined. The limit of detection was found as 9.00 × 10⁻⁹ mol dm⁻³. Lys-AuNCs displayed excellent selectivity and sensitivity in sensing of Cu²⁺ ions even in the presence of common interferences. The proposed method was successfully utilized to determine the concentrations of Cu²⁺ ions in tap water, sea water and drinking water samples. Moreover, the proposed method for sensing of Cu²⁺ ions in water samples were simple, rapid, cost-effective, selective, sensitive and easily performed.

Scheme 1 A schematic illustration about formation of Lys-AuNCs and detection for Cu²⁺ ions.

2 Materials and methods

2.1 Materials

Tetrachloroauric acid (HAuCl₄) and chicken egg white lysozyme (Lys) were acquired from Sigma-Aldrich, USA and used without further purification. All other reagents were of analytical grade and used as received. Water used in this investigation was doubly distilled over alkaline potassium permanganate using an all glass apparatus. All the measurements were carried out at room temperature (25 °C).

2.2 Instrumentation

The emission spectral studies were recorded with JASCO FP-6600 spectrofluorometer equipped with a 1.0 cm quartz cuvette. The excitation source was a 150 W xenon lamp and Lys-AuNCs was excited at 390 nm. The emission wavelength was monitored between 600 and 800 nm with a scanning speed of 200 nm min⁻¹. The emission and excitation slit widths used throughout the experiments were 5 and 10 nm, respectively. Absorption spectral measurements were performed on a JASCO V-630 UV-visible spectrophotometer. Quartz cuvettes of path length 1.0 cm were used to record the absorption spectra. The morphology of Lys-AuNCs was analyzed with High Resolution Transmission Electron Microscopy (HR-TEM) using JEOL JEM 2100 microscope instrument at an operating voltage of 200 kV. For HR-TEM measurements, the samples were prepared by dropping the Lys-AuNCs solution onto a carbon-coated copper grid. After drying, the sample was examined by HR-TEM. The particle size distribution histogram of Lys-AuNCs was assessed by counting 80 particles directly from HR-TEM images. The lifetime measurements were performed using time-correlated single photon counting (TCSPC) technique in a Horiba Jobin Yvon equipped with pulsed-diode excitation source of 390 nm. All the decays were collected at a magic-angle polarization using a Hamamatsu micro channel plate photomultiplier (2809U) as a detector. The fluorescence decay data was analyzed using IBH DAS6 software provided with the instrument. The quality of the fits was judged by analysing the chi-square (χ²) values and the distribution of the residues.

2.3 Synthesis of fluorescent Lys-AuNCs

All glassware were cleaned in freshly prepared aqua regia solution (HCl : HNO₃ = 3 : 1) and then rinsed by double distilled water. Lys-AuNCs was prepared according to the reported literature.¹⁶ Briefly, aqueous HAuCl₄ solution (4.00 × 10⁻³ mol dm⁻³, 2 mL) was added to Lys solution (16 mg mL⁻¹, 2 mL) under vigorous stirring. Five minutes later, about 0.2 mL of NaOH (1.00 mol dm⁻³) was added to adjust the acidity of solution at pH 12. After that, the reaction was allowed to proceed under vigorous stirring for 12 h at room temperature. The colour of the solution changed from light yellow to light brown indicating the formation of Lys-AuNCs. The reduction of Au³⁺ in HAuCl₄ to Au⁰ was achieved by tyrosine residues present in Lys at higher pH. The synthesized Lys-AuNCs were stored at 4 °C for further use.

2.4 Detection of Cu²⁺ using Lys-AuNCs as probe

A stock solution of Cu²⁺ ions (0.10 mol dm⁻³) was prepared in water and stored at 4 °C in the refrigerator. For detection of Cu²⁺ ions, different concentrations of Cu²⁺ ions were freshly prepared by pipetting an aliquot of the stock solution into 5 mL standard measuring flask containing 0.5 mL of Lys-AuNCs and finally made upto 5 mL with double distilled water. Lys-AuNCs and Cu²⁺ ions are mixed uniformly and allowed to equilibrate for 5 minutes and then transferred into a quartz cuvette for recording emission spectral data. The relative emission intensity can be described as ΔF = F₀ − F, where F₀ and F denote the emission intensity of Lys-AuNCs in the absence and presence of Cu²⁺ ions, respectively. To evaluate the selectivity of Lys-AuNCs towards Cu²⁺ ions, the fluorescence response to other common metal ions (Al³⁺, Mg²⁺, Mn²⁺, Ni²⁺, Cd²⁺, Fe²⁺, Fe³⁺, Ca²⁺, Pb²⁺, Zn²⁺, Na⁺ and K⁺) were also tested. All measurements were performed in triplicate at ambient conditions. Limit of detection (LOD) values were calculated from LOD = 3S/m, where S is the standard deviation of blank measurements (n = 10) and m is the slope of the calibration curve.³⁷

2.5 Real sample analysis

Drinking water and tap water were obtained from the Department of Chemistry, Bharathiar University, Coimbatore and sea water samples collected from the ocean near Puducherry beach, India were filtered three times through qualitative filter paper. Different concentrations of Cu²⁺ ions were spiked in the real water samples and analyzed using Lys-AuNCs as probe. The percentage recoveries were computed using the following equation (eqn (1)).³⁷

3 Results and discussion

3.1 Characterization of Lys-AuNCs

The formation of Lys-AuNCs was characterized by absorption, emission and HR-TEM measurements. The absorption, excitation and emission spectral behaviour of Lys-AuNCs are shown in Fig. 1. The absorption spectrum of Lys-AuNCs is illustrated in Fig. 1(A). It can be seen from Fig. 1(A) that the absorption spectrum of Lys-AuNCs did not exhibit obvious surface plasmon resonance (SPR) band at 520 nm indicating the formation of nanoclusters rather than larger sized nanoparticles. It has been already reported that the absence of SPR band in the AuNCs is due to the formation of nanoclusters inside the protein matrix.³⁸ The excitation and emission spectra recorded for Lys-AuNCs and are shown in Fig. 1(B). When the emission wavelength was fixed at 660 nm, Lys-AuNCs showed an excitation band at 390 nm (Fig. 1(B)(a)). The emission spectrum of Lys-AuNCs exhibits an emission maximum at 660 nm, when excited at 390 nm (Fig. 1(B)(b)). The inset of Fig. 1(B) shows that the Lys-AuNCs solution was light brown in color under visible light (Fig. 1(B)(i)), meanwhile, it emit bright-red luminescence under UV light at 365 nm (Fig. 1(B)(ii)). The morphological properties of Lys-AuNCs were also examined by HR-TEM. The HR-TEM image of Lys-AuNCs is displayed in Fig. 2. It is evident from the HR-TEM image that Lys-AuNCs are spherical in shape (Fig. 2(A)) with an average diameter of 2.5 ± 0.3 nm (Fig. 2(B)) with narrow size distribution and monodispersity.

Fig. 1 (A) UV-visible absorption spectrum of Lys-AuNCs. (B) The excitation (a) and emission (b) spectrum of Lys-AuNCs (inset: photographs of Lys-AuNCs under visible light (i) and ultraviolet light (ii)).

Fig. 2 HR-TEM image (A) and particle size distribution of Lys-AuNCs (B).

3.2 Detection of Cu²⁺ ions by Lys-AuNCs

The effect of increasing concentration of Cu²⁺ ions on the emission intensity of Lys-AuNCs is illustrated in Fig. 3. The emission spectrum of Lys-AuNCs in the absence of Cu²⁺ ions show an emission maximum at 660 nm, when excited at 390 nm (Fig. 3(a)). On successive addition of increasing concentrations of Cu²⁺ ions to the Lys-AuNCs, the emission intensity of Lys-AuNCs showed a progressive decrease and reached about 73% reduction in the intensity upon the addition of 12.00 × 10⁻⁷ mol dm⁻³ of Cu²⁺ ions. Recently, a similar kind of behaviour has been reported in the case of bovine serum albumin conjugated zinc oxide nanoparticles with Cu²⁺ ions.³⁹ It is believed that the excited electrons of Lys-AuNCs loses its energy as intersystem crossing caused by the paramagnetic behaviour of the Cu²⁺ bound to Lys-AuNCs resulting in the emission quenching of Lys-AuNCs.⁴⁰ Hence, decrease in emission intensity is attributed to the interaction between Cu²⁺ ions and Lys-AuNCs.

Fig. 3 Emission spectra of Lys-AuNCs at various concentrations of Cu²⁺. [Cu²⁺]: [a] 0.00, [b] 0.40 × 10⁻⁷, [c] 0.80 × 10⁻⁷, [d] 1.20 × 10⁻⁷, [e] 1.60 × 10⁻⁷, [f] 2.00 × 10⁻⁷, [g] 2.40 × 10⁻⁷, [h] 2.80 × 10⁻⁷, [i] 3.20 × 10⁻⁷, [j] 3.60 × 10⁻⁷, [k] 4.00 × 10⁻⁷, [l] 4.80 × 10⁻⁷, [m] 5.60 × 10⁻⁷, [n] 6.40 × 10⁻⁷, [o] 7.20 × 10⁻⁷, [p] 8.00 × 10⁻⁷, [q] 8.80 × 10⁻⁷, [r] 9.60 × 10⁻⁷, [s] 10.40 × 10⁻⁷, [t] 11.20 × 10⁻⁷ and [u] 12.00 × 10⁻⁷ mol dm⁻³.

3.3 Emission quenching of Lys-AuNCs by Cu²⁺ ions

The emission quenching of Lys-AuNCs by Cu²⁺ ions may occur either static (ground state complex formation) or dynamic (collisional) in nature. In order to ascertain the possible quenching mechanism of Lys-AuNCs in the presence of Cu²⁺ ions, the respective emission data were analyzed using Stern–Volmer equations (eqn (2) and (3)).⁴¹where, F₀ and F are the emission intensity of Lys-AuNCs in the absence and presence of quencher, respectively. K_SV is the Stern–Volmer quenching constant, [Q] is the concentration of quencher and τ₀ is the average lifetime of the Lys-AuNCs in the absence of Cu²⁺ ions (29.26 ns). The Stern–Volmer plot of Lys-AuNCs in the presence of various concentrations of Cu²⁺ ions are shown in Fig. 4. As can be seen from Fig. 4, the plot of F₀/F vs. [Cu²⁺] exhibited a good linear relationship within the investigated concentrations of Cu²⁺ ions. The linear Stern–Volmer plot is indicative of single class of quenching either static or dynamic in nature. The K_SV value obtained from the slope of the linear plot is 1.49 × 10⁶ dm³ mol⁻¹. The K_q value calculated as 5.09 × 10¹³ dm³ mol⁻¹ s⁻¹, is 1000-fold higher than the maximum value possible for diffusion controlled quenching of various kinds of quencher to fluorescence molecules (2.00 × 10¹⁰ dm³ mol⁻¹ s⁻¹),⁴¹ indicating that the probable quenching mechanism of Lys-AuNCs in the presence of Cu²⁺ ions is initiated by ground state complex formation rather than by dynamic collision.⁴¹ Moreover, time resolved fluorescence lifetime measurement was employed to distinguish static and dynamic quenching between Lys-AuNCs and Cu²⁺ ions. The fluorescence lifetime measurement of Lys-AuNCs in the absence and presence of Cu²⁺ ions are illustrated in Fig. S1.‡ The fluorescence decay of Lys-AuNCs in the absence and presence of Cu²⁺ ions are fitted to a bi-exponential function. It can be seen from Fig. S1(a)‡ that the fluorescence lifetime value of Lys-AuNCs is 0.77 ns (8%) and 31.74 ns (92%). Upon the addition of Cu²⁺ ions (4.00 × 10⁻⁷ mol dm⁻³) to Lys-AuNCs, the fluorescence lifetime of Lys-AuNCs is changed to 0.64 ns (9%) and 30.74 ns (91%) (Fig. S1(b)‡). Further, we have used average fluorescence lifetime value to get qualitative analysis. The absence of any significant changes in the average fluorescence lifetime of Lys-AuNCs (29.26 ns to 30.22 ns) in the presence of Cu²⁺ ions clearly indicates the formation of ground state complexation between Lys-AuNCs and Cu²⁺ ions.

Fig. 4 Stern–Volmer plot for Lys-AuNCs in the presence of different concentrations of Cu²⁺ ions. The error bars indicated the relative standard deviation of three repeated experiments.

3.4 Selectivity of the method

High selectivity is decisive in most scenarios, especially in practical applications. In order to estimate the selectivity of the present method, the interference from other environmentally relevant metal ions, including structural analogues and commonly coexistent physiological level species are also investigated. To evaluate the selectivity of the sensor, the effects of commonly interfering cations, such as Al³⁺, Mg²⁺, Mn²⁺, Ni²⁺, Cd²⁺, Fe²⁺, Fe³⁺, Ca²⁺, Pb²⁺, Zn²⁺, Na⁺ and K⁺ were measured. The selective sensing of Cu²⁺ ions by Lys-AuNCs were performed in the presence of 100-fold higher concentrations of other interfering cations. The relative emission intensity of Lys-AuNCs in the presence of Cu²⁺ and other interfering cations are shown in Fig. 5. It can be seen from Fig. 5 that competitive metal ions did not alter the relative emission intensity of Lys-AuNCs significantly. The drastic change in the relative emission intensity of Lys-AuNCs is associated only with the addition of Cu²⁺ ions. It was observed that common interfering cations did not interfere in the selective detection of Cu²⁺ ions, thereby, revealing excellent selectivity of this method. The excellent selectivity of Lys-AuNCs can probably be attributed to the strong interaction between the Lys-AuNCs and Cu²⁺ ions.

Fig. 5 Relative emission intensity of Lys-AuNCs in the presence of different individual metal ions. The error bars indicated the relative standard deviation of three repeated experiments.

3.5 Sensitivity of the method

The capability of this analytical system for quantitative detection of Cu²⁺ ions was evaluated under optimum experimental conditions. In order to evaluate the sensitivity of the system for Cu²⁺ detection, the emission intensity of Lys-AuNCs at 660 nm was monitored as a function of the concentration of Cu²⁺ cations. As evident from Fig. 3, the emission intensity of Lys-AuNCs at 660 nm decreased with the increase in concentration of Cu²⁺ cations. The relative emission intensity of Lys-AuNCs vs. concentration of Cu²⁺ ions is shown in Fig. 6. The calibration plot showed a good linear relationship with correlation coefficient of 0.9976 over the Cu²⁺ concentrations ranging from 0.40 × 10⁻⁷ to 4.00 × 10⁻⁷ mol dm⁻³. From the slope of the linear plot, the limit of detection (LOD) for Cu²⁺ ions was calculated as 9.00 × 10⁻⁹ mol dm⁻³ (S/N = 3), which is considerably lower than the maximum level of Cu²⁺ ions in drinking water permitted by the USEPA.²⁶ In this case, the interaction between Lys-AuNCs and Cu²⁺ ions reaches an equilibrium within 2 min. Therefore, Cu²⁺ ions can be determined quickly. The limit of detection obtained for Cu²⁺ in the present study was compared with the previously reported methods and the results are summarized in Table 1. This LOD was comparable with or better than those obtained using other nanomaterial-based sensors for Cu²⁺ cations.^39,42–50 The good sensitivity and selectivity with wide linearity for the detection of Cu²⁺ cations suggests great potential of Lys-AuNCs for application in bioassays.

Fig. 6 The plot of relative emission intensity (ΔF) vs. different concentrations of Cu²⁺ ions (inset: relative emission intensity of Lys-AuNCs vs. the Cu²⁺ concentrations from 0.00 to 4.00 × 10⁻⁷ mol dm⁻³). The error bars indicated the relative standard deviation of three repeated experiments.

Table 1 Comparison of proposed method with other reported methods for the determination of Cu²⁺ ions

Probe	Linear range (×10^−6 mol dm^−3)	Detection limit (×10^−9 mol dm^−3)	Sample matrix	References
Glutathione-capped ZnxHg1−xSe QDs	0.03–5.00	20 000.00	—	42
Gemini-coated CdSe/ZnS QDs	0.00–500.00	1100.00	—	43
Silica-coated CdSe/ZnS QDs	0.00–10.00	900.00	—	44
BSA–ZnO NPs	1.00–6.00	609.00	—	39
Cyclen-functionalized FPNs	1.00–30.00	340.00	—	45
BSA–AuNCs	0.50–100.00	300.00	Tap water and electroplating wastewater	46
MSNs–NM	0.00–150.00	280.00	—	47
GQDs	0.00–15.00	226.00	—	48
Glutathione–AuNCs	0.10–6.25	86.00	—	49
AuNCs@DTT	0.00–60.00	80.00	—	50
Lys-AuNCs	0.04–0.40	9.00	Tap water, drinking water, sea water	This work

---

3.6 Real sample analysis

Real sample analysis is vital for evaluating the performance of a newly designed sensor because of its possible influence from naturally existing substances. The practical application of this Lys-AuNCs fluorescent sensor was estimated through the determination of Cu²⁺ ions spiked in real water samples such as tap water, drinking water and sea water. These water samples were spiked with two known concentrations of Cu²⁺ ions (2.00 × 10⁻⁷ and 4.00 × 10⁻⁷ mol dm⁻³) and analyzed by the standard addition method. The results obtained from this nanosensor are summarized in Table 2. It can be seen from Table 2 that the results obtained for real water samples show good agreement with the Cu²⁺ ions spiked samples. The obtained recoveries in the range of 96.00% to 107.50% with small relative standard deviation (RSD) values (Table 2), suggest that the ingredients in real water samples do not cause serious interference in the detection of Cu²⁺ ions. Therefore, the proposed Lys-AuNCs based fluorescent nanosensor has potential applications in the detection of Cu²⁺ ions in environmental samples and biological systems.

Table 2 Determination of Cu²⁺ ions in different water samples using a standard addition method (n = 3)

Samples	Cu^2+ spiked × 10^−7 mol dm^−3	Cu^2+ found × 10^−7 mol dm^−3	Recovery (%)	Relative standard deviation (%)
Tap water	2.00	2.10	105.00	1.22
	4.00	3.96	99.00	2.16
Drinking water	2.00	1.97	98.50	0.72
	4.00	3.84	96.00	1.59
Sea water	2.00	2.07	103.50	1.19
	4.00	4.30	107.50	2.07

---

4 Conclusions

In summary, a simple and cost-effective detection method based on fluorescent Lys-AuNCs probes has been developed, which allows rapid and selective detection of Cu²⁺ ions. The emission spectral results confirmed that the successive addition of Cu²⁺ induced significant emission quenching of Lys-AuNCs. The outcome from the emission and time resolved fluorescence studies clearly revealed the existence of static quenching mechanism between Lys-AuNCs and Cu²⁺ ions. Furthermore, the selectivity assay reveals that this fluorescent nanosensor has good selectivity to Cu²⁺ over other common interfering metal ions. This assay had a wide linear range of 0.40 × 10⁻⁷ mol dm⁻³ to 4.00 × 10⁻⁷ mol dm⁻³ with limit of detection value of 9.00 × 10⁻⁹ mol dm⁻³ (S/N = 3). The practical application of the present method was demonstrated by determining Cu²⁺ in environmental water samples.

Acknowledgements

KS acknowledges Department of Science and Technology INSPIRE fellowship (SRF) (DST-INSPIRE Program), Government of India for the financial support (IF110497). MI acknowledges the University Grants Commission (UGC-MRP, Project no. 41-309/2012 (SR)) and DST-SERB project no. SB/EMEQ-062/2013, India for the financial support. The authors are grateful to PSG Institute of Advanced Studies, Coimbatore, for HR-TEM measurements.

References

1. W. Chen and S. Chen, Angew. Chem., Int. Ed., 2009, 48, 4386–4389.
2. L. Shang, S. J. Dong and G. U. Nienhaus, Nano Today, 2011, 6, 401–418.
3. S. Choi, R. M. Dickson and J. Yu, Chem. Soc. Rev., 2012, 41, 1867–1891.
4. Y. Lu and W. Chen, Chem. Soc. Rev., 2012, 41, 3594–3623.
5. A. M. Derfus, W. C. W. Chan and S. N. Bhatia, Nano Lett., 2004, 4, 11–18.
6. A. Retnakumari, S. Setua, D. Menon, P. Ravindran, H. Muhammed, T. Pradeep, S. Nair and M. Koyakutty, Nanotechnology, 2010, 21, 055103.
7. P. Maity, S. Xie, M. Yamauchi and T. Tsukuda, Nanoscale, 2012, 4, 4027–4037.
8. J. Zheng, C. Zhou, M. Yu and J. Liu, Nanoscale, 2012, 4, 4073–4083.
9. H. W. Li, Y. Yue, T. Y. Liu, D. Li and Y. Wu, J. Phys. Chem. C, 2013, 117, 16159–16165.
10. J. Sun and Y. Jin, J. Mater. Chem. C, 2014, 2, 8000–8011.
11. J. Zheng, P. R. Nicovich and R. M. Dickson, Annu. Rev. Phys. Chem., 2007, 58, 409–431.
12. R. Jin, Nanoscale, 2010, 2, 343–362.
13. X. Yuan, Z. Luo, Y. Yu, Q. Yao and J. Xie, Chem.–Asian J., 2013, 8, 858–871.
14. T. Zhou, Y. Huang, W. Li, Z. Cai, F. Luo, C. J. Yang and X. Chen, Nanoscale, 2012, 4, 5312–5315.
15. J. Liu, L. Lu, S. Xu and L. Wang, Talanta, 2015, 134, 54–59.
16. D. Lu, L. Liu, F. Li, S. Shuang, Y. Li, M. M. F. Choi and C. Dong, Spectrochim. Acta, Part A, 2014, 121, 77–80.
17. R. Kramer, Angew. Chem., Int. Ed., 1998, 37, 772–773.
18. S. Wanga, X. Wanga, Z. Zhang and L. Chen, Colloids Surf., A, 2015, 468, 333–338.
19. Y. F. Lee, T. W. Deng, W. J. Chiu, T. Y. Wei, P. Roy and C. C. Huang, Analyst, 2012, 137, 1800–1806.
20. M. E. A. Hellal, F. Hellal, Z. E. Khemissi, R. Jebali and M. Dachraoui, Bull. Environ. Contam. Toxicol., 2011, 86, 194–198.
21. C. Cervantes and F. G. Corona, FEMS Microbiol. Rev., 1994, 14, 121–137.
22. C. Molteni, H. K. Abicht and M. Solioz, Appl. Environ. Microbiol., 2010, 76, 4099–4101.
23. G. Grass, C. Rensing and M. Solioz, Appl. Environ. Microbiol., 2011, 77, 1541–1547.
24. K. J. Barnham, C. L. Masters and A. I. Bush, Nat. Rev. Drug Discovery, 2004, 3, 205–214.
25. S. A. Reddy, K. J. Reddy, S. L. Narayan and A. V. Reddy, Food Chem., 2008, 109, 654–659.
26. U.S. EPA, Aquatic life ambient freshwater quality criteria – copper: 2007 revision, U.S. Environmental Protection Agency, Washington, D.C, 2007, Report number: EPA-822/R-07–001.
27. B. Liao, W. Wang, X. Deng, B. He, W. Zeng, Z. Tang and Q. Liu, RSC Adv., 2016, 6, 14465–14467.
28. X. Lin, S. X. Li and F. Y. Zheng, RSC Adv., 2016, 6, 9002–9006.
29. K. M. Vengaian, C. D. Britto, K. Sekar, G. Sivaraman and S. Singaravadivel, RSC Adv., 2016, 6, 7668–7673.
30. Q. Huang, X. Lin, C. Lin, Y. Zhang, S. Hu and C. Wei, RSC Adv., 2015, 5, 54102–54108.
31. J. M. Lin, Y. Q. Huang, Z. b. Liu, C. Q. Lin, X. Ma and J. M. Liu, RSC Adv., 2015, 5, 99944–99950.
32. P. Lv, K. Yu, X. Tan, R. Zheng, Y. Ni, Z. Wang, C. Liu and W. Wei, RSC Adv., 2016, 6, 11256–11261.
33. S. Saracoglu and L. Elci, Anal. Chim. Acta, 2002, 452, 77–83.
34. C. Karunakaran, H. Zhang, J. P. Crow, W. E. Antholine and B. Kalyanaraman, J. Biol. Chem., 2004, 279, 32534–32540.
35. J. Djedjibegovic, T. Larssen, A. Skrbo, A. Marjanovic and M. Sober, Food Chem., 2012, 131, 469–476.
36. M. Pouzar, T. Cernohorsky, R. Bulanek and A. Krejcova, Chem. Listy, 2000, 94, 197–201.
37. H. Dai, Y. Shi, Y. Wang, Y. Sun, J. Hu, P. Ni and Z. Li, Sens. Actuators, B, 2014, 202, 201–208.
38. J. Xie, Y. Zheng and J. Y. Ying, J. Am. Chem. Soc., 2009, 131, 888–889.
39. Z. Chen and D. Wu, Sens. Actuators, B, 2014, 192, 83–91.
40. C. V. Durgadas, C. P. Sharma and K. Sreenivasan, Analyst, 2011, 136, 933–940.
41. J. R. Lakowicz, Principles of fluorescence spectroscopy, Springer Science + Business Media, New York, 3rd edn, 2006.
42. F. C. Liu, Y. M. Chen, J. H. Lin and W. L. Tseng, J. Colloid Interface Sci., 2009, 337, 414–419.
43. H. B. Li and X. Q. Wang, Sens. Actuators, B, 2008, 134, 238–244.
44. T. W. Sung and Y. L. Lo, Sens. Actuators, B, 2012, 165, 119–125.
45. J. Chen, Y. Li, W. Zhong, Q. Hou, H. Wang, X. Sun and P. Yi, Sens. Actuators, B, 2015, 206, 230–238.
46. D. Y. Cao, J. Fan, J. R. Qiu, Y. F. Tu and J. L. Yan, Biosens. Bioelectron., 2013, 42, 47–50.
47. J. Cui, S. Wang, K. Huang, Y. Li, W. Zhao, J. Shi and J. Gu, New J. Chem., 2014, 38, 6017–6024.
48. F. Wang, Z. Gu, W. Lei, W. Wang, X. Xia and Q. Hao, Sens. Actuators, B, 2014, 190, 516–522.
49. G. Zhang, Y. Li, J. Xu, C. Zhang, S. Shuang, C. Dong and M. M. F. Choi, Sens. Actuators, B, 2013, 183, 583–588.
50. H. Ding, C. Liang, K. Sun, H. Wang, J. K. Hiltunen, Z. Chen and J. Shen, Biosens. Bioelectron., 2014, 59, 216–220.

---

Footnotes

† This paper is dedicated to Professor R. Ramaraj on the occasion of his 60th birthday.

‡ Electronic supplementary information (ESI) available. See DOI: 10.1039/c6ra08325k

---