Dual-modal light scattering and fluorometric detection of lead ion by stimuli-responsive aggregation of BSA-stabilized copper nanoclusters

Abstract

A new and facilely prepared light scattering (LS) and fluorometric dual-mode sensor is developed for lead ion (Pb²⁺) detection with high sensitivity and selectivity by use of bovine serum albumin stabilized copper nanoclusters (BSA–Cu NCs). This assay relied on changes in optical properties due to Pb²⁺-responsive state transformation from nanoclusters to nanoparticles. BSA protected Cu NCs exhibit strong fluorescence emission and the sensing mechanism is based on the Pb²⁺-responsive aggregation induced size change of BSA–Cu NCs and the fluorescence quenching of Cu NCs. While upon addition of Pb²⁺, the complexation between BSA and the lead ions would lead to the aggregation of BSA–Cu NCs which subsequently results in light scattering signal enhancement. The detection limit of Pb²⁺ can be as low as 1 nM. In addition, the Cu NCs show a dramatic fluorescence turn-off as a result of the Pb²⁺-responsive aggregation process from the complex attached Pb²⁺ to the BSA–Cu NCs, with a detection limit of 10 nM. Both the turn-on light scattering assay and the turn-off fluorometric assay of the dual-mode sensing system exhibit high selectivity toward Pb²⁺ over interfering substances. Furthermore, we demonstrate the application of the present approach in water samples, which suggests its potential applications such as environmental analysis in the future.

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Introduction

Photoluminescent metal nanoclusters (NCs) with sizes ranging from about ten to a few hundred atoms, have been successfully synthesized in the past decades and have attracted extensive research interest.^1,2 Metal nanoclusters, as intermediates between metal atoms and nanoparticles, display molecule-like properties and are considered as promising substitutes to organic dyes and quantum dots.³ Luminescent NCs benefit from the ultra-small size, high biocompatibility, tunable fluorescent properties, and low toxicity. To date, several fluorescent noble-metal NCs have been reported (such as gold, silver NCs and alloy NCs). Particularly gold nanoclusters (Au NCs) and silver nanoclusters (Ag NCs) have drawn wide attention in recent years and have highly promising been used in imaging, catalysis, and sensing.^4–7 Compared with Au NCs and Ag NCs, only a few studies on the application of Cu NCs have been reported in biological and chemical sensing field.^8,9 However, their applications remain largely unexplored. Cu NCs are generally synthesized by template-assisted synthesis method with thiol ligands, carboxyl ligand, and biomolecules (e.g., proteins, single-stranded DNA and double-stranded DNA) commonly as templates.^8,10–13 Moreover, Cu NCs exhibit excellent fluorescent properties and are significantly cheaper due to abundant, economical and readily available synthetic materials for the preparation of Cu NCs as compared to the expensive starting material for noble-metal nanoclusters.⁹ Emerging as a new type of fluorescent nanomaterial, Cu NCs have been considered as a satisfactory candidate for sensing.

Lead is a kind of heavy metal element with serious contamination to environment and toxic effects on human health.^14,15 Lead compounds have been widely used in paint pigments, mining, storage batteries, water pipes, and gasoline additives, leading to increase the release of leads to the environment and human exposure in its aqueous medium.¹⁶ Lead causes toxicity to many organs and tissues, which is difficult to be detoxified and easily accumulated in human nervous and cardiovascular systems.¹⁵ The World Health Organization and Environmental Protection Agency (EPA) have set the maximum concentration limit of Pb²⁺ as 48 nM (10.0 ppb) in drinking water.^17,18 Traditional methods for the detection of lead ions are expensive, complicated and time-consuming,^19–22 therefore it is necessary to explore a low-cost, rapid and accurate detection technique of lead ions with high sensitivity and specificity.

Herein, we report a luminescent bovine serum albumin stabilized copper nanoclusters exhibiting strong luminescence at room temperature, which can serve as the first case of a dual-mode system of light scattering and fluorometric sensor for quantitatively detecting Pb²⁺ (Scheme 1). Upon the addition of Pb²⁺, the complexation between BSA and the lead ions would lead to the aggregation of BSA–Cu NCs which subsequently results in light scattering signal enhancement. With this, a simple and sensitive detection light scattering sensing for Pb²⁺ in aqueous solution was developed. Meanwhile, as the amounts of Pb²⁺ increase, the fluorescence intensity readout signal exhibited sharply decreasing as a result of the photo-induced electron transfer process from the complex attached Pb²⁺ to the BSA–Cu NCs. Owing to the above assays, BSA–Cu NCs were employed not only as a light scattering enhancing (turn-on) sensor but also as a fluorometric quenching (turn-off) sensor for the detection of Pb²⁺ in water samples. This approach holds great potential to broaden ways for assaying Pb²⁺ in real sample.

Scheme 1 Schematic illustration of the dual-mode system of light scattering turn-on and fluorescence turn-off sensor for detection of lead ions.

Experimental section

Materials

Bovine serum albumin (BSA), cupric sulfate (CuSO₄) was obtained from were from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Sodium hydroxide (NaOH), hydrochloric acid (HCl), nitric acid (HNO₃) and other metal salts or reagents were purchased from Shanghai Chemical Reagent Co., Ltd. (Shanghai, China). All reagents were of analytical grade, and used without further purification. Ultra-pure water was purified through a Millipore system used in preparing all solutions.

Apparatus

The fluorescence measurements were carried out on Jasco FP-6500 fluorescence spectrometer (Jasco, Japan). Light scattering spectra were obtained by scanning synchronously with the same excitation and emission wavelengths (namely, Δλ = 0) from 250 to 700 nm by a LS-50 luminescence spectrometer (PerkinElmer, USA). The UV-visible absorption spectra were carried out on a Shimadzu 2450 UV-visible spectrometer (Shimadzu, Japan). Fourier transform infrared (FTIR) spectroscopic measurements were performed on a NEXUS-670 Fourier transform spectrometer (Nicolet, USA). The high resolution transmission electron microscopy (HR-TEM) images were observed by JEM-2100 (JEOL, Japan). The circular dichroic (CD) spectra were recorded on a Jasco J-810 circular dichroism spectrometer (Jasco, Japan).

Synthesis of protein templated copper nanoclusters

All glassware used in the experiments were cleaned in a bath of freshly prepared Aqua Regia (HCl : HNO₃ volume ratio = 3 : 1), and rinsed thoroughly in ethanol and water prior to use. In a typical experiment, 2 mL CuSO₄ aqueous solution (20 mM) was added into the 10 mL BSA solution (15 mg mL⁻¹). After vigorous stirring for 3 minutes, 1.0 mL NaOH solution (1.0 M) was introduced, and then the mixture was allowed to stir for 8 h at 55 °C. The color of the mixture solution changed from blue to light violet. At last, the concentration of product was 3.33 mM (calculated by the number of copper atoms). The final solution was stored in refrigerator at 4 °C when before using.

Procedure for light scattering detection of Pb²⁺

In a typical test, 300 μL of as-prepared BSA–Cu NCs solution was diluted with phosphate buffer solution (50 mM, pH 7.0) and mixed with certain amount of Pb²⁺. The mixture was incubated at room temperature for 10 min. LS spectra were obtained by scanning synchronously with the same excitation and emission wavelengths (namely, Δλ = 0) from 250 to 700 nm by a LS-50 luminescence spectrometer.²³

Fluorescence quenching of BSA–Cu NCs by Pb²⁺

In a typical assay, 300 μL of as-prepared BSA–Cu NCs solution was diluted with phosphate buffer solution (50 mM, pH 7.0) and mixed with certain amount of Pb²⁺. The mixture was incubated at room temperature for 10 min and then fluorescence spectra were recorded under excitation at 324 nm.

Results and discussion

Characterization of the as-prepared BSA templated copper nanoclusters

Bovine serum albumin protected copper nanoclusters (BSA–Cu NCs) with blue-emitting were synthesized by a facile one-pot route in aqueous solution. The optical properties of BSA–Cu NCs were characterized by UV-vis absorption spectroscopy and fluorescence spectroscopy. The UV-vis absorption of the BSA–Cu NCs and BSA were given in Fig. 1a. It was demonstrated that BSA–Cu NCs exhibited a new absorption peak around 325 nm as compared to BSA with a strong absorption peak at 280 nm, showing the Cu NCs formation. The inserted photograph in Fig. 1a displays that the synthesized BSA–Au NCs solution was light violet in color under visible light (right photograph), while BSA solution was colorless (left photograph). The fluorescence emission and excitation spectrum of BSA–Cu NCs were investigated as shown in Fig. 1b. In aqueous solution, the BSA–Cu NCs displayed a strong fluorescence emission at 401 nm with an excitation peak at 324 nm. The TEM images of the BSA–Cu NCs were given in Fig. 1c. It was demonstrated that Cu NCs are well dispersed and about 2.8 nm in diameter. In addition, FT-IR spectra of BSA–Cu NCs and BSA were carried out. Fig. 1d reveals that we have successfully synthesized samples as the peaks of BSA at around 1500 cm⁻¹ disappears.

Fig. 1 Optical properties and TEM image of synthesized BSA–Cu NCs: (a) UV-vis absorption spectrum of pre-prepared BSA–Cu NCs. The inserted figures are the photographs of the BSA (left) and synthesized BSA–Cu NCs (right) under visible light. (b) Excitation and emission spectra of BSA–Cu NCs in aqueous solution. (c) TEM image of BSA–Cu NCs. (d) The FT-IR spectra of BSA–Cu NCs (black line) and BSA (red line).

Pb²⁺ detection using BSA–Cu NCs as light scattering sensor

To ensure that the proposed system can be used to detect Pb²⁺, we evaluated the light scattering signal changes from the interaction of the BSA–Cu NCs and Pb²⁺. Fig. 2a (black curve) shows the typical light scattering spectra of BSA–Cu NCs in aqueous solution. The light scattering spectrum of Cu NCs displayed a weak scattering peak at around 396 nm by scanning synchronously from 250 to 700 nm (Δλ = 0). Upon adding Pb²⁺ to the BSA–Cu NCs solution, the light scattering of BSA–Cu NCs was turn on greatly (Fig. 2a), denoting that the BSA–Cu NCs could serve as a highly efficient light scattering sensor to detect Pb²⁺. The specific feature that Pb²⁺ could enhance the light scattering signal of the system was then employed for highly sensitive detection of Pb²⁺. Fig. 2a depicted the typical LS response of BSA–Cu NCs upon the reaction with different concentrations of Pb²⁺. It appears significant enhancement of LS intensity of BSA–Cu NCs, which suggests that the introduction of Pb²⁺ produces larger scattering particles result in stronger light scattering signals. As shown, there is a maximum light scattering peak also located at 396 nm for the BSA–Cu NCs in the presence of Pb²⁺. With increasing the concentration of Pb²⁺, the intensity of the light scattering increase gradually. The enhanced value of LS intensity (I − I₀, where I₀ and I was the LS intensity of BSA–Cu NCs in absence and presence of Pb²⁺, respectively) versus the concentration of Pb²⁺ was plotted in figure of Fig. 2b. A good linear relationship between the LS intensity and the concentration of Pb²⁺ is obtained in the range from 3.0 nM to 21 nM. The detection limit was low as 1 nM (S/N = 3). The light scattering response proved to be very sensitive. Therefore, these results demonstrated that our system could be a simple and sensitive approach for Pb²⁺ sensing.

Fig. 2 Light scattering analysis for Pb²⁺: (a) the light scattering spectra of BSA–Cu NCs upon exposure to different concentrations of Pb²⁺ in 50 mM PBS buffer (pH 7.0). Concentrations of Pb²⁺ from bottom to top are 0, 3, 9, 15, and 21 nM. (b) Plot of light scattering intensity increase I − I₀ at 396 nm versus the concentration of Pb²⁺; detection limit of Pb²⁺ is calculated to be 1 nM on the basis of three times deviation.

Pb²⁺ detection using BSA–Cu NCs as fluorescent sensor

To demonstrate the utility of this approach, the dependence of the change of fluorescence of the BSA–Cu NCs on the concentration of added Pb²⁺ was also examined. Fig. 1b shows the typical excitation (red curve) and emission (black curve) spectra of BSA–Cu NCs in aqueous solution. The emission spectrum of BSA–Cu NCs displayed an emission peak at around 401 nm upon excitation at 324 nm. Upon adding Pb²⁺ to the BSA–Cu NCs solution, the fluorescence of BSA–Cu NCs was quenched greatly (Fig. 3a), denoting that the BSA–Cu NCs could serve as a highly efficient fluorescent probe to detect Pb²⁺. The specific feature that Pb²⁺ could quench the fluorescence of the BSA–Cu NCs was then employed for highly sensitive detection of Pb²⁺. As shown in Fig. 3a, the fluorescent intensity of BSA–Cu NCs at 401 nm decreased gradually with increasing concentrations of Pb²⁺, indicating that the fluorescence intensity of BSA–Cu NCs was highly dependent on the concentration of Pb²⁺. The decreased value of fluorescence intensity (F₀ − F, where F₀ and F was the fluorescence intensity of BSA–Cu NCs in absence and presence of Pb²⁺, respectively) versus the concentration of Pb²⁺ was plotted in figure of Fig. 3b. A good linear relationship between the fluorescence intensity and Pb²⁺ concentration over the range of 30–180 nM was obtained, with a detection limit of 10 nM. The fluorescence response proved to be very sensitive, thus the BSA–Cu NCs system could be serving as a very selective fluorescent probe for Pb²⁺ sensing. The detection limit was about 1 order of magnitude lower than that of fluorescence detection, which is attributed to much more sensitive enhancement of LS. Although the detection limit of LS assay was slightly higher than those of sensitive fluorescent sensors for Pb²⁺, it is still sensitive enough to monitor the Pb²⁺ for environmental analysis. Thus, the BSA–Cu NCs opens a new possibility for the construction of a sensitive “turn-off” LS and “turn-on” fluorescence dual-modal detection system.

Fig. 3 Fluorescence emission spectra analysis for Pb²⁺: (a) the fluorescence spectra of BSA–Cu NCs upon exposure to different concentrations of Pb²⁺ in 50 mM PBS buffer (pH 7.0). Concentrations of Pb²⁺ from top to bottom are 0, 30, 120, 210, 270, 330, and 390 nM. (b) Plot of fluorescence intensity decrease F₀ − F at 401 nm versus the concentration of Pb²⁺; detection limit of Pb²⁺ is calculated to be 10 nM on the basis of three times deviation.

Selectivity for Pb²⁺ detection

To investigate the selectivity of the BSA–Cu NCs sensing system, the response of the approach to other metal ions was investigated under the same conditions as in the case of Pb²⁺. The metal ions including Fe²⁺, Cd²⁺, Zn²⁺, Ca²⁺, Co²⁺, Mg²⁺, Ni²⁺, Na⁺, K⁺, and Ag⁺ were added into 0.6 μM BSA–Cu NCs solution. As shown in Fig. 4, both the LS enhancing (Fig. 4a) and the fluorescence quenching (Fig. 4b) efficiencies of Pb²⁺ to BSA–Cu NCs sensing system were significance, whereas no obvious optical signals changes were observed in the presence of other metal ions under the identical conditions. It demonstrates that the new proposed dual-modal sensing systems are selective for the detection of Pb²⁺ over other metal ions. Therefore, the BSA–Cu NCs could serve as a highly selective and reliable dual-modal sensor for Pb²⁺.

Fig. 4 Assessment of interference of other metal ions on the determination of Pb²⁺: selectivity assay results by light scattering method at 396 nm (a) or fluorescence technique at 401 nm (b). Conditions: the concentrations of metal ions are 0.3 μM. I₀ and I represent light-scattering intensities in the absence and presence of metal ion, respectively. F₀ and F represent fluorescence intensities in the absence and presence of metal ion, respectively.

Mechanism of Pb²⁺ induced LS enhancing and fluorescence quenching of BSA–Cu NCs

The mechanism of LS enhancing and fluorescence quenching of BSA–Cu NCs in the presence of Pb²⁺ has also been investigated. This assay relied on changes in optical properties due to the Pb²⁺ induced aggregation from nanoclusters to larger diameter nanoparticles. This transformation was confirmed by TEM characterization (Fig. 5a). We found that when Pb²⁺ was present in a solution of BSA–Cu NCs, it induced state transformation from BSA–Cu NCs into nanoparticles. The diameter of original Cu NCs was about 2.8 nm, while the diameter of the formed larger diameter nanoparticles became over 100 nm after the addition of Pb²⁺. The LS enhancing in the presence of Pb²⁺ can be attributed to the Cu NCs aggregation induced by the complexation between BSA and the Pb²⁺. BSA contains a high affinity site for Pb²⁺ ion and the binding involves carboxylate groups. We also observed that fluorescence signal changed gradually as increasing concentration of Pb²⁺ due to the concentration dependent aggregation process between the BSA–Cu NCs and the target Pb²⁺. The possible sensing mechanism was a photo induced electron transfer (PET) process between the BSA–Cu NCs and the target Pb²⁺. It has been also reported in previously literatures.²⁴ Thus, changes in these optical properties were used to construct a dual-modal assay of Pb²⁺.

Fig. 5 TEM and circular dichroism characterization of BSA–Cu NCs in the presence of Pb²⁺: (a) typical TEM images of synthesized BSA–Cu NCs in the presence of 10 mM Pb²⁺. Scale bar: 200 nm. (b) Circular dichroism (CD) spectra of BSA (black line), and BSA–Cu NCs in the absence (red line) and presence (green line) of 2 mM Pb²⁺ in 50 mM PBS buffer (pH 7.0). Conditions: the concentration of BSA and BSA–Cu NCs is 80 μM, respectively. All spectra were recorded after a reaction time of 10 min.

In addition, the conformational change of BSA was investigated by circular dichroism (CD) spectrometry. Native BSA displays CD features with minima at 209 and 222 nm, corresponding to the highly α-helical secondary structure (Fig. 5b). After the formation of Cu NCs, the peak at 209 nm in native BSA was shifted to 205 nm, and the peak at 222 nm gradually decrease, which reveals the loss of α-helix content and increase in β-sheet and random coil structure. The percentage of various conformations has been determined for both pure BSA and BSA–Au NCs by using CDNN software, which revealed 38.1% loss of α-helix structure. It turns up further blue shift to 202 nm after the addition of Pb²⁺ with 38.1% loss of α-helix structure (compared with pure BSA), which suggest that such unfolded BSA was very efficient in promoting the complexation between BSA–Au NCs and Pb²⁺.

Detection of Pb²⁺ in real samples

The applicability of the BSA–Cu NCs LS system based on Pb²⁺-responsive aggregation for detecting Pb²⁺ in a real sample was evaluated. A standard addition method was adopted to detect the concentration of Pb²⁺ in environmental water samples from Tongyu river water.^25–27 The analytical results were listed in Table 1. The detected Pb²⁺ content in water samples were derived from the standard curve and the regression equation. The results demonstrate that the proposed BSA–Cu NCs LS sensors can be used to analyze Pb²⁺ in environmental monitoring. Table 2 compares our present approach with other fluorescence, colorimetric, electrochemical and resonance scattering approaches for the detection of Pb²⁺ ions with respect to detection limit and linear range.^28–35 The BSA–Cu NCs sensor shows advantages such as lower limit of detection and simpler sensing system. Thus, it holds great practicality for Pb²⁺ detection in real environmental samples.

Table 1 Recoveries of lead ions spiked in river water by the developed LS assay

Samples	Proposed method (nM)	Added amounts (nM)	Found^a (nM)	Recovery (%)
a Average values are from three independent measurements.
1	Not found	10	9.75	97.5
2	Not found	15	15.16	101.1
3	Not found	20	20.56	102.8

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Table 2 Comparison of the analytical performance of the sensing systems for the detection of Pb²⁺ in terms of linear range and limit of detection

Method	Linear range	LODs	System	Reference
Fluorescence	0.02–1 μM	20 nM	DNA	28
Fluorescence	0–480 nM	18 nM	T30695, SYBR Green I	29
Fluorescence	3–50 nM	1 nM	Aptamer, TOTO-3, Tb^3+	30
Colorimetric	—	500 nM	DNAzyme, Au NPs	31
Colorimetric	0.03–0.3 μM	10 nM	DNAzyme, ABTS, hemin	32
Electrochemical	0.5–10 μM	300 nM	DNAzyme	33
Electrochemical	0.5 nM to 50 μM	0.5 nM	Hairpin DNA	34
Resonance scattering	1–120 nM	0.9 nM	TBA	35
Fluorescence	30–180 nM	10 nM	BSA–Cu NCs	This study
LS	3–21 nM	1 nM	BSA–Cu NCs	This study

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Conclusions

In summary, we have demonstrated herein a facile prepared, label-free, highly sensitive and selective light scattering and fluorometric dual-mode sensor for the measurement of lead ion with good analytical performance by using bovine serum albumin stabilized copper nanoclusters. It is simple in design and economic in operation. The method relies on the mechanism of concentration dependent Pb²⁺-responsive state transformation induced aggregation of BSA–Cu NCs. This dual modal assay allowed the determination of lead ion with more cost-effective, rapid, sensitive and a cheaper detection system in comparison with the traditional methods. More importantly, its usefulness for real samples applications has been successfully demonstrated. To the end, changing the template with other affinity functionalities, such as sulfhydryl, DNA, aptamers and so on, the metal nanoclusters-based sensing platform can be expended to the assay of a wide range of important compounds. The current approach has provided a new avenue for dual-mode test of BSA–Cu NCs based sensing platform and holds great potential for environmental monitoring, diagnostics and biosensors.

Acknowledgements

We are grateful for the financial support from the National Natural Science Foundation of China (grant No. 21575123, 21501146) and the Natural Science Foundation of Jiangsu Province (BK20150424, BK20140464).

Notes and references

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