Synthesis of ovalbumin-stabilized highly fluorescent gold nanoclusters and their application as an Hg²⁺ sensor

Abstract

Biomolecule-functionalized fluorescent gold nanoclusters (AuNCs) have attracted a lot of attention because of their good biocompatibility and considerable environmental/cost advantages. Recently, some proteins rich in tyrosine and cysteine have been proven to work as templates for the direct synthesis of AuNCs under alkaline conditions. However, the low quantum yield (QY) of AuNCs is still a restraining factor, which constrains its wide applications. In this study, highly fluorescent AuNCs have been synthesized in a basic aqueous solution using ovalbumin (OVA) as both a reducing and stabilizing agent. The QY of the ovalbumin-stabilized AuNCs (OVA@AuNCs) was found to be twice that of the reported BSA-stabilized AuNCs (BSA@AuNCs) under the same conditions. Moreover, the good pH stability and time stability of the OVA@AuNCs were examined. These properties will be helpful for AuNCs-based sensing and imaging. Further research revealed that the fluorescence of the OVA@AuNCs could be quenched by Hg²⁺ and it can be used as a sensor for sensitive Hg²⁺ detection with a detection limit of 10 nM.

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Introduction

Metal nanoclusters, composed of a few to tens of atoms, are a new type of luminescent nanomaterial that have attracted a great deal of interest.^1–3 When compared with nanoparticles, a distinct feature is that they have some characteristic properties such as photoluminescence, photostability, and Stokes shift. AuNCs with sizes smaller than 3 nm are a specific type of nanomaterial. Unlike the well-known large gold nanoparticles, AuNCs do not exhibit surface plasma resonance (SPR) absorption in the visible region. Instead of this, AuNCs can exhibit excited fluorescence under UV light. With advantages of long lifetimes, discrete electronic states and size-dependent fluorescence, AuNCs have attracted a lot of attention in bio-imaging and bioanalysis.^4–7

The preparation of AuNCs from Au³⁺ in the presence of small thiol compounds, such as 2-phenylethanethiol (PhCH₂CH₂SH), has been reported over the past decade.⁸ However, the low quantum yield (usually less than 1%), poor water dispersibility, and instability have limited their bioapplications. Over the past decade, two major categories for the preparation of stable, water dispersible AuNCs have been reported. The first category is an etching process using polymers,⁹ and the other category is a chemical reduction in the presence of thiol ligands.^10,11 For chemical reduction, chemicals, polymers, and biomolecules that act as capping agents are required for the preparation of stable and good fluorescent AuNCs. Biomolecules, such as peptides and proteins, can be used as structure-defined scaffolds to induce the nucleation and growth of AuNCs. In recent years, an interesting method has been developed to synthesize AuNCs using proteins as the sole reduction agent.^12–15 It has been suggested that proteins rich in tyrosine (Tyr) and cysteine (Cys) residues are critical to produce protein-stabilized AuNCs. This is because Tyr residues can reduce Au(III) ions at alkaline pH, and Cys residues, similar to thiol-protected AuNCs, are able to stabilize AuNCs. The synthesis of protein-stabilized AuNCs is a simple, one-pot synthetic route and the proteins used are often commercially available. Moreover, the protein-stabilized AuNCs synthesized have good biocompatibility, stability, and considerable environmental/cost advantages. However, the QY of the protein-AuNCs synthesized is still low in comparison with Ag nanoclusters (QY: 95% in ethanol),¹⁶ which limits the wide application of protein-stabilized AuNCs in bio-imaging and bioanalysis. Therefore, it is still necessary to develop new synthetic methods for protein-stabilized AuNCs to enhance the QY.

Inspired by previous study, we wondered whether other proteins, which are rich in Cys and Tyr residues, could also be applied to synthesize fluorescent AuNCs. After several attempts, we found that AuNCs could be synthesized using OVA acting as both the reducing and stabilizing agent. OVA is an N-linked glycoprotein derived from chicken egg white that is made up of 385 amino acids. It is a strong candidate for the synthesis of protein-stabilized AuNCs, because OVA contain amino acid residues rich with 6 Cys and 10 Tyr. The preparation of OVA@AuNCs was easily carried out by mixing OVA and chloroauric acid (HAuCl₄) under basic conditions. The pH stability, time stability of OVA@AuNCs and the effectiveness of fluorescent-based heavy metal ion sensing were examined. Moreover, the OVA@AuNCs synthesized had a high QY (∼12%), which was found to be twice that of the reported BSA-stabilized AuNCs (BSA@AuNCs) (QY: ∼6%). In this study, we also demonstrated that the highly fluorescent AuNCs could be designed and used as a Hg²⁺ sensor with high sensitivity.

Experimental section

Chemicals and materials

HAuCl₄, OVA, sodium hydroxide (NaOH), horseradish peroxidase (HRP), manganese chloride, potassium chloride, lead chloride, zinc chloride, cobalt chloride, nickel chloride, ferric chloride, ferrous chloride, cadmium chloride, copper chloride, and silver nitrate were purchased from Sigma-Aldrich Chemical Co. (St. Louis, USA). Bovine serum albumin (BSA), hydrochloric acid, barium chloride, magnesium chloride, calcium chloride, and mercuric acetate were obtained from Sinopharm Chemical Reagent Co., Ltd (Shanghai, China). Water used throughout all the experiments was purified using a Milli-Q system (Branstead, USA) to a specific resistance of >18 MΩ cm.

Synthesis of ovalbumin-stabilized fluorescent gold nanoclusters

The OVA@AuNCs sample was prepared according to the following procedure: First, 100 μL of 250 μM OVA aqueous solution and 100 μL of 1.5 mM HAuCl₄ aqueous solution were mixed under vigorous stirring at room temperature. After mixing, NaOH was added into the mixed solution to adjust the pH to 12.65, and the mixture was incubated at 37 °C on a constant temperature shaking table at 250 rpm for 10.5 h. After the reaction was complete, the mixture was transferred in a 30 kD ultrafiltration device and centrifuged under 12 000 rpm for 20 min to remove the unreacted reagents and terminate the reaction. Finally, the OVA@AuNCs were redissolved in ultrapure water and stored at 4 °C. BSA@AuNCs and HRP@AuNCs were synthesized using the same method.

Characterization of the ovalbumin-stabilized fluorescent gold nanoclusters

Fluorescence spectra of the OVA@AuNCs synthesized were obtained using a Hitachi F-7000 Fluorescence Spectrophotometer. The excitation spectra were obtained by fixing the emission wavelength at 650 nm, whereas the emission spectra were obtained by fixing the excitation wavelength at 490 nm. Data points were recorded with an interval of 1 nm. Transmission electron microscopy (TEM) was performed on a JEOL JEM-2100 high resolution transmission electron microscope at 200 kV.

Hg²⁺ detection using the ovalbumin-stabilized fluorescent gold nanoclusters

Different concentrations of mercuric acetate (aqueous solution or spiked in lake water, which was collected from Meilan Lake of Shanghai) and the prepared OVA@AuNCs were mixed with an equivalent volume and the mixture was incubated at ambient temperature for 5 min. Subsequently, 50 μL of the mixtures were used to obtain the fluorescent spectra.

To examine the selectivity of Hg²⁺ detection using the OVA@AuNCs, other ions (i.e. Ca²⁺, Na⁺, K⁺, Mg²⁺, Mn²⁺, Ba²⁺, Ag⁺, Cd²⁺, Cu²⁺, Zn²⁺, Fe²⁺, Fe³⁺, Co⁺, Ni²⁺, and Pb²⁺) were used instead of Hg²⁺.

Results and discussion

To find potential candidates for the preparation of protein-stabilized AuNCs, some common proteins were tried in the synthesis of AuNCs following the procedure of Xie et al.¹² The related data are not shown here. After several attempts, we found that OVA could be used as both the reducing and stabilizing agent to synthesize AuNCs. In fact, it was not unexpected that Au(III) ions could be reduced by OVA because it contains 10 Tyr residues and possibly other residues with reduction capability. Next, the synthetic conditions used to prepare the OVA@AuNCs, such as reaction time and pH, were optimized in our study.

The effect of reaction time on the synthesis of ovalbumin-stabilized fluorescent gold nanoclusters

To obtain stable OVA@AuNCs with high QY, the reaction time for preparing the OVA@AuNCs was investigated. It was observed from Fig. 1 that the fluorescence intensity of the OVA@AuNCs was gradually increased over time until 10.5 h wherein it reached a maximum. It indicated that Au³⁺ in HAuCl₄ was reduced to Au⁰ by tyrosine residues in OVA and the AuNCs were formed in situ. However, the results showed that the fluorescence intensity of the OVA@AuNCs started to decrease after 10.5 h. It was thought that the continuous growth of AuNCs destroyed the spatial structure of OVA so that the microenvironment for the formation of AuNCs was changed. Therefore, to maintain the high fluorescence of the OVA@AuNCs synthesized, an ultrafiltration experiment was performed to remove the redundant NaOH and HAuCl₄, and terminate the reaction after incubation for 10.5 h.

Fig. 1 The effect of reaction time on the synthesis of OVA@AuNCs at pH 12.65; the excitation wavelength was 490 nm. (A) The fluorescence responses of the OVA@AuNCs at different reaction times, which varied from 0 h to 14 h. (B) The evolution of the fluorescence intensity of the OVA@AuNCs with reaction time. (C) The fluorescence of OVA@AuNCs at various reaction times under UV light.

The effect of pH on the synthesis of ovalbumin-stabilized fluorescent gold nanoclusters

The gold cluster of 0 valence could be produced at high pH wherein Au⁺ can be reduced to Au⁰ by the Tyr residues. So the addition of NaOH was critical to form the OVA@AuNCs. Fig. 2 showed that the fluorescence intensity of the OVA@AuNCs gradually increased with an increase in the pH value from 9 to 12.65. However, when NaOH was not added, or under acidic conditions upon the addition of HCl, the fluorescence of the AuNCs disappeared and the characteristic peak of the OVA@AuNCs at 650 nm was also not detected, which indicated that there was no formation of AuNCs. As a consequence, the OVA@AuNCs were synthesized only in an alkaline solution at a pH value above 9. However, considering that the spatial structure of OVA may be destroyed in a strong alkali environment, a pH value above 12.65 was not considered in our experiment. Therefore, a pH of 12.65 was chosen as the optimal reaction condition for the synthesis of the OVA@AuNCs.

Fig. 2 The effect of pH on the synthesis of OVA@AuNCs. (A) The fluorescence response of the OVA@AuNCs at different pH with the pH value varied from 4 to 12.65. (B) The evolution of the fluorescence intensity of OVA@AuNCs with pH. (C) The fluorescence of OVA@AuNCs at different pH under UV light.

Preparation and characterization of the ovalbumin-stabilized fluorescent gold nanoclusters

Based on the abovementioned results, the fluorescent OVA@AuNCs were synthesized by mixing OVA and HAuCl₄ at pH 12.65 with further incubation at 37 °C for 10.5 h. The formation of the OVA@AuNCs was first confirmed by high resolution transmission electron microscopy (HRTEM). As shown in Fig. 3, typical spherical particles with an average diameter of about 3.8 nm can be observed. As for the production of fluorescence, it has been reported that both BSA and HRP can be used as the reducing and stabilizing agents.^12,14 Therefore, the OVA@AuNCs were compared with BSA@AuNCs and HRP@AuNCs, and the results shown in Fig. 4. As shown in Fig. 4, the maximum emission peak of the OVA@AuNCs was observed at 650 nm and the maximum excitation peak was at 490 nm (Fig. 4C). The same results can be observed in the BSA@AuNCs and HRP@AuNCs systems (Fig. 4A and B). However, the fluorescence emission peak intensity of the OVA@AuNCs was higher than the fluorescence emission peak intensity of the BSA@AuNCs and HRP@AuNCs at 650 nm. The high fluorescence of OVA@AuNCs was observed from the image under UV light (Fig. 4D).

Fig. 3 TEM image of the as-prepared OVA@AuNCs.

Fig. 4 (A) Fluorescence spectra of the synthesized BSA@AuNCs. (B) Fluorescence spectra of the synthesized HRP@AuNCs. (C) Fluorescence spectra of the synthesized OVA@AuNCs. (D) Photograph of the different protein-stabilized AuNCs in solution under the visible light on the left and the fluorescence of the different protein-stabilized AuNCs in solution under UV light on the right.

The excellent fluorescence properties of the synthesized OVA@AuNCs attracted our attention. Therefore, the photoluminescence quantum yield (QY) of the OVA@AuNCs was calculated. Rhodamine B was chosen as a standard substance to measure the quantum yield with reference to the literature.¹⁷ The calculated QY of the OVA@AuNCs was about 12%, and the QY of BSA@AuNCs and HRP@AuNCs were 5.5% and 6.7%, respectively. The QY of BSA@AuNCs was consistent with that reported in the literature.⁹ The high QY of OVA@AuNCs will be helpful for applications in bio-imaging and bioanalysis.

The stability of the synthesized ovalbumin-stabilized fluorescent gold nanoclusters

Stability is often considered as an important indicator of the practicability of nanomaterials. So the stability of the synthesized OVA@AuNCs was taken into consideration in our experiments. First, the impacts of storage time on the stability of the synthesized OVA@AuNCs were investigated. The synthesized OVA@AuNCs were redissolved in ultrapure water and stored at 4 °C. The results in Fig. 5 illustrate that the synthesized OVA@AuNCs could be stored at 4 °C for at least 15 days. In addition, we tested the effect of pH on the stability of the synthesized OVA@AuNCs. As shown in Fig. 6, although the pH value was varied from 2 to 12.65, the fluorescence intensity of the synthesized OVA@AuNCs was not significantly changed over 15 days. Therefore, the results demonstrated that the synthesized OVA@AuNCs can be used in samples with different acidity. In conclusion, the synthesized OVA@AuNCs are stable, which is favourable for further utilization and exploitation.

Fig. 5 The effect of storage time on the stability of the synthesized OVA@AuNCs. The synthesized OVA@AuNCs were stored at 4 °C and the storage time was varied from 1 day to 15 days. (A) The fluorescence response of the OVA@AuNCs with different storage time. (B) The evolution of the fluorescence intensity of the OVA@AuNCs with storage time. (C) The fluorescence of the OVA@AuNCs with storage time under UV light.

Fig. 6 The effect of pH on the stability of the synthesized OVA@AuNCs. The synthesized OVA@AuNCs were redissolved in ultrapure water at a specific pH and the solution was incubated at 4 °C for 30 min. The fluorescence intensity was recorded after incubation. (A) The fluorescence response of the OVA@AuNCs, which were redissolved in solution at different pH. (B) The evolution of the fluorescence intensity of the OVA@AuNCs at different pH values. (C) The fluorescence of OVA@AuNCs at different pH under UV light.

Hg²⁺ detection using the as-prepared ovalbumin-stabilized fluorescent gold nanoclusters as fluorescent probes

Hg²⁺ pollution is highly toxic to the endocrine system, brain and kidneys by interaction with the thiol groups found in proteins and aminophospholipids.^18,19

Some methods have been developed for the detection of Hg²⁺.^20–23 However, new strategies with improved sensitivity and usability are still urgently needed.

First, in next set of experiments, we measured the modulation of the photoluminescence intensity of the OVA@AuNCs in the presence of various metal ions. The metal ions tested included Hg²⁺, Zn²⁺, Cd²⁺, Co²⁺, Pb²⁺, Ni²⁺, Fe²⁺, Fe³⁺, Ag⁺, Cu²⁺, Na⁺, K⁺, Mg²⁺, Ca²⁺, Ba²⁺, and Mn²⁺. As shown in Fig. 7A, 2 mM of Na⁺, K⁺, Mg²⁺, Mn²⁺, Ca²⁺, and Ba²⁺ did not significantly change the fluorescence of the OVA@AuNCs in solution. On the contrary, we found that the fluorescence emission intensity of OVA@AuNCs was obviously enhanced by Zn²⁺ and Cd²⁺ in Fig. 7B. It has been reported that chelation between the fluorescent molecule and the metal ions enhanced the fluorescence.²⁴ However, the enhancement was gradually weakened when the concentration of Cd²⁺ was more than 500 μM.

Fig. 7 (A–C) The effect of different metal ions on the fluorescence of synthesized OVA@AuNCs. (D) The relative fluorescence (I/I₀) of OVA@AuNCs at λ_ex = 490 nm in the presence of 10 μM of Na⁺, K⁺, Mg²⁺, Mn²⁺, Ca²⁺, Ba²⁺, Ag⁺, Cd²⁺, Cu²⁺, Zn²⁺, Fe²⁺, Fe³⁺, Co²⁺, Ni²⁺, Pb²⁺, and Hg²⁺ at pH 12.65. I₀ and I are the photoluminescence intensities of the OVA@AuNCs in the absence and presence of the abovementioned ions.

In addition, the fluorescence of OVA@AuNCs can be quenched by Cd²⁺. Unlike Cd²⁺, the fluorescence enhancement of OVA@AuNCs by Zn²⁺ reached equilibrium when the concentration was as high as 500 μM. Further research revealed that several metal ions (Co²⁺, Pb²⁺, Ni²⁺, Fe²⁺, Fe³⁺, Ag⁺, and Cu²⁺) could quench the fluorescence of the synthesized OVA@AuNCs. However, the results indicated that the fluorescence of OVA@AuNCs cannot be quenched completely by these metal ions except Fe³⁺. Moreover, only high concentrations of Fe³⁺ could completely quench the fluorescence of the prepared OVA@AuNCs (Fig. 7C). When compared with other metal ions, a strong decrease in the fluorescence was observed in the presence of 10 μM of Hg²⁺ (Fig. 7D), which was attributed to the specific and strong interactions between Hg²⁺ and Au⁺.^25–27 The mechanism can be explained using a photo-induced electron transfer process.^28,29

Finally, we designed a method for the detection of Hg²⁺ in an aqueous solution based on the induced fluorescence quenching of the OVA@AuNCs. It was found that there was a linear correlation between the fluorescence intensity and the concentration of Hg²⁺ over the range of 0–10 μM; the curve-fitting equation was y = −0.0094x + 106, R² > 0.99 (Fig. 8, black curve), and the corresponding limit of detection (LOD) was 10 nM. In comparison, the commonly used BSA@AuNCs were not as sensitive as the OVA@AuNCs. The sensitivity was 1.5 a.u./μM (calculated from the slope of the red linear curve in Fig. 8), ca. 6 times lower than that of OVA@AuNCs. The detection of Hg²⁺ in real samples using OVA@AuNCs was also investigated. As shown in Table 1, lake water samples, which had been spiked with different concentrations of Hg²⁺, were studied. The results showed that an acceptable recovery and relative standard deviation (RSD) could be obtained, suggesting the potential application of our methodology for environmental monitoring.

Fig. 8 Quenching efficiency plotted with respect to the Hg²⁺ concentration. The concentration of Hg²⁺ was 0–10 μM.

Table 1 The detection of Hg²⁺ in lake water

Sample number	Added (μM)	Found (μM)	Recovery (%)	RSD (%) (n = 3)
1	0.1	0.94	94%	6.6
2	1	1.06	106%	4.3
3	10	8.97	90%	6.9

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Possible reaction mechanism

We have developed a synthesis of ovalbumin-stabilized highly fluorescent gold nanoclusters. Herein, we have tried to explain the reaction mechanism for the high fluorescence produced by the OVA@AuNCs. Recent studies have demonstrated that Tyr or Tyr residues in peptides can reduce Au(III) ions through their phenolic groups, and the reduction capability of Tyr can be greatly improved by adjusting the reaction pH above the pK_a (∼10).³⁰ Therefore, it was expected that Au(III) ions could be reduced by OVA because it contains 10 Tyr residues and possibly other residues with reduction capability. BSA contains 21 Tyr residues; however, the QY of the OVA@AuNCs was higher than the QY of BSA@AuNCs in our experiments. It was thought that Au(III) ions were reduced by other residues with reduction capability, such as tryptophan, because tryptophan could also reduce Au(III) ions at alkaline pH above the pK_a as previously reported.³¹

Conclusions

In our study, OVA-stabilized fluorescent AuNCs with a red emission at 650 nm in an aqueous solution were synthesized using OVA as both the reducing and stabilizing agent. The OVA@AuNCs had a higher QY than BSA@AuNCs and HRP@AuNCs. Moreover, the synthesized OVA@AuNCs were stable at different pH values in solution and can be stored at 4 °C for at least 15 days. Finally, the fluorescence intensity of the as-prepared OVA@AuNCs was sensitive to Hg²⁺ and decreased as the concentration of Hg²⁺ increased. The calibration graphs were linear over the range of 0–10 μM and the corresponding LOD was 10 nM.

Acknowledgements

This study is supported by the National Natural Science Foundation of China (Grant No. 31200742).

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