Facile Synthesis of Ultrastable Fluorescent Copper Nanoclusters and Their Cellular Imaging Application

Copper nanoclusters (Cu NCs) are generally formed by several to dozens of atoms. Because of wide range of raw materials and cheap prices, Cu NCs have attracted scientists’ special attention. However, Cu NCs tend to undergo oxidation easily. Thus, there is a dire need to develop a synthetic protocol for preparing fluorescent Cu NCs with high QY and better stability. Herein, we report a one-step method for preparing stable blue-green fluorescent copper nanoclusters using glutathione (GSH) as both a reducing agent and a stabilizing agent. High-resolution transmission electron microscopy (HRTEM), X-ray photoelectron spectroscopy (XPS) and electrospray ionization mass spectrometer (ESI-MS) were used to characterize the resulting Cu NCs. The as-prepared Cu NCs@GSH possess an ultrasmall size (2.3 ± 0.4 nm), blue-green fluorescence with decent quantum yield (6.2%) and good stability. MTT results clearly suggest that the Cu NCs@GSH are biocompatible. After incubated with EB-labeled HEK293T cells, the Cu NCs mainly accumulated in nuclei of the cells, suggesting that the as-prepared Cu NCs could potentially be used as the fluorescent probe for applications in cellular imaging.


Characterization
The fluorescence spectra were measured with an Enspire Multimode Plate Reader (PerkinElmer, Shanghai, China). The optical adsorption spectra were recorded on an UV2550 Spectrophotometer (SHIMADZU, Shanghai, China). High-resolution transmission electron microscopy (HRTEM) images were obtained with a Tecnai G 2 F30 (FEI, Shanghai, China) at an accelerating voltage of 200 kV. The X-ray photoelectron spectroscopy (XPS) were measured by AXIS-ULTRA DLD (Shimadzu, Shanghai, China). The mass spectra were obtained with a micro TOF-Q electrospray ionization mass spectrometer (ESI-MS) (Bruker Daltonics, Shanghai, China). The optical density (OD) of the mixture was measured at 490 nm with a microplate absorbance reader (VersaMax (Molecular Devices), Shanghai, China). Cellular imaging was performed on a Confocal microscope (Olympus IX 81 + FV1000, Shanghai, China) using an excitation wavelength of 380 nm. All the experiments were conducted at room temperature, if not stated otherwise.

Synthesis of Blue-Green-Emitting Cu NCs@GSH
The GSH-stabilized CuNCs were prepared by a thermo-reduced method. In a typical experiment, GSH (1 mL, 9 mM) was mixed with CuCl 2 (8.9 mL, 50 mM), and the solution became cloudy. Next, NaOH solution (100 µL, 1 M) was added dropwise with vigorous stirring until the mixture solution changed to be clear. Then the mixture was kept stirring at 80 • C for 24 h until the color changed from pale blue to purple. The purple solution of Cu NCs was then gradually cooled down to room temperature and stored in refrigerator at 4 • C. The resulting concentrated Cu NCs@GSH were precipitated by addition of isopropanol, collected through centrifugal at 10,000 rpm, and then redispersed in water. After 3 purification cycles, and finally the Cu NCs@GSH were dispersed in 10 mL water for further application.

Cell Culture and Cytotoxicity Assay
HeLa cells were cultured in Dulbecco's modified Eagle's medium (DMEM) including high glucose supplemented with 10% fetal bovine serum (FBS), 100 U of penicillin and 100-µg/mL streptomycin at 5% CO 2 , 37 • C. The cell viability was determined by the MTT assay according to the manufacturer's instructions. Briefly, the HeLa cells were seeded in a 96-well plate in cell medium overnight. After being incubated with different concentrations of Cu NCs@GSH (0, 10, 50, 100, 200 and 400 µg/mL) for 12 h, 10 µL of MTT solution (5 mg/mL) was added to each well. After 4 h of incubation, 100 µL of DMSO was added to each well. The CuNCs@GSH are synthesized in aqueous phase, whose solubility depends on the ligand GSH. Moreover, the solubility of GSH is 500 g/L at pH 8.0-8.5.

Cellular Imaging
The human embryonic kidney cells HEK293T were grown in DMEM supplemented with 10% FBS at 37 • C and 5% CO 2 for 24 h. The cells were then washed with phosphate-buffered saline (PBS), followed by incubation with Cu NCs (100 µL, 250 µg/mL) for 2 h at 37 • C and then washed with PBS three times, followed by incubation with EB solution (100 µL, 2 µg/mL) for 15 min. Cellular imaging was then performed on a Confocal microscope (Olympus IX 81 + FV1000) using an excitation wavelength of 380 nm and 583 nm after the cells were washed with PBS three times.

Synthesis of Cu NCs@GSH
The GSH-stabilized blue-green fluorescent Cu NCs were prepared according to Scheme 1. The as-synthesized Cu NCs exhibited decent quantum yield (QY = 6.2% with quinine sulfate in 1-M H 2 SO 4 as a reference) and excellent stability. The GSH was chosen as both the scaffold and reducing agent to protect the clusters possibly due to the strong interaction of the Cu 2+ and appropriately placed cysteine residues in GSH during the encapsulation process [35][36][37]. Due to the biocompatibility of GSH, GSH-functionalized Cu NCs were found to be suitable for bio-applications due to their bioactive surface. This synthetic method can be categorized as a green one because no toxic chemical/solvent was used in this synthesis process and the reduction was induced thermally in the presence of GSH to produce Cu NCs@GSH, which were then used for cellular imaging. It is worth noticing that the reaction can be easily upscaled to produce more than 750 mL Cu NCs in a single synthesis step with decent fluorescence emission ( Figure S1), which demonstrates the cost-effective and large-scale production of fluorescent metal nanoclusters.
Nanomaterials 2020, 10, x; doi: FOR PEER REVIEW www.mdpi.com/journal/nanomaterials noticing that the reaction can be easily upscaled to produce more than 750 mL Cu NCs in a single synthesis step with decent fluorescence emission ( Figure S1), which demonstrates the cost-effective and large-scale production of fluorescent metal nanoclusters. Scheme 1. Schematic illustration of thermo-reduced synthesis of fluorescent Cu NCs@GSH.

Optimization Results for Reaction Conditions
We examined the effect of various synthetic parameters to produce highly fluorescent and stable Cu NCs including the concentration of GSH, NaOH, reaction temperature and the reaction time.
In the systematic optimization process, the volume of GSH (1 mL), CuCl2 (8.9 mL), NaOH (0.1 mL) were kept constant to keep the same total volume, i.e., 10 mL, for each optimization reaction, the variation parameter was concentration. Firstly, we examined the effect of concentration of GSH to produce stable and fluorescent Cu NCs. As shown in Figure 1A,B, it was observed that with an increase in the concentration of GSH, the fluorescence intensity of maximum emission peak at 497 nm increased sharply and then decreased slowly while the color of solution turned from brown to purple and finally to little green (the absorption spectra are shown in Figure S7), which may be due to the lower concentration of GSH that was not sufficient to protect Cu NCs well. At higher concentration of GSH, the nucleation of the Cu NCs was trapped by the capping reagent, which reduced the chance of collision and prevented the NCs from growing [38]. Hence, by controlling the amount of GSH, the blue-green (maximum emission wavelength at 497 nm) fluorescent Cu NCs@GSH can be obtained and the highest blue-green fluorescence intensity was achieved by using 9-mM GSH. The final concentration of GSH is 0.9 mM. NaOH was not only helpful to make the cloudy suspension of GSH and Cu 2+ clear, but it can also enhance the reducing capability of -SH of GSH [24]. In Figure  1C, the highest fluorescence intensity of Cu NCs was achieved when the concentration of NaOH was 1 M. The final concentration of NaOH is 10 mM. The lower (0.25 M, 0.5 M) and higher concentrations (2 M, 4 M) of NaOH were not helpful to further improve fluorescence intensity of Cu NCs. We also investigated the effect of temperature and reaction time to obtain the most stable and highly fluorescent Cu NCs. Figure 1D shows the effect of temperature on the fluorescence intensity of Cu NCs at the prolonged reaction time. Figure 1D and Figure 2 also show that 80 °C is the optimum temperature for the synthesis of highly fluorescent Cu NCs. It may due to the fact that a high temperature is not only helpful to overcome the energy barrier of the reduction process, but is also helpful to enhance the reaction rate of both NCs growth and digestion, therefore facilitating the structure optimization of Cu NCs [1,[39][40][41][42]. The reducing ability of GSH at lower/room temperature Scheme 1. Schematic illustration of thermo-reduced synthesis of fluorescent Cu NCs@GSH.

Optimization Results for Reaction Conditions
We examined the effect of various synthetic parameters to produce highly fluorescent and stable Cu NCs including the concentration of GSH, NaOH, reaction temperature and the reaction time. In the systematic optimization process, the volume of GSH (1 mL), CuCl 2 (8.9 mL), NaOH (0.1 mL) were kept constant to keep the same total volume, i.e., 10 mL, for each optimization reaction, the variation parameter was concentration. Firstly, we examined the effect of concentration of GSH to produce stable and fluorescent Cu NCs. As shown in Figure 1A,B, it was observed that with an increase in the concentration of GSH, the fluorescence intensity of maximum emission peak at 497 nm increased sharply and then decreased slowly while the color of solution turned from brown to purple and finally to little green (the absorption spectra are shown in Figure S7), which may be due to the lower concentration of GSH that was not sufficient to protect Cu NCs well. At higher concentration of GSH, the nucleation of the Cu NCs was trapped by the capping reagent, which reduced the chance of collision and prevented the NCs from growing [38]. Hence, by controlling the amount of GSH, the blue-green (maximum emission wavelength at 497 nm) fluorescent Cu NCs@GSH can be obtained and the highest blue-green fluorescence intensity was achieved by using 9-mM GSH. The final concentration of GSH is 0.9 mM. NaOH was not only helpful to make the cloudy suspension of GSH and Cu 2+ clear, but it can also enhance the reducing capability of -SH of GSH [24]. In Figure 1C, the highest fluorescence intensity of Cu NCs was achieved when the concentration of NaOH was 1 M. The final concentration of NaOH is 10 mM. The lower (0.25 M, 0.5 M) and higher concentrations (2 M, 4 M) of NaOH were not helpful to further improve fluorescence intensity of Cu NCs. We also investigated the effect of temperature and reaction time to obtain the most stable and highly fluorescent Cu NCs. Figure 1D shows the effect of temperature on the fluorescence intensity of Cu NCs at the prolonged reaction time. Figures 1D and  2 also show that 80 • C is the optimum temperature for the synthesis of highly fluorescent Cu NCs. It may due to the fact that a high temperature is not only helpful to overcome the energy barrier of the reduction process, but is also helpful to enhance the reaction rate of both NCs growth and digestion, therefore facilitating the structure optimization of Cu NCs [1,[39][40][41][42]. The reducing ability of GSH at lower/room temperature (25 • C) was too low to achieve a decent yield of Cu NCs due to the slow reaction kinetics. However, by increasing the reaction temperature to 90 • C resulted in the drop of fluorescence intensity, which may be attributed to the aggregation of the ultrasmall Cu NCs into larger Cu NPs at higher temperature.  (25 °C) was too low to achieve a decent yield of Cu NCs due to the slow reaction kinetics. However, by increasing the reaction temperature to 90 °C resulted in the drop of fluorescence intensity, which may be attributed to the aggregation of the ultrasmall Cu NCs into larger Cu NPs at higher temperature.

Characterization of As-prepared Cu NCs@GSH
The as-synthesized Cu NCs@GSH were well dispersed in water and emitted an intense blue-green fluorescence under 365 nm irritation ( Figure 3D). Figure 3A shows that the maximum excitation wavelength and emission wavelength are located at 372 nm and 497 nm, respectively. Meanwhile, as the blank control, the pure Cu 2+ , pure GSH and their mixture show no fluorescence ( Figure S2). In Figure 3B, there is an obvious absorption peak around 600 nm, which may be attributed to the aggregation of Cu NCs@GSH (in Figure S3) and not the formation of large size Cu NPs. The location of emission peak remained unchanged even upon various excitation wavelengths ( Figure 3C), implying that it was real fluorescence and not light scattering [43][44][45] and the relatively uniform surface state [46,47]. After freeze-drying, the lavender powder was obtained, which showed no fluorescence by irradiating at 365 nm while the strong blue-green fluorescence of Cu NCs@GSH can be recovered after their redispersion in water ( Figure S4). The QY of as-prepared Cu NCs@GSH in aqueous solution was calculated to 6.2% using quinine sulfate (QY = 0.54 in 1 M H2SO4) as the reference [48]. It was also observed that by dispersing the Cu NCs in ethanol aqueous mixture, with increasing volume ratio of ethanol to water, the fluorescence intensity was gradually enhanced by increasing ethanol content ( Figure S5), indicating that the Cu NCs@GSH exhibit the aggregation-induced emission enhancement (AIEE), which agrees with previous reports [49][50][51][52]. More important, the as-synthesized Cu NCs@GSH exhibited excellent stability (Figure 4).

Characterization of As-Prepared Cu NCs@GSH
The as-synthesized Cu NCs@GSH were well dispersed in water and emitted an intense blue-green fluorescence under 365 nm irritation ( Figure 3D). Figure 3A shows that the maximum excitation wavelength and emission wavelength are located at 372 nm and 497 nm, respectively. Meanwhile, as the blank control, the pure Cu 2+ , pure GSH and their mixture show no fluorescence ( Figure S2). In Figure 3B, there is an obvious absorption peak around 600 nm, which may be attributed to the aggregation of Cu NCs@GSH (in Figure S3) and not the formation of large size Cu NPs. The location of emission peak remained unchanged even upon various excitation wavelengths ( Figure 3C), implying that it was real fluorescence and not light scattering [43][44][45] and the relatively uniform surface state [46,47]. After freeze-drying, the lavender powder was obtained, which showed no fluorescence by irradiating at 365 nm while the strong blue-green fluorescence of Cu NCs@GSH can be recovered after their redispersion in water ( Figure S4). The QY of as-prepared Cu NCs@GSH in aqueous solution was calculated to 6.2% using quinine sulfate (QY = 0.54 in 1 M H 2 SO 4 ) as the reference [48]. It was also observed that by dispersing the Cu NCs in ethanol aqueous mixture, with increasing volume ratio of ethanol to water, the fluorescence intensity was gradually enhanced by increasing ethanol content ( Figure S5), indicating that the Cu NCs@GSH exhibit the aggregation-induced emission enhancement (AIEE), which agrees with previous reports [49][50][51][52]. More important, the as-synthesized Cu NCs@GSH exhibited excellent stability ( Figure 4).
As shown in Figure 4A, the fluorescence intensity of Cu NCs remained the same even in the solution with high ionic strength. The Cu NCs also exhibited good resistance to oxidation for that the addition of H 2 O 2 influenced the fluorescence intensity very slightly ( Figure 4B). After exposure to 372-nm excitation light for 7000 s, the fluorescence intensity of Cu NCs decreased by 13.4%, exhibiting better photostability than that organic dye Rhodamine 6G ( Figure 4C). The storage under 4 • C for 3 months and 6 months reduced the fluorescence intensity of Cu NCs only by 5.5% and 16.5%, respectively, exhibiting their decent stability over a long period of time that would be very beneficial for their potential bio-applications ( Figure 4D).   As shown in Figure 4A, the fluorescence intensity of Cu NCs remained the same even in the solution with high ionic strength. The Cu NCs also exhibited good resistance to oxidation for that the    As shown in Figure 4A, the fluorescence intensity of Cu NCs remained the same even in the solution with high ionic strength. The Cu NCs also exhibited good resistance to oxidation for that the The FTIR spectrum shows a typical absorption band of the carboxyl group at 1712 cm −1 that corresponds to the weak interaction of the glycine residues of GSH with Cu NCs ( Figure 5A). Compared with the pure GSH alone, the characteristic peak of SH at 2525 cm −1 , disappeared in the GSH-stabilized Cu NCs, suggesting the interaction between thiol group and Cu NCs [49]. XPS analysis showed that the binding energy of Cu 2p 3/2 and Cu 2p 1/2 were located at 932.9 eV and 952.4 eV ( Figure 5B) indicating that Cu NCs are indeed composed of both Cu(0) and Cu(I) species, and the existence of Cu(I) also supports their improved stability and quantum yield [23,24]. Furthermore, there was no peak displayed around 942.0 eV, demonstrating the absence of Cu(II) in Cu NCs [52]. In addition, the S 2p3/2 peak located at 166.3 eV confirmed the covalent interaction of Cu NCs with the SH group ( Figure 5E) [53]. The peaks of S(1s), C(1s), N(1s), Na, O(1s), Cu (2p), and Na(1s) are clearly visible in the spectrum (Figure 5F), further indicating that Cu NCs are stabilized by GSH [54]. The exact atomic composition of Cu NCs was determined using ESI-MS analysis. From the MS spectrum ( Figure 5C), the highest peak located at 1942.7 could be attributed to the structure composition of [Cu 11 (GSH) 4 +Na+8H]. The HRTEM image ( Figure 5D) clearly showed the formation of uniform Cu NCs with an average diameter of 2.3 ± 0.4 nm.

Cytotoxicity Assay and Cellular Imaging
Prior to the biologic applications, the cytotoxicity of the Cu NCs@GSH was studied by the MTT method. As shown in Figure 6, after different concentrations of Cu NCs@GSH were incubated with HeLa cells for 12 h, there was no obvious decrease of the cell viability, but the slight cell growth. These results clearly suggest that the Cu NCs@GSH are biocompatible and suitable for potential bioimaging applications. In order to explore the role of Cu NCs in the field of fluorescent bioimaging, we chose human embryonic kidney cells HEK293T in this study (Figure 7). After incubation with Cu NCs, all the cells showed clear cells morphology and a bright blue fluorescence from the intracellular region ( Figure 7A,B), while no fluorescence was observed from the control experiment under the similar conditions ( Figure S6). EB is a commonly used organic dye in tissue labeling due to its specific binding to nuclei. Comparing the images of EB-labeled HEK293T cells ( Figure 7C) and the overlap of the above three images ( Figure 7D), it can be seen that the Cu NCs were also mainly accumulated in nuclei of the cells, suggesting that the as-prepared Cu NCs could potentially be used

Cytotoxicity Assay and Cellular Imaging
Prior to the biologic applications, the cytotoxicity of the Cu NCs@GSH was studied by the MTT method. As shown in Figure 6, after different concentrations of Cu NCs@GSH were incubated with HeLa cells for 12 h, there was no obvious decrease of the cell viability, but the slight cell growth. These results clearly suggest that the Cu NCs@GSH are biocompatible and suitable for potential bioimaging applications. In order to explore the role of Cu NCs in the field of fluorescent bioimaging, we chose human embryonic kidney cells HEK293T in this study (Figure 7). After incubation with Cu NCs, all the cells showed clear cells morphology and a bright blue fluorescence from the intracellular region ( Figure 7A,B), while no fluorescence was observed from the control experiment under the similar conditions ( Figure S6). EB is a commonly used organic dye in tissue labeling due to its specific binding to nuclei. Comparing the images of EB-labeled HEK293T cells ( Figure 7C) and the overlap of the above three images ( Figure 7D), it can be seen that the Cu NCs were also mainly accumulated in nuclei of the cells, suggesting that the as-prepared Cu NCs could potentially be used as the fluorescent probe for applications in cellular imaging.

Conclusions
In summary, a facile synthetic protocol is developed for the preparation of highly fluorescent Cu NCs using GSH as both a reducing and stabilizing agent, which can be upscaled easily. The as-synthesized Cu NCs exhibit bright blue-green fluorescence, high QY and excellent stability. Moreover, the Cu NCs exhibit low cytotoxicity and, therefore, possess great potential for cellular imaging to study various biologic processes.
Supplementary Materials: The following are available online at www.mdpi.com/xxx/s1. Figure S1: The photographs of Cu NCs under ordinary daylight (A) and UV-irradiation (B); Figure S2: Fluorescence emission spectra of GSH, Cu 2+ solution and mixture of GSH and Cu 2+ ; Figure S3: A high resolution TEM image of Cu

Conclusions
In summary, a facile synthetic protocol is developed for the preparation of highly fluorescent Cu NCs using GSH as both a reducing and stabilizing agent, which can be upscaled easily. The as-synthesized Cu NCs exhibit bright blue-green fluorescence, high QY and excellent stability. Moreover, the Cu NCs exhibit low cytotoxicity and, therefore, possess great potential for cellular imaging to study various biologic processes.
Supplementary Materials: The following are available online at www.mdpi.com/xxx/s1. Figure S1: The photographs of Cu NCs under ordinary daylight (A) and UV-irradiation (B); Figure S2: Fluorescence emission spectra of GSH, Cu 2+ solution and mixture of GSH and Cu 2+ ; Figure S3: A high resolution TEM image of Cu NCs@GSH ; Figure S4

Conclusions
In summary, a facile synthetic protocol is developed for the preparation of highly fluorescent Cu NCs using GSH as both a reducing and stabilizing agent, which can be upscaled easily. The as-synthesized Cu NCs exhibit bright blue-green fluorescence, high QY and excellent stability. Moreover, the Cu NCs exhibit low cytotoxicity and, therefore, possess great potential for cellular imaging to study various biologic processes.

Supplementary Materials:
The following are available online at http://www.mdpi.com/2079-4991/10/9/1678/s1. Figure S1: The photographs of Cu NCs under ordinary daylight (A) and UV-irradiation (B); Figure S2: Fluorescence emission spectra of GSH, Cu 2+ solution and mixture of GSH and Cu 2+ ; Figure S3: A high resolution TEM image of Cu NCs@GSH ; Figure S4: (A) Fluorescence excitation (dash line) and emission (solid line) spectra of Cu NCs@GSH in water (blue) and solid (black) state; (B) The photographs of Cu NCs@GSH solid and redispersed in water under daylight (above) and 365-nm UV light (below), Figure S5: