# Green Synthesis of Fluorescent Ag Nanoclusters for Detecting Cu2+ Ions and Its “Switch-On” Sensing Application for GSH

Authors: Yunpeng Shang; Hui Gao; Lei Li; Chaoqun Ma; Jiao Gu; Chun Zhu; Zichen Yang; Chengwei Wang; Ye Zhang; Guoqing Chen
Journal: Journal of Spectroscopy
 (2021)

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## Abstract

Herein, we prepared the L-histidine- (His-) protected silver nanoclusters (Ag NCs) by the microwave synthesis method. The synthesis process was rapid, facile, and environmentally friendly. Under 356 nm excitation, the as-prepared Ag NCs exhibited the blue fluorescence, and the fluorescence emission peak was located at 440 nm. The Ag NCs could successfully detect trace copper (Cu2+) ions in the aqueous solution and the limit of detection (LOD) was as low as 0.6 pM. Interestingly, the Ag NCs showed a different pH-dependent selectivity for both Cu2+ and iron (Fe3+) ions with no responses to other heavy metal ions. Furthermore, the as-fabricated fluorescent sensing system was utilized to detect glutathione (GSH, the LOD was 0.8 nM) by using the “switch-on” fluorescence recovery of Ag NCs through adding glutathione (GSH) to the Cu2+-Ag NCs solution.


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## Body

## 1. Introduction

Since heavy metal ions would combine with other toxins in water to produce toxic substances, they could cause serious water pollution leading to a destructive effect on the environment and human health. Among them, iron (Fe3+) and copper (Cu2+) ions are two of the important heavy metal ions. For children and adults, ingest excessive Fe3+ will lead to acute or chronic iron poisoning [1–3]. Excessive Cu2+ has toxic effects on brain neurons and causes serious diseases such as Alzheimer’s disease, Wilson’s disease, and Parkinson’s disease [4–6].Conventional instrumental analysis methods required expensive instrumentation and complicated sample preparations for the trace determination of heavy metal ions [7]. Therefore, finding a fast, sensitive, and selective alternative method is one of the most important research subjects. In recent years, fluorescent nanomaterials have been widely used to develop sensitive fluorescent sensing probes due to their high sensitivity, high selectivity, and wide measurement range [8–13]. Silver nanoclusters (Ag NCs), as novel fluorescent nanomaterials, have been widely used as fluorescent sensing probes in sensing, biorecognition, chemical detection, and catalysis because of their high stability, good water solubility, easy modification, good biocompatibility, and strong bleaching resistance [14–17]. Various preparation methods of Ag NCs including UV-light mediated synthesis and microwave synthesis have been proposed [18–20]. The Ag NCs synthesized with different stabilizing ligands such as DNA, protein, peptide, and amino acid possess different optical properties and application areas [21–26].For the detection, many methods of detection of Cu2+ using the Ag NCs as the probe have been reported. Jing Liu et al. synthesized Ag NCs with PMAA for the detection of Cu2+, and the limit of detection (LOD) was 100 nM [27]. Na Xiao et al. synthesized Ag NCs with glutathione for the detection of Cu2+, and the LOD was 27 nM [28]. Elaheh Babaee et al. synthesized a dual-emissive ratiometric nanohybrid probe for the detection of Cu2+, and the LOD was 7.0 nM [9]. The above-mentioned methods could detect very low concentrations of Cu2+, but it is still of great significance to sensitive detection of Cu2+.In this research, we prepared an Ag NCs fluorescent probe for the detection of Cu2+ and Fe3+ by changing the pH value. The Ag NCs were synthesized using histidine as stabilizer and reductant by microwave synthesis method. Under 356 nm excitation, the Ag NCs solution showed the blue fluorescence with the fluorescent emission peak at 440 nm. The fluorescence intensity response of Ag NCs to the Cu2+ and Fe3+ was used as the detection signal. A simple pH-tuning method could achieve the selective detection of Cu2+ and Fe3+. According to this method, the Ag NCs selectively respond to Cu2+ and Fe3+ at pH = 4.3 and 7, respectively. In addition, as an important tripeptide, glutathione (GSH) has always been a popular detection material [35–37]. Since GSH with thiol bonds could easily form strong complexation with Cu2+ [29], we built a fluorescent sensing system to detect GSH. The fluorescence of the Ag NCs was recovered by forming a strong complex of GSH and Cu2+. It has application prospects in water pollution and dairy product detection.


## 2. Materials and Methods

### 2.1. Chemicals

Silver nitrate (AgNO3, AR), L-histidine (His, BR), nitric acid (HNO3, AR), hydrochloric acid (HCl, AR), copper chloride (CuCl2, AR), barium chloride (BaCl2, AR), calcium chloride (CaCl2, AR), chromium chloride (CrCl3, AR), iron chloride (FeCl3, AR), potassium chloride (KCl, AR), magnesium chloride (MgCl2, AR), manganese chloride (MnCl2, AR), sodium chloride (NaCl, AR), lead chloride (PbCl2, AR), aluminum chloride (AlCl3, AR), sodium dihydrogen phosphate (NaH2PO4, AR), and disodium hydrogen phosphate (Na2HPO4, AR) were purchased from Sinopharm Group Chemical Reagent Co., Ltd. Glutathione reduced (GSH, 99%) and ferrous chloride tetrahydrate (FeCl2·4H2O, AR) were purchased from MACKLIN. Ultrapure water was used throughout all experiments.


### 2.2. Synthesis of Ag NCs

In the experiment, all glassware was thoroughly washed with freshly prepared aqua regia (HCl/HNO3, 3 : 1) and rinsed with ultrapure water prior. In a typical synthesis, an aqueous solution of AgNO3 (0.25 M, 1 mL) was mixed with an aqueous solution of L-histidine (0.125 M, 100 mL) and stirred quickly at room temperature for 10 min. Then the mixture solution was heated (700 W, 2450 MHz) in a microwave oven for 8 min. The color of the solution changed from colorless to light yellow. The solution was then allowed to cool to the ambient temperature before further purification by the dialysis (the dialysis membrane with a molecular mass of 500 Da) for 24 h. The dialyzed solution was freeze-dried to obtain Ag NCs powder and stored in the fridge for further use.


### 2.3. Apparatus and Characterization

Fluorescence emission spectra and excitation spectra were recorded on FLS920 (Edinburgh, Livingston, England). The UV-Vis absorption spectra were measured on the UV-Vis spectrophotometer (Shimadzu, Suzhou, China). The X-ray photoelectron spectra (XPS) were recorded on an ESCALAB 250xi X-ray photoelectron spectrometer (Waltham, MA, USA). Transmission electron microscopy (TEM) image was gained from a JEOL 2100F microscope (Peabody, MA, USA) operating at a maximum acceleration voltage of 200 kV. The domestic microwave oven was from Midea, Guangdong, China.


### 2.4. Quantum Yield Measurements

The quantum yield (QY) of the Ag NCs in water was determined by the reference method. The reference standard material is quinine sulfate (QY = 55% in 0.1 M H2SO4). And the formula of relative QY is as follows:(1)ϕx=AsIs⋅IxAx⋅nx2ns2⋅ϕs,where ϕx is the QY of the Ag NCs and A and I are the absorbance and the integral intensity (excited at 330 nm) of the Ag NCs. n is the refractive index (1.33 in water). The subscript “s” represents quinine sulfate, and “x” represents the Ag NCs. We prepared a series of different concentrations of the Ag NCs and quinine sulfate solution (adjusting the concentration so that their absorbance was between 0 and 0.2) to make the results more accurate.


### 2.5. Detection of Cu2+ and Fe3+ Ions

All fluorescence measurement conditions were set as follows: the excitation and emission slit were 4 nm and 6 nm, respectively, the excitation wavelength was 356 nm, and emission was recorded from 380 nm to 600 nm. The typical detection method of Cu2+ and Fe3+ ions was as follows. The Ag NCs powder was dispersed in water at a concentration of 10 mg/mL for further use. The pH value of the Ag NCs fluorescence probe solution was set at 4.3 for Cu2+ and 7.0 for Fe3+. Then a certain amount of Cu2+ and Fe3+ solution was added, and the fluorescence spectrum was recorded 30 minutes later. The ratio of I0 and I (I0/I or I/I0) was considered to plot the calibration curve and prediction of spiked concentrations, where I0 and I were the fluorescence intensity in the absence and presence of ions. For the selectivity and interference studies, an identical concentration (100 μM) of the stock solution of other metal ions (i.e., Na+, Mg2+, Ca2+, Mn2+, Cu2+, Cd2+, Pb2+, Ba2+, Fe3+, Fe2+, Al3+, and Cr3+) was mixed with the probe solution under the same conditions and recorded the fluorescence spectrum 30 minutes later.


### 2.6. Detection of GSH

All fluorescence measurement conditions were set as above. The typical detection method of GSH was as follows. The Ag NCs solution was prepared with a pH = 4.3; then the stock solution of Cu2+ (1 mM) was mixed. After 30 minutes, we added a certain amount of GSH solution with different concentrations and recorded the fluorescence spectrum 30 minutes later. The difference between I and I0 (I – I0) was considered; that is, the calibration curve and prediction could be drawn, where I0 and I were the fluorescence intensity of the Cu2+-Ag NCs probe system in the absence and presence of GSH.


## 2.1. Chemicals

Silver nitrate (AgNO3, AR), L-histidine (His, BR), nitric acid (HNO3, AR), hydrochloric acid (HCl, AR), copper chloride (CuCl2, AR), barium chloride (BaCl2, AR), calcium chloride (CaCl2, AR), chromium chloride (CrCl3, AR), iron chloride (FeCl3, AR), potassium chloride (KCl, AR), magnesium chloride (MgCl2, AR), manganese chloride (MnCl2, AR), sodium chloride (NaCl, AR), lead chloride (PbCl2, AR), aluminum chloride (AlCl3, AR), sodium dihydrogen phosphate (NaH2PO4, AR), and disodium hydrogen phosphate (Na2HPO4, AR) were purchased from Sinopharm Group Chemical Reagent Co., Ltd. Glutathione reduced (GSH, 99%) and ferrous chloride tetrahydrate (FeCl2·4H2O, AR) were purchased from MACKLIN. Ultrapure water was used throughout all experiments.


## 2.2. Synthesis of Ag NCs

In the experiment, all glassware was thoroughly washed with freshly prepared aqua regia (HCl/HNO3, 3 : 1) and rinsed with ultrapure water prior. In a typical synthesis, an aqueous solution of AgNO3 (0.25 M, 1 mL) was mixed with an aqueous solution of L-histidine (0.125 M, 100 mL) and stirred quickly at room temperature for 10 min. Then the mixture solution was heated (700 W, 2450 MHz) in a microwave oven for 8 min. The color of the solution changed from colorless to light yellow. The solution was then allowed to cool to the ambient temperature before further purification by the dialysis (the dialysis membrane with a molecular mass of 500 Da) for 24 h. The dialyzed solution was freeze-dried to obtain Ag NCs powder and stored in the fridge for further use.


## 2.3. Apparatus and Characterization

Fluorescence emission spectra and excitation spectra were recorded on FLS920 (Edinburgh, Livingston, England). The UV-Vis absorption spectra were measured on the UV-Vis spectrophotometer (Shimadzu, Suzhou, China). The X-ray photoelectron spectra (XPS) were recorded on an ESCALAB 250xi X-ray photoelectron spectrometer (Waltham, MA, USA). Transmission electron microscopy (TEM) image was gained from a JEOL 2100F microscope (Peabody, MA, USA) operating at a maximum acceleration voltage of 200 kV. The domestic microwave oven was from Midea, Guangdong, China.


## 2.4. Quantum Yield Measurements

The quantum yield (QY) of the Ag NCs in water was determined by the reference method. The reference standard material is quinine sulfate (QY = 55% in 0.1 M H2SO4). And the formula of relative QY is as follows:(1)ϕx=AsIs⋅IxAx⋅nx2ns2⋅ϕs,where ϕx is the QY of the Ag NCs and A and I are the absorbance and the integral intensity (excited at 330 nm) of the Ag NCs. n is the refractive index (1.33 in water). The subscript “s” represents quinine sulfate, and “x” represents the Ag NCs. We prepared a series of different concentrations of the Ag NCs and quinine sulfate solution (adjusting the concentration so that their absorbance was between 0 and 0.2) to make the results more accurate.


## 2.5. Detection of Cu2+ and Fe3+ Ions

All fluorescence measurement conditions were set as follows: the excitation and emission slit were 4 nm and 6 nm, respectively, the excitation wavelength was 356 nm, and emission was recorded from 380 nm to 600 nm. The typical detection method of Cu2+ and Fe3+ ions was as follows. The Ag NCs powder was dispersed in water at a concentration of 10 mg/mL for further use. The pH value of the Ag NCs fluorescence probe solution was set at 4.3 for Cu2+ and 7.0 for Fe3+. Then a certain amount of Cu2+ and Fe3+ solution was added, and the fluorescence spectrum was recorded 30 minutes later. The ratio of I0 and I (I0/I or I/I0) was considered to plot the calibration curve and prediction of spiked concentrations, where I0 and I were the fluorescence intensity in the absence and presence of ions. For the selectivity and interference studies, an identical concentration (100 μM) of the stock solution of other metal ions (i.e., Na+, Mg2+, Ca2+, Mn2+, Cu2+, Cd2+, Pb2+, Ba2+, Fe3+, Fe2+, Al3+, and Cr3+) was mixed with the probe solution under the same conditions and recorded the fluorescence spectrum 30 minutes later.


## 2.6. Detection of GSH

All fluorescence measurement conditions were set as above. The typical detection method of GSH was as follows. The Ag NCs solution was prepared with a pH = 4.3; then the stock solution of Cu2+ (1 mM) was mixed. After 30 minutes, we added a certain amount of GSH solution with different concentrations and recorded the fluorescence spectrum 30 minutes later. The difference between I and I0 (I – I0) was considered; that is, the calibration curve and prediction could be drawn, where I0 and I were the fluorescence intensity of the Cu2+-Ag NCs probe system in the absence and presence of GSH.


## 3. Results and Discussion

### 3.1. Characterization of Ag NCs

The fluorescent Ag NCs were prepared by the microwave method with the His as the reductant and stabilizer. The product exhibited a light-yellow solution. In the synthesis process of the Ag NCs, Ag+ was reduced to Ag0 by the imidazolyl groups of the His, and the carboxyl group played an important role in protecting the silver core [8]. All reactants were nontoxic, and the synthesis method was a simple and green process, as shown in Scheme 1. TEM image showed that the Ag NCs were the monodisperse spheres with an average particle size of 4.15 ± 0.8 nm (Figure 1). In the HR-TEM image, obvious lattice fringes could be seen with a lattice distance of 2.27 Å (Figure S1).Scheme 1
Schematics of the formation of the Ag NCs.Figure 1
(a) TEM images of the Ag NCs. (b) The particle size distribution histogram of the Ag NCs.
(a)(b)By XPS, surface composition and elemental analysis for the Ag NCs were characterized. The four peaks of the Ag NCs at 285, 368, 400, and 531 eV (Figure2(a)) could be attributed to C1s, Ag3d, N1s, and O1s, respectively. The results showed that the Ag NCs were mainly composed of C, O, N, and Ag. As shown in Figure 2(b), the two peaks at about 367.8 and 373.9 eV were assigned to the Ag 3d levels. These two peaks could be deconvoluted into four distinct component peaks. The peaks at 368.8 eV (3d5/2) and 375.2 eV (3d3/2) shown by green line and light blue line were assigned to Ag+. The other peaks at 367.7 eV (3d5/2) and 373.8 eV (3d3/2) shown by red and dark blue lines were assigned to Ag0. Ag0 and Ag+ could be the core and Ag ions on the surface of the Ag NCs, respectively. Ag ions played an important role in adsorbing the His to stabilize the Ag NCs. The N1s XPS spectrum could be deconvoluted into three distinct component peaks (Figure 2(c)). The peaks at 398 eV, 400 eV, and 408.1 eV shown by red line, green line, and blue line were attributed to C-N, N-H, and -NO2. The peak that appeared at 408.1 eV showed that some of the amino groups of L-histidine reduced Ag+ to Ag0 and the amino groups were oxidized to -NO2 groups [26].Figure 2
(a) XPS survey spectrum of the Ag NCs. (b-c) High-resolution XPS spectra of Ag3d and N1s, respectively.
(a)(b)(c)


### 3.2. The Optical Properties of the Ag NCs

Optical properties including UV-Vis absorption, fluorescence excitation and emission, and time-resolved fluorescence spectra of the Ag NCs were investigated. As shown in Figure3(a), the absorption peak at about 300 nm of AgNO3 disappeared, while a new absorption band appeared at 250–300 nm. In addition, the absorption peak near 411 nm was the characteristic surface Plasmon resonance (SPR) peak of large-size silver nanoparticles [30, 31]. There was no absorption peak emerging around 411 nm, as Figure 3(a) shows. Therefore, it could prove that the product was the Ag NCs with a core diameter of less than 2 nm. The fluorescence spectra of the Ag NCs solution, the His solution, and the solution mixed with His and AgNO3 were compared under the excitation at 365 nm. The fluorescence of the His solution and the solution mixed with His and AgNO3 could not be observed under the same excitation (Figure S2). Therefore, the Ag NCs were the fluorescence substance in the solution. As shown in Figure 3(b), the fluorescence emission reached the maximum at 356 nm excitation with the emission maximum at 440 nm. In addition, the Ag NCs solution under natural light had no obvious luminescence, while under ultraviolet (365 nm), it exhibited the blue fluorescence. Using quinine sulfate in 0.1 M H2SO4 as a reference, the QY was 5.2%. Time-resolved fluorescence spectrum (Figure 3(c)) showed that the fluorescence lifetime of the Ag NCs was 1.13 ns (13.87%), 3.98 ns (51.57%), and 8.25 ns (34.56%), and the average lifetime was 5.06 ns (Table S1).Figure 3
(a) UV-Vis absorption spectrum of His (black), AgNO3 (red), and the Ag NCs (blue) solution. (b) The PL excitation and emission spectra of the Ag NCs. The inset in (b) is the Ag NCs solution under natural light (left) and under ultraviolet (right). (c) Time-resolved fluorescence spectrum of the Ag NCs.
(a)(b)(c)


### 3.3. Effects of Solution pH on Detection of Cu2+ and Fe3+ by the Ag NCs

The influence of the solution pH was examined to optimize the sensing conditions. It was found that the sensitivity and selectivity of the Ag NCs detection of Cu2+ and Fe3+ were dependent on the pH value. The Ag NCs had a high response to Cu2+ at pH = 4.3 (Figure 4(a)) and to Fe3+ at pH = 7 (Figure 4(b)). And at those two pH value conditions, the effect of other metal ions on the fluorescence intensity of the Ag NCs was very small. Therefore, pH = 4.3 and pH = 7 were selected as the detection condition for Cu2+ and Fe3+ ions, respectively. The results showed that the selectivity to Cu2+ and Fe3+ could be achieved by controlling the pH value of the Ag NCs fluorescent probe solution without adding other chelating agents.Figure 4
Selectivity patterns of the Ag NCs to Cu2+ at pH = 4.3 (a) and Fe3+ at pH = 7 (b). All metal ions concentrations were 100 μM.
(a)(b)After adding Cu2+ and Fe3+ ions, the Ag NCs solution exhibited different responses under different pH value. It could be attributed to the different binding mechanism between the two ions and His. According to the previous reports, His presented a strong binding affinity with Cu2+ via coordinating Cu2+ through the amino group and the imidazole ring [32]. The terminal amino and the imidazole nitrogen donors could form a six-membered chelate. In the presence of excess ligand, bis(ligand) complex could be formed in slightly acidic samples. When the pH increased, the amino group and the imidazole ring were deprotonated [33]. It could decrease the Cu2+-binding affinity with His. In addition, the Fe3+ could coordinate with carboxyl groups of the surface ligand. In acidic media, the carboxyl groups were easily protonated at a low pH value. Hence, neutral media were the most suitable condition for Ag NCs detecting Fe3+ [8, 34]. In alkaline media, OH− and Fe3+ (Cu2+) could form precipitation. It could result in complexing capability getting weak. Hence, when pH = 4.3 and 7, the Ag NCs showed a high response to Cu2+ and Fe3+, respectively.


### 3.4. Calibration Curves and Detection Limits for Sensing Cu2+ and Fe3+

The above-mentioned optimal pH conditions for the Cu2+ and Fe3+ were then employed to construct their calibration curves based on the as-prepared Ag NCs. When the concentration of Cu2+ was less than 1 × 10−6 M (Figure 5(a)), the fluorescence intensity gradually increased with the increasing concentration of the Cu2+. When the concentration of Cu2+ was more than 1 × 10−3 M, the fluorescence intensity decreased with the increasing of the concentration of Cu2+ (Figure S3). Figure 5 showed that the fluorescence emission intensity ratio was sensitively and proportionately increased with an increasing concentration of Cu2+ at 440 nm. A linear relationship from 1 × 10−12∼1 × 10−6 M could be described (I/I0) = 0.0155 [log C] + 1.294 (R2 = 0.9897). And the LOD for Cu2+ was as low as 0.6 pM.Figure 5
(a) Fluorescence response of the Ag NCs upon addition of different concentrations of Cu2+ from bottom to top: 0.1 × 10−12 M, 1 × 10−11 M, 1 × 10−10 M, 1 × 10−9 M, 1 × 10−8 M, 1 × 10−7 M, and 1 × 10−6 M. (b) The linear calibration ranges of the fluorescence intensity ratio to Cu2+ concentrations.
(a)(b)When the pH = 7 (Figure6(a)), the fluorescence intensity decreased with the increasing concentration of Fe3+ ions. The fluorescence intensity ratio of the Ag NCs at pH = 7 (Figure 6(b)) possessed a linear relationship with the concentration of Fe3+ from 100∼1000 μM with a regression equation of (I0/I) = 0.0008 [CFe3+] + 0.9353 (R2 = 0.9944). The LOD for Fe3+ is 9.8 μM.Figure 6
(a) Fluorescence response of the Ag NCs upon addition of different concentrations of Fe3+ from top to bottom: 0 mM, 0.1 mM, 0.2 mM, 0.4 mM, 0.6 mM, 0.8 mM, 1 mM, and 5 mM. (b) The linear calibration ranges of the fluorescence intensity ratio to Fe3+ concentrations.
(a)(b)The Ag NCs showed different response mechanism for the above two Cu2+ concentration ranges. The fluorescence of Ag NCs was enhanced at low concentration (under 1 μM) of Cu2+ and quenched at high concentration (over 1 mM) of Cu2+. The different response of the Ag NCs toward the two kind Cu2+ concentration ranges could be attributed to the different binding degree of Cu2+ with His. As shown in Figure S4, after adding two concentrations of Cu2+ (1 mM and 1 nM), the lifetimes of the Ag NCs samples changed. It proved that the fluorescence dynamic quenching of the Ag NCs due to the Cu2+ was binding to His. The proposed mechanisms for the interaction of the Ag NCs by two concentrations of Cu2+ were further confirmed by the analysis of UV-Vis absorption spectrum and TEM images. As shown in Figure 7, when the concentration of Cu2+ was 1 mM, the aggregation of the Ag NCs was indicated because the absorbance of the absorption peak at 250–300 nm was enhanced. The aggregation of the Ag NCs in the presence of Cu2+ (Figure S5(a)) could be attributed to the interaction between the His shell of the Ag NCs and Cu2+. The quenched fluorescence could be attributed to aggregation of the Ag NCs by the simple coordination of Cu2+ with the amino groups of the His ligand. When the concentration of Cu2+ was 1 nM, it was indicated that the Ag NCs were without aggregation because the absorption spectra at 250–300 nm were unchanged (Figure 7). It could be seen that the Ag NCs still have good dispersion in the presence of Cu2+ (Figure S5(b)). This might be due to the fact that the low concentration of Cu2+ was not enough to induce the Ag NCs aggregation. In addition, the absorption peak of the π bond groups at 200 nm decreased (Figure 7). It could be attributed to the addition of Cu2+ which only improved the spatial structure of the functional groups on the surface of the Ag NCs. It was supposed that the fluorescence intensity was enhanced due to the spatial structural change of the Ag NCs.Figure 7
UV-Vis absorption spectrum of the Ag NCs upon addition of different concentrations of Cu2+.


### 3.5. Application to Water Samples

As mentioned above, the fluorescence of Ag NCs was enhanced in the presence of the Cu2+ at a low concentration without interference from other ions. Therefore, the proposed probe could be used for the detection of Cu2+ in real samples. To verify this, mineral water samples spiked with different concentrations of Cu2+ were examined. Table 1 summarizes the obtained results for the prediction of the concentrations of Cu2+. As shown in Table 1, there was an excellent percent recovery for Cu2+ in the mineral water samples. Therefore, the probe displayed high capability for the determination of the copper ions in real samples.Table 1
Application of the probe for Cu2+ in real samples (data are average of three replicate measurements).
SampleCu2+ (pM)Recovery (%)AddedFoundMineral water11.07 (±0.1)107100103 (±11)1031000010073 (±110)101


### 3.6. Comparison of the Ag NCs with Previously Reported Fluorescent Nanoprobes

The merits such as linear range, detection limit, and selectivity of the proposed Ag NCs for Cu2+ and Fe3+ are summarized in Table 2. Compared with other fluorescent probes reported for the determination of two ions, according to Table 2, the reported probes’ selective determination of two ions in the aqueous solution was to mask one of these ions with a masking agent such as EDTA. In most cases, EDTA as an efficient Cu2+ and Fe3+ chelating agent could not be used to selectively determine Cu2+ and Fe3+.Table 2
Comparison of Ag NCs probe for the detection of Cu2+ and Fe3+ with previously reported fluorescent-based probes in aqueous solution.
ProbeDetected ionLinear rangeLOD (nM)SelectivityRefAg NCsCu2+—10With no selectivity over ions even with the help of EDTA[38]Hg2+0∼100 nM5Au-Ag NCsCu2+0.5 nM∼2.5μM0.3Selective determination of Hg2+ by masking Cu2+ with EDTA[39]Hg2+0.2 nM∼2.5μM0.1Ag NCsCu2+0.1 nM∼20μM28Masking Hg2+ with thymine and masking Cu2+ with potassium pyrophosphate[40]Hg2+0.1 nM∼10μM35Ag NCsCu2+0∼1μM2.8Masking Cu2+ with EDTA for selective determination of Hg2+[41]Hg2+0.01∼0.5μM1.0Ag NCsFe3+0.5μM∼20 μM0.12—[42]Au NCs and Au/Ag NCsFe3+5μM∼1 mM1.11Masking Fe3+ with EDTA for selective determination of Hg2+[34]Hg2+5 nM∼5μM1.56Ag NCsCu2+1 pM∼1 mM0.6Switching the selectivity by controlling solution pH (pH = 4.3 for Cu2+ determination and pH = 7 for Fe3+ determination)This workFe3+100∼1000μM9.8This work proposed a method for selective determination of Cu2+ and Fe3+ only by controlling the solution pH value. For example, in the acidic media, the easier protonation of carboxyl groups at low pH value resulted in the inability of Fe3+ to coordinate with carboxyl groups. Therefore, Fe3+ could not disturb the determination of Cu2+ ions.


### 3.7. Calibration Curves and Detection Limits for Sensing of GSH

Since GSH is a very important antioxidant, Cu2+-Ag NCs sensing system was further utilized for the detection of GSH. A “switch-on” effect on recovery of the fluorescence of Ag NCs could be expressed in this sensing system. A wide detection range and low detection limit could be obtained by this method. As shown in Figure 8, the fluorescence intensity of the Ag NCs was obviously recovered at 440 nm. A linear relationship for GSH was obtained from 1 nM to 8 mM with a regression equation of (I – I0) = 12107.6–1234.49(−log10 (C)) (R2 = 0.9888), where I0 and I were the fluorescence intensity of the Cu2+-Ag NCs probe system in the absence and presence of GSH. The LOD for GSH was 0.8 nM.Figure 8
(a) GSH from bottom to top: 0 nM, 1 nM, 5 nM, 10 nM, 50 nM, 100 nM, 500 nM, 1μM, 10 μM, 100 μM, 500 μM, 800 μM, 1 mM, 5 mM, and 8 mM. (b) The linear calibration ranges of the fluorescence recovery to GSH concentrations.
(a)(b)


## 3.1. Characterization of Ag NCs

The fluorescent Ag NCs were prepared by the microwave method with the His as the reductant and stabilizer. The product exhibited a light-yellow solution. In the synthesis process of the Ag NCs, Ag+ was reduced to Ag0 by the imidazolyl groups of the His, and the carboxyl group played an important role in protecting the silver core [8]. All reactants were nontoxic, and the synthesis method was a simple and green process, as shown in Scheme 1. TEM image showed that the Ag NCs were the monodisperse spheres with an average particle size of 4.15 ± 0.8 nm (Figure 1). In the HR-TEM image, obvious lattice fringes could be seen with a lattice distance of 2.27 Å (Figure S1).Scheme 1
Schematics of the formation of the Ag NCs.Figure 1
(a) TEM images of the Ag NCs. (b) The particle size distribution histogram of the Ag NCs.
(a)(b)By XPS, surface composition and elemental analysis for the Ag NCs were characterized. The four peaks of the Ag NCs at 285, 368, 400, and 531 eV (Figure2(a)) could be attributed to C1s, Ag3d, N1s, and O1s, respectively. The results showed that the Ag NCs were mainly composed of C, O, N, and Ag. As shown in Figure 2(b), the two peaks at about 367.8 and 373.9 eV were assigned to the Ag 3d levels. These two peaks could be deconvoluted into four distinct component peaks. The peaks at 368.8 eV (3d5/2) and 375.2 eV (3d3/2) shown by green line and light blue line were assigned to Ag+. The other peaks at 367.7 eV (3d5/2) and 373.8 eV (3d3/2) shown by red and dark blue lines were assigned to Ag0. Ag0 and Ag+ could be the core and Ag ions on the surface of the Ag NCs, respectively. Ag ions played an important role in adsorbing the His to stabilize the Ag NCs. The N1s XPS spectrum could be deconvoluted into three distinct component peaks (Figure 2(c)). The peaks at 398 eV, 400 eV, and 408.1 eV shown by red line, green line, and blue line were attributed to C-N, N-H, and -NO2. The peak that appeared at 408.1 eV showed that some of the amino groups of L-histidine reduced Ag+ to Ag0 and the amino groups were oxidized to -NO2 groups [26].Figure 2
(a) XPS survey spectrum of the Ag NCs. (b-c) High-resolution XPS spectra of Ag3d and N1s, respectively.
(a)(b)(c)


## 3.2. The Optical Properties of the Ag NCs

Optical properties including UV-Vis absorption, fluorescence excitation and emission, and time-resolved fluorescence spectra of the Ag NCs were investigated. As shown in Figure3(a), the absorption peak at about 300 nm of AgNO3 disappeared, while a new absorption band appeared at 250–300 nm. In addition, the absorption peak near 411 nm was the characteristic surface Plasmon resonance (SPR) peak of large-size silver nanoparticles [30, 31]. There was no absorption peak emerging around 411 nm, as Figure 3(a) shows. Therefore, it could prove that the product was the Ag NCs with a core diameter of less than 2 nm. The fluorescence spectra of the Ag NCs solution, the His solution, and the solution mixed with His and AgNO3 were compared under the excitation at 365 nm. The fluorescence of the His solution and the solution mixed with His and AgNO3 could not be observed under the same excitation (Figure S2). Therefore, the Ag NCs were the fluorescence substance in the solution. As shown in Figure 3(b), the fluorescence emission reached the maximum at 356 nm excitation with the emission maximum at 440 nm. In addition, the Ag NCs solution under natural light had no obvious luminescence, while under ultraviolet (365 nm), it exhibited the blue fluorescence. Using quinine sulfate in 0.1 M H2SO4 as a reference, the QY was 5.2%. Time-resolved fluorescence spectrum (Figure 3(c)) showed that the fluorescence lifetime of the Ag NCs was 1.13 ns (13.87%), 3.98 ns (51.57%), and 8.25 ns (34.56%), and the average lifetime was 5.06 ns (Table S1).Figure 3
(a) UV-Vis absorption spectrum of His (black), AgNO3 (red), and the Ag NCs (blue) solution. (b) The PL excitation and emission spectra of the Ag NCs. The inset in (b) is the Ag NCs solution under natural light (left) and under ultraviolet (right). (c) Time-resolved fluorescence spectrum of the Ag NCs.
(a)(b)(c)


## 3.3. Effects of Solution pH on Detection of Cu2+ and Fe3+ by the Ag NCs

The influence of the solution pH was examined to optimize the sensing conditions. It was found that the sensitivity and selectivity of the Ag NCs detection of Cu2+ and Fe3+ were dependent on the pH value. The Ag NCs had a high response to Cu2+ at pH = 4.3 (Figure 4(a)) and to Fe3+ at pH = 7 (Figure 4(b)). And at those two pH value conditions, the effect of other metal ions on the fluorescence intensity of the Ag NCs was very small. Therefore, pH = 4.3 and pH = 7 were selected as the detection condition for Cu2+ and Fe3+ ions, respectively. The results showed that the selectivity to Cu2+ and Fe3+ could be achieved by controlling the pH value of the Ag NCs fluorescent probe solution without adding other chelating agents.Figure 4
Selectivity patterns of the Ag NCs to Cu2+ at pH = 4.3 (a) and Fe3+ at pH = 7 (b). All metal ions concentrations were 100 μM.
(a)(b)After adding Cu2+ and Fe3+ ions, the Ag NCs solution exhibited different responses under different pH value. It could be attributed to the different binding mechanism between the two ions and His. According to the previous reports, His presented a strong binding affinity with Cu2+ via coordinating Cu2+ through the amino group and the imidazole ring [32]. The terminal amino and the imidazole nitrogen donors could form a six-membered chelate. In the presence of excess ligand, bis(ligand) complex could be formed in slightly acidic samples. When the pH increased, the amino group and the imidazole ring were deprotonated [33]. It could decrease the Cu2+-binding affinity with His. In addition, the Fe3+ could coordinate with carboxyl groups of the surface ligand. In acidic media, the carboxyl groups were easily protonated at a low pH value. Hence, neutral media were the most suitable condition for Ag NCs detecting Fe3+ [8, 34]. In alkaline media, OH− and Fe3+ (Cu2+) could form precipitation. It could result in complexing capability getting weak. Hence, when pH = 4.3 and 7, the Ag NCs showed a high response to Cu2+ and Fe3+, respectively.


## 3.4. Calibration Curves and Detection Limits for Sensing Cu2+ and Fe3+

The above-mentioned optimal pH conditions for the Cu2+ and Fe3+ were then employed to construct their calibration curves based on the as-prepared Ag NCs. When the concentration of Cu2+ was less than 1 × 10−6 M (Figure 5(a)), the fluorescence intensity gradually increased with the increasing concentration of the Cu2+. When the concentration of Cu2+ was more than 1 × 10−3 M, the fluorescence intensity decreased with the increasing of the concentration of Cu2+ (Figure S3). Figure 5 showed that the fluorescence emission intensity ratio was sensitively and proportionately increased with an increasing concentration of Cu2+ at 440 nm. A linear relationship from 1 × 10−12∼1 × 10−6 M could be described (I/I0) = 0.0155 [log C] + 1.294 (R2 = 0.9897). And the LOD for Cu2+ was as low as 0.6 pM.Figure 5
(a) Fluorescence response of the Ag NCs upon addition of different concentrations of Cu2+ from bottom to top: 0.1 × 10−12 M, 1 × 10−11 M, 1 × 10−10 M, 1 × 10−9 M, 1 × 10−8 M, 1 × 10−7 M, and 1 × 10−6 M. (b) The linear calibration ranges of the fluorescence intensity ratio to Cu2+ concentrations.
(a)(b)When the pH = 7 (Figure6(a)), the fluorescence intensity decreased with the increasing concentration of Fe3+ ions. The fluorescence intensity ratio of the Ag NCs at pH = 7 (Figure 6(b)) possessed a linear relationship with the concentration of Fe3+ from 100∼1000 μM with a regression equation of (I0/I) = 0.0008 [CFe3+] + 0.9353 (R2 = 0.9944). The LOD for Fe3+ is 9.8 μM.Figure 6
(a) Fluorescence response of the Ag NCs upon addition of different concentrations of Fe3+ from top to bottom: 0 mM, 0.1 mM, 0.2 mM, 0.4 mM, 0.6 mM, 0.8 mM, 1 mM, and 5 mM. (b) The linear calibration ranges of the fluorescence intensity ratio to Fe3+ concentrations.
(a)(b)The Ag NCs showed different response mechanism for the above two Cu2+ concentration ranges. The fluorescence of Ag NCs was enhanced at low concentration (under 1 μM) of Cu2+ and quenched at high concentration (over 1 mM) of Cu2+. The different response of the Ag NCs toward the two kind Cu2+ concentration ranges could be attributed to the different binding degree of Cu2+ with His. As shown in Figure S4, after adding two concentrations of Cu2+ (1 mM and 1 nM), the lifetimes of the Ag NCs samples changed. It proved that the fluorescence dynamic quenching of the Ag NCs due to the Cu2+ was binding to His. The proposed mechanisms for the interaction of the Ag NCs by two concentrations of Cu2+ were further confirmed by the analysis of UV-Vis absorption spectrum and TEM images. As shown in Figure 7, when the concentration of Cu2+ was 1 mM, the aggregation of the Ag NCs was indicated because the absorbance of the absorption peak at 250–300 nm was enhanced. The aggregation of the Ag NCs in the presence of Cu2+ (Figure S5(a)) could be attributed to the interaction between the His shell of the Ag NCs and Cu2+. The quenched fluorescence could be attributed to aggregation of the Ag NCs by the simple coordination of Cu2+ with the amino groups of the His ligand. When the concentration of Cu2+ was 1 nM, it was indicated that the Ag NCs were without aggregation because the absorption spectra at 250–300 nm were unchanged (Figure 7). It could be seen that the Ag NCs still have good dispersion in the presence of Cu2+ (Figure S5(b)). This might be due to the fact that the low concentration of Cu2+ was not enough to induce the Ag NCs aggregation. In addition, the absorption peak of the π bond groups at 200 nm decreased (Figure 7). It could be attributed to the addition of Cu2+ which only improved the spatial structure of the functional groups on the surface of the Ag NCs. It was supposed that the fluorescence intensity was enhanced due to the spatial structural change of the Ag NCs.Figure 7
UV-Vis absorption spectrum of the Ag NCs upon addition of different concentrations of Cu2+.


## 3.5. Application to Water Samples

As mentioned above, the fluorescence of Ag NCs was enhanced in the presence of the Cu2+ at a low concentration without interference from other ions. Therefore, the proposed probe could be used for the detection of Cu2+ in real samples. To verify this, mineral water samples spiked with different concentrations of Cu2+ were examined. Table 1 summarizes the obtained results for the prediction of the concentrations of Cu2+. As shown in Table 1, there was an excellent percent recovery for Cu2+ in the mineral water samples. Therefore, the probe displayed high capability for the determination of the copper ions in real samples.Table 1
Application of the probe for Cu2+ in real samples (data are average of three replicate measurements).
SampleCu2+ (pM)Recovery (%)AddedFoundMineral water11.07 (±0.1)107100103 (±11)1031000010073 (±110)101


## 3.6. Comparison of the Ag NCs with Previously Reported Fluorescent Nanoprobes

The merits such as linear range, detection limit, and selectivity of the proposed Ag NCs for Cu2+ and Fe3+ are summarized in Table 2. Compared with other fluorescent probes reported for the determination of two ions, according to Table 2, the reported probes’ selective determination of two ions in the aqueous solution was to mask one of these ions with a masking agent such as EDTA. In most cases, EDTA as an efficient Cu2+ and Fe3+ chelating agent could not be used to selectively determine Cu2+ and Fe3+.Table 2
Comparison of Ag NCs probe for the detection of Cu2+ and Fe3+ with previously reported fluorescent-based probes in aqueous solution.
ProbeDetected ionLinear rangeLOD (nM)SelectivityRefAg NCsCu2+—10With no selectivity over ions even with the help of EDTA[38]Hg2+0∼100 nM5Au-Ag NCsCu2+0.5 nM∼2.5μM0.3Selective determination of Hg2+ by masking Cu2+ with EDTA[39]Hg2+0.2 nM∼2.5μM0.1Ag NCsCu2+0.1 nM∼20μM28Masking Hg2+ with thymine and masking Cu2+ with potassium pyrophosphate[40]Hg2+0.1 nM∼10μM35Ag NCsCu2+0∼1μM2.8Masking Cu2+ with EDTA for selective determination of Hg2+[41]Hg2+0.01∼0.5μM1.0Ag NCsFe3+0.5μM∼20 μM0.12—[42]Au NCs and Au/Ag NCsFe3+5μM∼1 mM1.11Masking Fe3+ with EDTA for selective determination of Hg2+[34]Hg2+5 nM∼5μM1.56Ag NCsCu2+1 pM∼1 mM0.6Switching the selectivity by controlling solution pH (pH = 4.3 for Cu2+ determination and pH = 7 for Fe3+ determination)This workFe3+100∼1000μM9.8This work proposed a method for selective determination of Cu2+ and Fe3+ only by controlling the solution pH value. For example, in the acidic media, the easier protonation of carboxyl groups at low pH value resulted in the inability of Fe3+ to coordinate with carboxyl groups. Therefore, Fe3+ could not disturb the determination of Cu2+ ions.


## 3.7. Calibration Curves and Detection Limits for Sensing of GSH

Since GSH is a very important antioxidant, Cu2+-Ag NCs sensing system was further utilized for the detection of GSH. A “switch-on” effect on recovery of the fluorescence of Ag NCs could be expressed in this sensing system. A wide detection range and low detection limit could be obtained by this method. As shown in Figure 8, the fluorescence intensity of the Ag NCs was obviously recovered at 440 nm. A linear relationship for GSH was obtained from 1 nM to 8 mM with a regression equation of (I – I0) = 12107.6–1234.49(−log10 (C)) (R2 = 0.9888), where I0 and I were the fluorescence intensity of the Cu2+-Ag NCs probe system in the absence and presence of GSH. The LOD for GSH was 0.8 nM.Figure 8
(a) GSH from bottom to top: 0 nM, 1 nM, 5 nM, 10 nM, 50 nM, 100 nM, 500 nM, 1μM, 10 μM, 100 μM, 500 μM, 800 μM, 1 mM, 5 mM, and 8 mM. (b) The linear calibration ranges of the fluorescence recovery to GSH concentrations.
(a)(b)


## 4. Conclusions

In this research, L-histidine-protected Ag NCs were prepared by the simple synthesis process. The Ag NCs were the monodisperse spheres with an average particle size of 4.15 nm. The Ag NCs could be utilized for selective detection of Cu2+ and Fe3+ by simply adjusting the pH value of the Ag NCs solution without using the masking agent. And a fluorescent sensing system was further built for the detection of GSH. Based on the above method, a low LOD of the Cu2+ and GSH could be obtained.



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