**3. Results and Discussion**

## *3.1. Characterization of MPA-InP/ZnS QDs*

As shown in Figure 1, the synthesized MPA-InP/ZnS QDs were spherical and well dispersed in water solution. The particle size of MPA-InP/ZnS QDs was distributed in a relatively narrow range of 1.87–5.86 nm, with an average size of 3.21 nm (inset in Figure 1a). The MPA-InP/ZnS QDs had an obvious lattice with a lattice spacing of 0.23 nm, as shown in Figure 1b, and the charge couple device (CCD) image revealed that InP/ZnS QDs have round shape and excellent light-emitting characteristics.

**Figure 1.** TEM (**a**) and HRTEM images (**b**) of MPA-InP/ZnS QDs (inset: particle size distribution (**i**) and CCD image of MPA-InP/ZnS QDs (**ii**)). TEM: transmission electron microscopy, HRTEM: high resolution transmission electron microscope, MPA-InP/ZnS QDs: mercaptopropionic acid capped InP/ZnS quantum dots, CCD: charge couple device.

FT-IR spectra were obtained to investigate the functional groups on the surface of MPA-InP/ZnS QDs. As shown in Figure 2a, the FT-IR spectra of MPA (red curve) displays that the absorption bands at 3142 cm<sup>−</sup><sup>1</sup> and 1701 cm<sup>−</sup><sup>1</sup> are attributed to the stretching vibration of the O–H and the C=O of COOH, respectively. The FT-IR spectra of MPA-InP/ZnS QDs (black curve) shows the absorption band at 3396 cm<sup>−</sup><sup>1</sup> is attributed to the stretching vibration of the O–H band of COOH. The peaks at 1640, 1568, 1486, and 1396 cm<sup>−</sup><sup>1</sup> are caused by the stretching vibrations of the C=O, C–H, –NH, and –OH bonds, respectively. The presence of carboxyl hydroxyl and amine groups was demonstrated on the surface of the MPA-InP/ZnS QDs [23], which are beneficial to the homogenous and stable dispersion of MPA-InP/ZnS QDs in water [24] and indicating the InP/ZnS QDs are successfully capped by MPA. XPS spectra were recorded to investigate the surface elements of the MPA-InP/ZnS QDs. As shown in Figure 2b, the XPS spectrum of MPA-InP/ZnS

QDs shows three peaks at 284.8, 402.4, and 532.0 eV, which correspond to the C1s, N1s, and O1s orbitals with relative atomic percentages of 73.96%, 5.01%, and 21.03%, respectively. The high-resolution C1s and O1s XPS spectra, as shown in Figure 2c,d, demonstrate the presence of C–C/C–H, C–O–H/C–O–N, O–H, and C=O, which agrees with the FT-IR spectra. The XPS spectra shows that the MPA-InP/ZnS QDs contain a large number of carboxyl groups, which further indicates that the InP/ZnS QDs are successfully capped by MPA.

**Figure 2.** FT-IR spectrum (**a**), XPS patterns (**b**), C1s XPS spectrum (**c**), and O1s XPS spectrum (**d**) of MPA-InP/ZnS QDs. XPS: X-ray photoelectron spectroscopy, C1s XPS: X-ray photoelectron spectroscopy of 1s orbital electron peak of carbon atom, O1s XPS: X-ray photoelectron spectroscopy of 1s orbital electron peak of oxygen atom, MPA-InP/ZnS QDs: mercaptopropionic acid capped InP/ZnS quantum dots.

The fluorescence excitation and emission spectra of the MPA-InP/ZnS QDs were obtained to investigate the fluorescence properties. As shown in Figure 3a, the MPA-InP/ZnS QDs show an emission wavelength at 520 nm with an excitation wavelength of 250 nm. The MPA-InP/ZnS QDs are pale yellow in daylight and exhibit strong yellow color under UV light (365 nm), as shown in the insert of Figure 3a. An obvious emission peak located at approximately 520 nm with excitation wavelength scanning from 200 nm to 300 nm can be observed in Figure 3b, indicating that the emission is independent of the excitation wavelength. The quantum yield of purified MPA-InP/ZnS QDs was measured by a fluorescence spectrometer with an integrating sphere to be 12.05%.

**Figure 3.** The absorption spectrum, excitation, and emission spectroscopy (**a**). (insert: photos of MPA-InP/ZnS QDs solutions under sunlight (**i**) and UV light (365 nm)) (**ii**) Emission spectroscopy with different excitation of MPA-InP/ZnS QDs (**b**). MPA-InP/ZnS QDs: mercaptopropionic acid capped InP/ZnS quantum dots.

#### *3.2. Detection of Cu2+ by Utilizing the MPA-InP/ZnS QDs*

The influence of pH, the concentration of MPA-InP/ZnS QDs, and the reaction time were investigated to optimize the conditions of reaction conditions for the detection of Cu2+. As shown in Figure 4a, the ratio of the fluorescence quenching of the MPA-InP/ZnS QDs with Cu2+ (50 nM, 100 μL) added in PBS buffer at different pH values (2–10) was recorded, and the fluorescence was quenched most obviously at pH = 8.0. Thus, the value of 8.0 was used as the optimum pH. Figure 4b shows that the fluorescence intensity of MPA-InP/ZnS QDs at a concentration of 14 nM was the strongest, which means that the quenching degree may be the greatest in the presence of same concentration of Cu2+. In order to investigate this issue further, the fluorescence intensity of MPA-InP/ZnS QDs in the concentration of 6 nM, 10 nM, 14 nM, and 18 nM in the presence of different concentrations of Cu2+ were measured. As shown in Figure S1 of Supplementary Materials, in the concentrations of Cu2+ of 0–1000 nmol/L, the MPA-InP/ZnS QDs solution with a concentration of 14 nM has the greatest degree of fluorescence quenching. The results indicated that 14 nM is the best concentration. Furthermore, the time-based fluorescence behavior of the MPA-InP/ZnS QDs with added Cu2+ was studied, and it is shown in Figure 4c that the fluorescence was stable after 12 min. Therefore, we chose 12 min as the incubation time for the complete reaction of MPA-InP/ZnS QDs and Cu2+. All the following experiments were performed under the optimum conditions.

The fluorescence intensity of MPA-InP/ZnS QDs solutions with different concentrations of Cu2+ was measured at λex = 250 nm to explore the sensitivity in terms of detecting Cu2+ in water. Figure 5a shows a remarkable decrease in fluorescence intensity at 520 nm with increasing concentrations of Cu2+. The insets of Figure 5a shows photos of MPA-InP/ZnS QD solutions with different concentrations of Cu2+ irradiated by a UV lamp (365 nm). The color of the MPA-InP/ZnS QDs solution under UV light gradually changed from extremely bright yellow to pale yellow until colorless with increasing Cu2+ concentration. The Figure 5b shows a good linear relationship (R<sup>2</sup> = 0.94) between the F0/F (F0 is the initial fluorescence intensity of MPA-InP/ZnS QDs, and F is the fluorescence intensity of MPA-InP/ZnS QDs after adding Cu2+) and concentration of Cu2+ in the range of 0–1000 nM. Moreover, the insert of Figure 5b shows that the fluorescence intensity of the MPA-InP/ZnS QDs at low concentrations of Cu2+ (0–50 nM) is quenched more rapidly, which means that the Cu2+ detection is extremely sensitive at low concentrations. In the concentration range of 0–50 nM, the calibration curve has better linearity (R<sup>2</sup> = 0.99), and the limit of detection (LOD) of the prepared MPA-InP/ZnS QDs calculated by the Formula 3σ/x was 0.22 nM [18].

**Figure 4.** The fluorescence intensity of MPA-InP/ZnS QDs without and with Cu2+ in different pH and the fluorescence-quenching rate of MPA-InP/ZnS QDs after adding Cu2+ (**a**), the fluorescence intensity of MPA-InP/ZnS QDs in different concentrations (**b**), and the changes of fluorescence intensity of MPA-InP/ZnS QDs with added copper over time (**c**). MPA-InP/ZnS QDs: mercaptopropionic acid capped InP/ZnS quantum dots.

**Figure 5.** The fluorescence spectra of MPA-InP/ZnS QDs with different added concentrations of Cu2+ (0 nM, 3 nM, 5 nM, 10 nM, 15 nM, 20 nM, 30 nM, 50 nM, 100 nM, 150 nM, 200 nM, 250 nM, 300 nM, 400 nM, 600 nM, and 1000 nM) (**a**), (insert: photos of MPA-InP/ZnS QDs solutions with different concentrations of Cu2+ under 365 nm UV light) and the relationship between the F0/F and the concentrations of Cu2+ (**b**), (insert: the relationship between the F0/F and low concentrations of Cu2+ (0–50 nM)). MPA-InP/ZnS QDs: mercaptopropionic acid capped InP/ZnS quantum dots.

The stability and selectivity are important indicators for evaluating the practicability and feasibility of probes. Figure 6a shows that the changes in the fluorescence intensity of the MPA-InP/ZnS QDs solution at indoor environment are less than 8% within seven days, indicating the perfect stability of the MPA-InP/ZnS QDs probe. Furthermore, the influence of the potentially competing metal ions Na+, Mg2+, Al3+, K+, Ca2+, Co2+, Mn2+, Fe3+, Ba2+, Cd2+, Pb2+, and Ag+ (500 nM for each) was studied. As shown in Figure 6b, compared with

Cu2+, only Ag+ and Fe3+ can quench the fluorescence of InP/ZnS QDs. The fluorescencequenching degree of InP/ZnS QDs are 7.25% and 7.77% in the concentration of 500 nM of Ag+ and Fe3+, respectively. While in the presence of 50 nM Cu2+, the fluorescencequenching degree of InP/ZnS QDs is 39.2%, indicating the fluorescence quenching of MPA-InP/ZnS QDs by other metal ions is almost negligible. The ion selectivity is dependent on the intrinsic affinity between the analyte and the surface ligands [25], and the MPA ligand has a high affinity constant with Cu2+ [26], which may be the reason of MPA-InP/ZnS QDs has higher selectivity to Cu2+ than other metal ions. These experimental results show the excellent sensitivity, high selectivity, and good anti-influence of the MPA-InP/ZnS QDs probe for the detection of trace Cu2+ in water.

**Figure 6.** The stability (**a**) and the selectivity (**b**) of the fluorescent probe.

#### *3.3. Fluorescence-Quenching Mechanism of MPA-InP/ZnS QDs*

The fluorescence-quenching mechanism of quantum dots is complicated, generally including inner filter effect (IFE), fluorescence resonance energy transfer (FRET), photoinduced electron transfer (PET), static quenching effect (SQE), and dynamic quenching. The FRET, PET, and dynamic quenching can cause the fluorescence lifetime of the fluorophore to decay after adding a quencher, while IFE and SQE cannot [27]. As shown in Figure 7a, the fluorescence decay lifetimes of the MPA-InP/ZnS QDs without and with Cu2+ were 1.07 ns and 1.02 ns, respectively; these values were almost unchanged, indicating that FRET, PET, and dynamic quenching do not occur between MPA-InP/ZnS QDs and Cu2+. Moreover, IFE can be confirmed by UV-Vis absorption spectrum because IFE requires a certain degree of overlap between the excitation or emission band of the fluorophore and the absorption band of the UV-Vis spectrum of the quencher [28]. As shown in Figure 7b, the absorption band of Cu2+ in the range of 200 nm to 270 nm with a peak at 205 nm was observed, and the absorption band of Cu2+ overlaps with the excitation peak of the MPA-InP/ZnS QDs at 250 nm, indicating that the IFE may exist in the quenching mechanism. To further confirm the quenching mechanism, a typical Stern–Volmer diagram was constructed:

$$F\_0/F = 1 + K\_{SV}[Q] = 1 + K\_{\mathcal{q}} \pi\_0[Q] \tag{1}$$

where *F*0 is the fluorescence intensity of MPA-InP/ZnS QDs, *F* is the fluorescence intensity observed after adding Cu2+, *KSV* is the quenching Stern–Volmer constant, and [*Q*] is the concentration of the Cu2+, *Kq* is the dynamic quenching Stern–Volmer constant, and *τ*0 is the lifetime of the InP/ZnS QDs. The static quenching can be judged by *KSV* in different temperatures. The value of *KSV* decreases with the increase of temperature in the SQE process [29]. Obviously, as shown in Figure 7c, with the increasing temperature from 20 ◦C to 40 ◦C, the slope of the curves that are *KSV* decreases, which confirms the existence of SQE. Moreover, the maximum *Kq* of collision quenching is 2 × 10<sup>10</sup> L mol−<sup>1</sup> s<sup>−</sup><sup>1</sup> [30]. In addition, the *Kq* of 20 ◦C, 30 ◦C, and 40 ◦C in this paper are 2.34 × 1015, 1.31 × 1015, and 1.21 × 1015, respectively, which are much larger. The results further confirm the existence of SQE.

**Figure 7.** The fluorescence lifetime decay curves of MPA-InP/ZnS QDs without and with Cu2+ (**a**); the absorption spectrum of Cu2+, excitation, and emission spectra of MPA-InP/ZnS QDs (**b**); the calibration curves between F0/F and the concentrations of Cu2+ under different temperature (20 ◦C, 30 ◦C, and 40 ◦C) (**c**). MPA-InP/ZnS QDs: mercaptopropionic acid capped InP/ZnS quantum dots.

#### *3.4. Detection of Cu2+ in Real Samples*

The applicability and accuracy of MPA-InP/ZnS QDs for the detection of Cu2+ in environmental water samples and drinking samples were investigated. As shown in Table 1, spiked detections were carried out in pure water of Watsons; the recovery was 93.64–120.91%, and the relative standard deviation (RSD) (*n* = 3) was below 1.02%. The results of the detection of Cu2+ in different water samples using our prepared MPA-InP/ZnS QDs probes and ICP-MS are listed in Table 2 and Figure S2. The results of the two methods demonstrate that the MPA-InP/ZnS QDs probes are highly accurate and have the ability to detect trace amounts of Cu2+ in real water samples.

The performance of MPA-InP/ZnS QDs probes was also compared with several previously reported studies on the detection of Cu2+ in water. As shown in Table 3 and Figure S3, the detection range of fluorescent probes for Cu2+ can reach hundreds of micromoles; however, the LOD is difficult to reach for the nanomolar level. As the comparison, the detection range of our probe is 0–1000 nM with the LOD of 0.22 nM, which exhibited superior sensing performance, especially in the detection of trace Cu2+. Our work shows promising prospects in the highly sensitive detection of trace Cu2+ in real water.

**Table 1.** Determination results of copper ions (Cu2+) in pure water of Watsons samples (*n* = 3).



**Table 2.** Detection of Cu2+ using this method and ICP-MS (*n* = 3).

**Table 3.** Comparison of the performance of fluorescent MPA-InP/ZnS QDs probes for the detection of Cu2+.


MPA-InP/ZnS QDs: mercaptopropionic acid capped InP/ZnS quantum dots.
