Preparation of a Red−Emitting, Chitosan−Stabilized Copper Nanocluster Composite and Its Application as a Hydrogen Peroxide Detection Probe in the Analysis of Water Samples

Hydrogen peroxide (H2O2) is an important reactive oxygen species that mediates a variety of physiological functions in biological processes, and it is an essential mediator in food, pharmaceutical, and environmental analysis. However, H2O2 can be dangerous and toxic at certain concentrations. It is crucial to detect the concentration of H2O2 in the environment for human health and environmental protection. Herein, we prepared the red-emitting copper nanoclusters (Cu NCs) by a one-step method, with lipoic acid (LA) and sodium borohydride as protective ligands and reducing agents, respectively, moreover, adding chitosan (CS) to wrap LA−Cu NCs. The as-prepared LA−Cu NCs@CS have stronger fluorescence than LA−Cu NCs. We found that the presence of H2O2 causes the fluorescence of LA−Cu NCs@CS to be strongly quenched. Based on this, a fluorescent probe based on LA−Cu NCs@CS was constructed for the detection of H2O2 with a limit of detection of 47 nM. The results from this research not only illustrate that the as--developed fluorescent probe exhibits good selectivity and high sensitivity to H2O2 in environmental water samples but also propose a novel strategy to prepare red-emitting copper nanoclusters (Cu NCs) by a one-step method.


Introduction
Copper nanoclusters (Cu NCs), which have attracted much attention as functionalized green metal nanomaterials, have been widely studied and applied in the fields of fluorescent probes, biosensing, and cell imaging [1][2][3]. Different from traditional fluorescent materials (such as semiconductor quantum dots, organic dyes, and polymer microspheres), Cu NCs with ultrasmall size endow them with diversified functions, tunable fluorescence, low biological toxicity, and low cost [4][5][6] also prompt Cu NCs to possess broad application prospects. At present, Cu NCs can be synthesized by many generally adopted methods, such as the chemical reduction method, template method, and electrochemical method [7][8][9], and the prepared Cu NCs show fluorescence emission that ranges from blue to red light [10]. However, most Cu NCs show blue fluorescent emission with a short emission wavelength and a small Stokes shift. The penetrating ability of the sample is weak, and there is background signal interference, which partly damages biological samples and limits the applicability of Cu NCs as fluorescent probes for highly sensitive detection and biological analysis [11,12]. Therefore, the development of red fluorescent Cu NCs with stable performance and a large Stokes shift has always been the focus of researchers.
Chitosan (CS), a glycosaminoglycan, is a product of partial deacetylation of chitin, with a wide range of sources, and the main biomass sources are fungi, animal bones, and the shells of shrimps and crabs [13][14][15]. As a multifunctional polymer material, CS has various excellent properties, such as biocompatibility, biodegradability, antibacterial activity, and nontoxicity [16,17], and is regarded as a good applicable raw material in many fields, Chitosan (CS), a glycosaminoglycan, is a product of partial deacetylation of chitin, with a wide range of sources, and the main biomass sources are fungi, animal bones, and the shells of shrimps and crabs [13][14][15]. As a multifunctional polymer material, CS has various excellent properties, such as biocompatibility, biodegradability, antibacterial activity, and nontoxicity [16,17], and is regarded as a good applicable raw material in many fields, such as the food industry, pharmaceutical industry, cosmetics, and biotechnology [18][19][20]. A valuable feature of CS is that its chemical structure contains many intrinsic oxygen and nitrogen functional groups, which can be used as the starting point for covalent modification or chitosan chain cross-linking. However, the poor solubility limits the further application of CS, and many studies have modified CS to prepare derivatives to improve its solubility. At present, CS can be modified by adjusting the degree of deacetylation and viscosity, introducing hydrophilic groups, and changing the solvent pH (weakly acidic) [21][22][23]. In addition, CS can self-assemble and combine with nanomaterials such as graphene and metal nanomaterials [24,25] through covalent bonds and hydrogen bonds, which not only expands the application of CS but also provides functionalized properties.
The proposal of the aggregation-induced emission (AIE) effect fundamentally solved the problem of luminescence quenching caused by aggregation in solution, and the majority of researchers have performed much work on AIE [26][27][28]. The AIE enhancement effect is also an effective means for many metal nanoclusters to enhance fluorescence. In the aggregated state, intramolecular movement is restricted, which increases the coplanarity of the fluorescent molecules and contributes to fluorescence emission [29]. Several past studies [30][31][32] have shown that metal clusters can also produce AIE. Combined with these studies and our experimental results, we reasonably believe that the reason for the fluorescence of LA−Cu NCs@CS is related to the AIE effect of Cu NCs. After the coating of LA−Cu NCs with chitosan, the connection between Cu NCs and its ligand is closer, to produce AIE and emit strong red fluorescence. In this study, with lipoic acid (LA) as the ligand and sodium borohydride (NaBH4) as the reducing agent, LA−protected Cu NCs (LA−Cu NCs) were synthesized by a one-step method. The prepared LA−Cu NCs showed red fluorescence emission, but the stability was poor, the yield was low, and the NCs were difficult to separate and purify (Scheme 1). After adding CS, the dispersed LA−Cu NCs showed a state of aggregation, and the fluorescence intensity was increased by four times, showing AIE enhancement characteristics. Based on this, LA−Cu NCs@CS nanocomposites were prepared, and their related properties were explored. Finally, a fluorescent probe was constructed to realize the selective and sensitive detection of H2O2 in environmental water samples, and the limit of detection (LOD) was 47 nM in the detection range of 0.2-128 µ M.

Materials
Lipoic acid (LA) and chitosan (CS) were purchased from Aladdin Reagent Co., Ltd.  6 ] 3− , CO 3 2− , and PO 4 3− ) solutions were prepared by their respective crystal salts with the same concentration (32 µM). The tap water and lake water samples were randomly taken from the local tap water and Sanyuan Lake of Yantai University. All experimental water in this study was produced from a UPT−II−10T ultrapure water purifier (18.2 MΩ•cm). All chemical reagents were not further purified unless otherwise specified.

Apparatus
Scanning electron microscope (TEM) images were taken through a TSM-7900F electron microscope (JEOL Ltd., Tokyo, Japan). Transmission electron microscopy (TEM) and High resolution transmission electron microscope (HRTEM) images were taken through a JEM−2010 transmis XCsion electron microscope with an accelerating voltage of 200 kV (JEOL Ltd., Tokyo, Japan). Ultraviolet-visible absorption (UV-Vis) spectroscopy was obtained by a PerkinElmer Lambda 365 spectrometer (Shimfusa, Japan) with a wavelength interval of 5 nm. Fourier transform infrared (FT−IR) spectroscopy was performed using a Nicolet 5700 FT−IR spectrometer (Thermo Fisher Scientific, Waltham, MA, USA). X-ray photoelectron spectroscopy (XPS) was carried out using a Thermo ESCALAB−250 (Thermo Fisher Scient-ific, Waltham, MA, USA). The fluorescent spectrum was measured by an F−2700 spectrophotometer (Hitachi, Japan). Energy dispersive X-ray spectrometry (EDS) and EDS mapping elemental analysis were measured by an Ultim Extreme detector (Oxford Instruments Technology, Shanghai, China).

Preparation of LA−Cu NCs
Seventy−two milligrams (0.35 mmol) of LA and 14 mg (0.37 mmol) of NaBH 4 were accurately weighed into 16 mL ultrapure water and stirred thoroughly for 5 min at room temperature. Then, 700 µL of 25 mM Cu(NO 3 ) 2 •3H 2 O was added to the above-mixed solution, and the color of the solution gradually changed from colorless to yellow-brown. After that, the dissolved oxygen in the mixed solution was pumped out with a vacuum pump, and then high-purity nitrogen was injected. The step was repeated three times to ensure that the oxygen was completely removed. Finally, the above-mixed solution was stirred and reacted for 6 h in an ice water bath (5-10 • C) to obtain a clear yellow-brown LA−Cu NC solution, which exhibited weak red fluorescence under 365 nm UV light. The prepared LA−Cu NC solution was stored at 4 • C for subsequent use.

Preparation of LA−Cu NCs@CS
The operation was to add 1.6 mL 1 mg/mL CS−1% acetic acid solution (ratio of CuNCs and CS solutions = 10:1 v/v) to the preprepared LA−Cu NC solution, and there was a clear yellow precipitate in the solution. After vigorously stirring for 30 min at room temperature, a large amount of aggregated precipitate was generated, which emitted obvious red fluorescence under irradiation with 365 nm UV light. After that, the same volume of absolute ethanol was added to the above-mixed solution, and ultrasonic treatment was performed for 5 min to uniformly disperse the yellow precipitate. Then, the mixture was centrifuged at 8000 rpm for 8 min, washed three times with anhydrous ethanol, and dried in a rotary evaporator at 50 • C for 1 h. The yellow powder obtained was the pure product of LA−Cu NCs@CS, which was stored at 4 • C for future use.

Construction of Fluorescent Probe Based on LA−Cu NCs@CS
First, an anhydrous ethanol solution of LA−Cu NCs@CS (2 mg mL −1 , pH = 7.40) was prepared, and then a series of H 2 O 2 aqueous solutions with different concentrations (0.8, 2, Biosensors 2023, 13, 361 4 of 12 8, 16, 32, 64 and 128 µM) were added to 1 mL of LA−Cu NCs@CS solution. After reacting at room temperature for 5 min, the fluorescence emission spectrum was measured with fluorescence spectrophotometry. As the concentration of H 2 O 2 increased, the intensity of the fluorescence spectrum showed a gradually decreasing trend, a linear curve was drawn, and a linear regression equation was obtained by fitting, which was a model of the fluorescent probe.
In addition, under optimized conditions, different methodological verifications of the fluorescent probe were also carried out. Selectivity: Explore the interference of different cations and anions on the fluorescence of LA−Cu NCs@CS; Stability: the fluorescence intensity changes of LA−Cu NCs@CS within a certain time; Reproducibility: Three parallel experiments were performed to verify that the LA−Cu NCs@CS fluorescent probe has a good response to H 2 O 2 .

Detection of H 2 O 2 Using LA−Cu NCs@CS
To investigate the performance of the fluorescent probe in actual detection, different actual water samples were analyzed. After filtering the tap water and lake water samples with a 0.22 µm filter membrane, the fluorescent probe was used to test the tap water and lake water samples to find no H 2 O 2 , and then the spiked recovery method was used for H 2 O 2 determination. Three groups of H 2 O 2 spiked solutions of different concentrations (0.8, 8, 32 µM) were added to the treated tap water and lake water samples, and the fluorescence emission spectrum was tested after incubating at room temperature for 5 min. Three experiments were performed in parallel, and the relative standard deviation (RSD) was calculated. The measured fluorescence intensity value was substituted into the constructed linear regression equation to calculate the final spiked recovery rate.   Figures 1A and S2). In the presence of CS, LA−Cu NCs appeared to aggregate in an aqueous solution, which was also confirmed by TEM. LA−Cu NCs@CS did show an aggregate state in Figure 1B. The HRTEM image ( Figure 1C) showed that the single crystal lattice of LA−Cu NCs@CS was approximately 0.33 nm (3.3 Å), which corresponded to the 102 planes of Cu [30], and the average size was 3.5 nm ( Figure 1D). The characterization of the morphology above showed that the successful combination of CS and LA−Cu NCs, the formation of an aggregated state, and the size distribution had not changed significantly.

Optical Performance Analysis
As shown in Figure 2A, the UV-vis spectra showed that synthesized LA−Cu NCs had a broad absorption shoulder at 250~300 nm; LA−Cu NCs@CS had a strong and broad absorption peak at approximately 300 nm. The change in the spectrum proved that the encapsulation of the LA−Cu NCs by CS caused variation in the molecular structure of the light−absorbing group, which led to a shift in the absorption peak. In the FT−IR spectra ( Figure 2B

Optical Performance Analysis
As shown in Figure 2A, the UV-vis spectra showed that synthesized LA−Cu NCs had a broad absorption shoulder at 250~300 nm; LA−Cu NCs@CS had a strong and broad absorption peak at approximately 300 nm. The change in the spectrum proved that the encapsulation of the LA−Cu NCs by CS caused variation in the molecular structure of the light−absorbing group, which led to a shift in the absorption peak. In the FT−IR spectra ( Figure 2B  XPS was employed to perform chemical element and valence analysis. The XPS total spectrum showed that there were five peaks of C 1 s, N 1 s, O 1 s, S 2p, and Cu 2p under the corresponding binding energy, corresponding to the existence of C, N, O, S, and Cu elements in LA−Cu NCs@CS ( Figure 2C), which was consistent with expectations. In the Cu 2p spectrum (Figure 2D), the characteristic peaks at 932.2 eV and 952.3 eV were attributed to Cu 2p3/2 and Cu 2p1/2 of Cu(0), respectively. In addition, there was no obvious absorption peak at 943.5 eV, which indicated that Cu(II) in LA−Cu NCs@CS was almost nonexistent and had been completely reduced. The difference in binding energy between Cu(0) and Cu(I) was only approximately 0.1 eV, indicating that the valence state of Cu in LA−Cu NCs@CS may be 0 or +1 [33]. In addition, the EDS spectrum clearly shows the  Figure 2C), which was consistent with expectations. In the Cu 2p spectrum (Figure 2D), the characteristic peaks at 932.2 eV and 952.3 eV were attributed to Cu 2p3/2 and Cu 2p1/2 of Cu(0), respectively. In addition, there was no obvious absorption peak at 943.5 eV, which indicated that Cu(II) in LA−Cu NCs@CS was almost nonexistent and had been completely reduced. The difference in binding energy between Cu(0) and Cu(I) was only approximately 0.1 eV, indicating that the valence state of Cu in LA−Cu NCs@CS may be 0 or +1 [33]. In addition, the EDS spectrum clearly shows the inclusion of C, N, O, S, and Cu elements ( Figure S1), which was consistent with the XPS spectrum test result, and the mapping element distribution of the characteristic elements of N, O, S, and Cu was also very uniform. In addition, EDS Mapping analysis of LA−Cu NCs@CS was performed to further verify the successful synthesis of the materials. As shown in Figure S1, the characteristic spectra of Cu, N, S and other elements can be seen from the figure, indicating that the elements in LA−Cu NCs@CS are closely combined.
As shown in Figure 3A, under different excitation wavelengths (350-440 nm), LA−Cu NCs@CS had the maximum fluorescence emission when the excitation wavelength was 400 nm. At the optimal excitation wavelength, the excitation and emission spectra of LA−Cu NCs and LA−Cu NCs@CS showed that the fluorescence emission intensity of LA−Cu NCs was relatively weak, and the fluorescence intensity became approximately four times that of the original after adding CS ( Figure 3B). After CS encapsulated LA−Cu NCs to form LA−Cu NCs@CS, it triggered the aggregation of fluorophores and increased the fluorescence intensity. Figure 3C shows the fluorescence decay spectrum of LA−Cu NCs, whose fluorescence lifetime was 77.94 ns, as calculated by the weighted average method [34]. After adding CS, the fluorescence lifetime calculated by the same method was 58.03 ns ( Figure 3D), which was less than that without CS. This result indicated that the presence of CS can enhance the fluorescence intensity of LA−Cu NCs.

Optimization of Conditions
During the synthesis process, the effects of different reaction conditions on the fluorescence performance of LA−Cu NCs@CS were determined, including the ratio of raw materials, reaction time, pH, and reaction temperature. Figure 4A shows the fluorescence emission intensity of the different molar ratios of LA and Cu 2+ (1:1, 5:1, 10:1, 20:1, and 30:1) and the volume ratios (1:1, 5:1, 10:1, 20:1, and 30:1) of the total volume of the LA−Cu NC solution under excitation at 400 nm. As the ratio of LA increased, the fluorescence intensity gradually increased, reaching a maximum of 20:1. At the same time, the fluorescence

Optimization of Conditions
During the synthesis process, the effects of different reaction conditions on the fluorescence performance of LA−Cu NCs@CS were determined, including the ratio of raw materials, reaction time, pH, and reaction temperature. Figure 4A shows the fluorescence Biosensors 2023, 13, 361 7 of 12 emission intensity of the different molar ratios of LA and Cu 2+ (1:1, 5:1, 10:1, 20:1, and 30:1) and the volume ratios (1:1, 5:1, 10:1, 20:1, and 30:1) of the total volume of the LA−Cu NC solution under excitation at 400 nm. As the ratio of LA increased, the fluorescence intensity gradually increased, reaching a maximum of 20:1. At the same time, the fluorescence intensity was the largest when the ratio of the total volume of the LA−Cu NC solution to the volume of the CS solution was 10:1. The appropriate reaction time was not only conducive to the benign growth of LA−Cu NCs@CS molecules but could also avoid the excessive growth of the reaction time that would affect the fluorescence performance. The fluorescence emission intensity at different reaction times in Figure 4B shows that the fluorescence intensity was the maximum at 6 h, which was the time for the final reaction to prepare LA−Cu NCs@CS. The initial pH value of the synthesized LA−Cu NCs@CS was 4.14. When the pH was adjusted by adding 1 M NaOH, it was found that the fluorescence intensity changed little at pH 4-6, and the fluorescence intensity decreased significantly when the pH value was greater than 6 ( Figure 4C), which indicated that the weakly acidic environment (pH = 4-6) was suitable for the synthesis of LA−Cu NCs@CS. The effect of reaction temperature on fluorescence was also further optimized. As shown in Figure 4D, as the reaction temperature increased, the fluorescence intensity decreased greatly, which demonstrated that low temperature was more suitable for the synthesis of LA−Cu NCs@CS, and the final preparation of LA−Cu NCs@CS was carried out in an ice bath environment (5-10 • C).

Fluorescence Response of LA−Cu NCs@CS in the Presence of H2O2
H2O2 is an important and abundant reactive oxygen species in organisms that play an important role in maintaining the physiological balance of cells in the body, and H2O2 has a wide range of applications in the food, environment, pharmaceutical, and textile industries. However, excessive discharge of sewage and waste liquid containing a large amount of H2O2 will cause harm to the environment and water resources. At present, the analysis and monitoring of H2O2 have always been the focus of related research. In this study, it is found that the presence of H2O2 will cause the fluorescence of the LA−Cu NCs@CS solution to be significantly reduced. Therefore, the construction of a probe based on LA−Cu NCs@CS will show good prospects in the detection of H2O2. Figure 5A and Table S1 show the changes in fluorescence intensity after adding a series of concentrations (0.2, 0.8, 2, 8, 16, 32, 64, and 128 µ M) of H2O2 to LA−Cu NCs@CS for 5 min at room temperature. With increasing H2O2 concentration, the fluorescence in-  be significantly reduced. Therefore, the construction of a probe based on LA−Cu NCs@CS will show good prospects in the detection of H 2 O 2 . Figure 5A and Table S1 show the changes in fluorescence intensity after adding a series of concentrations (0.2, 0. 8,2,8,16,32,64, and 128 µM) of H 2 O 2 to LA−Cu NCs@CS for 5 min at room temperature. With increasing H 2 O 2 concentration, the fluorescence intensity of LA−Cu NCs@CS showed a declining trend, and the greater the H 2 O 2 concentration, the higher the fluorescence quenching degree. Figure 5B shows the linear relationship between the H 2 O 2 concentration and LA−Cu NCs@CS fluorescence intensity ratio ((F 0 − F)/F 0 ). Where F 0 and F are the fluorescence intensities of the LA−Cu NCs@CS solution without H 2 O 2 and with different concentrations of H 2 O 2 , respectively. There was a good linear relationship between LA−Cu NCs@CS and the fluorescence intensity ratio. The linear equation was (F 0 − F)/F 0 = 0.00378C + 0.00578 (R 2 = 0.9941), where C is the H 2 O 2 concentration. The LOD of the probe for the H 2 O 2 response was 47 nM (S/N = 3). In addition, to reflect the good detection performance of this method for H 2 O 2 , the detection of H 2 O 2 by different methods was compared with the methods mentioned in this study. As shown in Table 1, compared with other nanomaterials [35][36][37][38][39], this method had a lower LOD for H 2 O 2 detection. The above results showed that the fluorescent probe based on LA−Cu NCs@CS had a good performance and could achieve sensitive detection of H 2 O 2 . Moreover, this result was obtained by using three different hydrogen peroxide probes on the same day, which also proved that the probe had little difference in the detection performance of hydrogen peroxide during the day with acceptable RSD (2.21%).

Stability and Selectivity
Stability and selectivity experiments were performed separately to evaluate the applicability of the probe based on LA−Cu NCs@CS. Figure 6A shows the fluorescence intensity changes of LA−Cu NCs and LA−Cu NCs@CS prepared under the same conditions at different periods after storage at room temperature for approximately five days. In the first 20 h, the fluorescence intensity of LA−Cu NCs (red line) was reduced by approximately half, and the fluorescence intensity of LA−Cu NCs@CS (black line) decreased slowly within 20 h. Even after 128 h, the fluorescence intensity was also greater than the initial value of LA−Cu NCs. From this point of view, after adding CS to LA−Cu NCs, CS  carbon quantum dots 0.5~50 0.2 [35] gold nanoclusters capped by hemoglobin 0.5~700 0.21 [36] Fe, N−incorporated carbon nanotubes 0.1~100 0.068 [37] naphthalene backbone and a boric acid ester 1~250 0.7 [38] sulfonated porphyrin 1~8 0.32 [39] LA−Cu NCs@CS 0.2~128 0.047 This work

Stability and Selectivity
Stability and selectivity experiments were performed separately to evaluate the applicability of the probe based on LA−Cu NCs@CS. Figure 6A shows the fluorescence intensity changes of LA−Cu NCs and LA−Cu NCs@CS prepared under the same conditions at different periods after storage at room temperature for approximately five days. In the first 20 h, the fluorescence intensity of LA−Cu NCs (red line) was reduced by approximately half, and the fluorescence intensity of LA−Cu NCs@CS (black line) decreased slowly within 20 h. Even after 128 h, the fluorescence intensity was also greater than the initial value of LA−Cu NCs. From this point of view, after adding CS to LA−Cu NCs, CS might have a certain stabilizing and protective effect on LA−Cu NC molecules, and the electron−donating groups (−NH 2 and −OH) contained in CS were also beneficial to the fluorescence emission of LA−Cu NCs. It is worth noting that the difference in inner−day fluorescence intensity is slightly larger, and the relevant RSDs are between 3.77% and 4.84%. In addition, the red fluorescence intensity of the powder products of LA−Cu NCs@CS was much higher than that of LA−Cu NCs (inset a) under ultraviolet light. It is worth noting that the fluorescence intensity of the LA−Cu NCs@CS powder product under ultraviolet light hardly changed after storage for 2 months (inset b). Therefore, the addition of CS to LA−Cu NCs not only enhanced the fluorescence but also improved the stability, which provided a guarantee for practical applications. Under optimized conditions, the interference of different metal ions and anions with the same concentration (32 µ M) on the response of the LA−Cu NCs@CS system to H2O2 was explored separately. Figure 6B shows that only the fluorescence response of H2O2 had a significant change, and different metal ions and anions had almost no effect on the H2O2 probe test process, which indicated that the fluorescent probe based on LA−Cu NCs@CS had good anti-interference ability for H2O2 detection.

Actual Detection of H2O2
To verify the feasibility of the prepared fluorescent probe for H2O2 detection, we tested tap water and lake water samples and the presence of H2O2 was not detected. Then, the recovery rate was calculated using the spiked recovery method. First, the tap water and lake water were filtered with 0.22 µ m membranes, and then the filtered tap water and lake water were used to prepare H2O2 with different concentrations (0.8, 8, and 32 µ M) as the spiked samples. Finally, the linear equation of (F0-F)/F0 and H2O2 concentration was employed for the calculation of the final recovery rate. The spiked experiment was performed three times in parallel, and the results are shown in Table 2. The recovery rate of tap water was 95.88-98.44%, the recovery rate of lake water was 94.38-102.5%, and the total RSD ranged from 2.9% to 4.5%.  Under optimized conditions, the interference of different metal ions and anions with the same concentration (32 µM) on the response of the LA−Cu NCs@CS system to H 2 O 2 was explored separately. Figure 6B shows that only the fluorescence response of H 2 O 2 had a significant change, and different metal ions and anions had almost no effect on the H 2 O 2 probe test process, which indicated that the fluorescent probe based on LA−Cu NCs@CS had good anti-interference ability for H 2 O 2 detection.

Actual Detection of H 2 O 2
To verify the feasibility of the prepared fluorescent probe for H 2 O 2 detection, we tested tap water and lake water samples and the presence of H 2 O 2 was not detected. Then, the recovery rate was calculated using the spiked recovery method. First, the tap water and lake water were filtered with 0.22 µm membranes, and then the filtered tap water and lake water were used to prepare H 2 O 2 with different concentrations (0.8, 8, and 32 µM) as the spiked samples. Finally, the linear equation of (F 0 -F)/F 0 and H 2 O 2 concentration was employed for the calculation of the final recovery rate. The spiked experiment was performed three times in parallel, and the results are shown in Table 2. The recovery rate of tap water was 95.88-98.44%, the recovery rate of lake water was 94.38-102.5%, and the total RSD ranged from 2.9% to 4.5%. Accordingly, the fluorescent probe based on LA−Cu NCs@CS had good sensitivity and selectivity, which opened up a new path for the analysis and monitoring of H 2 O 2 in actual environmental water samples.

Conclusions
In summary, we report a novel method that is used to construct a probe for detecting H 2 O 2 . The as-synthesized LA−Cu NCs@CS had good nanomorphology and superior luminescence properties. The linear range of the method was from 0.2 µM to 128 µM, and the limit of detection was 47 nM. In addition, this probe exhibits strong anti-interference ability in water samples with excellent selectivity and reproducibility. Furthermore, the synthesis of LA−Cu NCs@CS was simple and environmentally friendly, inexpensive, and served as a reference for the quantitative analysis of H 2 O 2 in natural water and other water samples.
Supplementary Materials: The following supporting information can be downloaded at: https:// www.mdpi.com/article/10.3390/bios13030361/s1, Figure S1: The EDS image of LA−CuNCs@CS; Figure S2: The diameter distribution histogram of LA−Cu NCs.; Table S1: The corresponding fluorescence intensity under different concentrations of H 2 O 2 from Figure 5A. Equation (S1): The equation to calculate the LOD.
Funding: This work was financially supported by the National Natural Science Foundation of China (21778047), and Natural Science Foundation of Shandong Province (ZR2021MB024).
Institutional Review Board Statement: Not applicable.