A New Cellulose-Based Fluorescent Probe for Specific and Sensitive Detection of Cu2+ and Its Applications in the Analysis of Environmental Water

In this work, a novel fluorescent probe CMC−GE−AQ with an effective sensitive detection ability for Cu2+ was synthesized and constructed by using carboxymethyl cellulose (CMC) as the skeleton and 8-aminoquinoline (AQ) as the fluorophore. This probe exhibited a highly specific “turn-off” fluorescence response to Cu2+, and the fluorescence color changed from bright orange to colorless after adding Cu2+. The probe could selectively detect Cu2+ in a complex environment and its detection limit (LOD), the binding constant (Ka) and the numbers of binding sites (n) were calculated to be 6.4 × 10−8 mol L−1, 1.7 × 106 mol−1 L and 1.2, respectively. The sensing detection mechanism was confirmed by X-ray photoelectron spectroscopy (XPS) and density functional theory (DFT) calculations. In addition, the probe CMC−GE−AQ was successfully applied to detect Cu2+ in real water samples, and CMC−GE−AQ-based fluorescent microspheres can serve as a convenient tool for the detection of Cu2+.


Introduction
Cu 2+ , as the third essential trace element in the human body, plays an important role in biological processes by participating in the activities of cells [1]. However, high level of Cu 2+ in the body can cause great harm to human health, such as depression, diarrhea, memory loss and other symptoms, and can even lead to Hashimoto's disease, fibrocystic breast disease, Alzheimer's disease, Wilson's disease, etc. [2,3]. Furthermore, a high concentration of Cu 2+ is a common water pollutant, with toxicity, non-degradation and bioaccumulation [4,5], which can enter the food chain and into the human body through water circulation. Therefore, the detection of Cu 2+ is necessary for environmental protection and human health.
Carboxymethyl cellulose (CMC), as a kind of water-soluble natural polymer, is usually found in the form of sodium salts with the advantages of excellent biocompatibility, nontoxicity, good biodegradability and easy modification [20]. Due to the presence of many active oxygen-containing groups (carboxyl and hydroxyl), CMC is also an ideal carrier in the field of multifunctional modifications and constructing diverse fluorescent materials [21][22][23][24], For example, Fan prepared fluorescent hydrogels and aerogel hybrid materials by covalent coordination of lanthanide ions (Eu 3+ or Tb 3+ ) with carboxyl groups of CMC, which can be used to the detection of Fe 3+ [23]. Ye synthesized a fluorescent probe CMC/Tb(III) for detecting Mn 2+ in aqueous solution [24]. Thus, it can be seen that CMC-based fluorescent probes have great application prospects due to their high sensitivity and selectivity, good processability, and operability.
In this work, a novel carboxymethyl cellulose-based fluorescent probe CMC−GE−AQ toward Cu 2+ detection was prepared. This probe was designed by immobilizing AQ onto CMC with epichlorohydrin. The CMC−GE−AQ solution exhibited bright orange fluorescence, and the fluorescence was immediately quenched after the addition of Cu 2+ . CMC−GE−AQ could detect Cu 2+ with high sensitivity. The detection limit (LOD), the binding constant (K a ) and the numbers of binding sites (n) were calculated to be 6.4 × 10 −8 mol L −1 , 1.7 × 10 6 mol −1 L and 1.2, respectively. In addition, CMC−GE−AQ could monitor Cu 2+ in real water samples, and CMC−GE−AQ-based fluorescent microspheres were successfully prepared for the detection of Cu 2+ .

Preparation of Carboxymethyl Cellulose Glycidyl Ether (CMC−GE)
First, 2 g CMC−Na was completely dissolved in 40 mL deionized water, and the pH was adjusted to 14 with 30 wt% NaOH solution. Then, 8 mL of epichlorohydrin (ECH) was added dropwise into the CMC−Na solution to react at 60 • C for 3 h. After the reaction, the mixture was separated out by ethanol and filtered, and the residues were washed with ethanol and distilled water until neutrality to obtain CMC−GE. The epoxy group of CMC−GE was determined according to the literature [31].

Preparation of Probe (CMC−GE−AQ)
First, 2 g CMC−GE was added to 75 mL of water, and the pH of CMC−GE solution was adjusted to 12. Then, 18 g AQ was dissolved in a certain amount of THF, and the AQ solution was added into the CMC−GE solution, accompanied by stirring. The mixture reacted at 65 • C for 6 h. The reacted mixture was separated out by ethanol and filtered, the residue was washed with ethanol and distilled water to remove the unreacted AQ completely (the filtrate was colorless and no fluorescence). The remainder was 3-(quinolin-8-amino)-2-hydroxypropyl carboxymethyl cellulose ether (CMC−GE−AQ).

Characterization
The structural analyses of samples were recorded by the FT-IR spectra with a scan range from 4000 to 650 cm −1 using an infrared spectrometer (VERTEX 80 V, Bruker). Thirty-two scans were recorded with a resolution of 4 cm −1 .
The XPS spectra were performed in an X-ray photoelectron spectrometer (AXIS Ultra-DLD, Shimadzu, Kyoto, Japan) using a monochromatized Al Ka X-ray source (1486.6 eV). The spectral acquisition range was from 1100 to 0 eV, with an energy of 160 eV and a scan step of 1.0 eV. The high-resolution XPS spectra and quantitative analysis data of CMC−GE−AQ of C 1s, O 1s and N 1s were recorded. The quantitative relative sensitivity factors (RSF) of C 1s, O 1s and N 1s were 0.278, 0.780 and 0.477, respectively.
The UV-Vis absorption spectra of CMC−GE−AQ were determined by a Shimadzu UV-2450 spectrophotometer, and the scan range was from 225 to 350 nm.
The fluorescence spectra of testing samples were recorded by fluorescence spectrophotometer (LS 55, PE Co., Norwalk, CT, USA); the excitation wavelength was at 425 nm and excitation slit was at 7 nm.
The color changes of testing samples were observed under a 365 nm UV lamp. The Cu 2+ concentrations were measured by atomic absorption spectrometry (TAS−990AFG, Beijing, China).
Gaussian 09 program calculated using the B3LYP function with the 6−31G* set. The surface morphologies of CMC−GE−AQ-based fluorescent microspheres were determined with field emission scanning electron microscopy (FESEM) using a Regulus 8100 (Hitachi, Tokyo, Japan) at 100 magnifications.
The detection limit of CMC−GE−AQ for Cu 2+ was calculated by using emission titration datum. The emission spectra of the original CMC−GE−AQ were measured 20 times. Then, the relationship between the fluorescence emission intensity of the CMC−GE−AQ at 579 nm and the concentration of Cu 2+ was plotted. Each fluorescence emission intensity was measured 3 times. The detection limit (LOD) was calculated as Equation (1): where σ is the standard deviation of the emission intensity of the original CMC−GE−AQ, and s is the slope between the fluorescence emission intensity and the concentration of Cu 2+ .

Preparation of CMC−GE−AQ-based Microspheres
The CMC−GE−AQ-based microspheres were prepared according to the literature [32]. When 0.63 g CMC−GE−AQ was completely dissolved in 12.5 mL of 30 wt% NaOH solution, the CMC−GE−AQ solution was added into the mixture of liquid paraffin (50 mL), Span 80 (0.2 g), t-butanol (0.7 mL) and CCl 4 (0.7 mL) dropwise, accompanied by high-speed blending. Then, 2.5 mL of epichlorohydrin (ECH) was added dropwise into the mixture to react at 60 • C for 6 h. After the reaction, the mixture was separated out by ethanol and filtered, and the residues were washed with ethanol and distilled water until neutrality to obtain CMC−GE−AQ-based microspheres.

Synthesis and Characterization of CMC−GE−AQ
The synthetic route of CMC−GE−AQ is shown in Scheme 1. The reaction between the hydroxyl group of CMC−Na and ECH usually required alkaline conditions [33]. The fluorophore group was introduced onto the CMC−GE chain by the ring-opening reaction of epoxy groups with amino groups of AQ [34].
trations were determined by atomic absorption spectrometry for comparison.

Preparation of CMC−GE−AQ−Based Microspheres
The CMC−GE−AQ−based microspheres were prepared according to the literature [32]. When 0.63 g CMC−GE−AQ was completely dissolved in 12.5 mL of 30 wt% NaOH solution, the CMC−GE−AQ solution was added into the mixture of liquid paraffin (50 mL), Span 80 (0.2 g), t−butanol (0.7 mL) and CCl4 (0.7 mL) dropwise, accompanied by high−speed blending. Then, 2.5 mL of epichlorohydrin (ECH) was added dropwise into the mixture to react at 60 °C for 6 h. After the reaction, the mixture was separated out by ethanol and filtered, and the residues were washed with ethanol and distilled water until neutrality to obtain CMC−GE−AQ−based microspheres.

Synthesis and Characterization of CMC−GE−AQ
The synthetic route of CMC−GE−AQ is shown in Scheme 1. The reaction between the hydroxyl group of CMC−Na and ECH usually required alkaline conditions [33]. The fluorophore group was introduced onto the CMC−GE chain by the ring−opening reaction of epoxy groups with amino groups of AQ [34].
The FT−IR spectra of CMC−GE−AQ, CMC−GE, CMC−Na and AQ are compared in Figure 1. The FT−IR spectra of AQ showed that absorptions at 3450 and 3350 cm −1 were primary amine stretching [35]. Absorptions at 3430 and 2925 cm −1 were found in CMC−GE−AQ, CMC−GE and CMC−Na. The former was O−H stretching vibration, and the latter was −CH2− stretching vibration. The absorptions of CMC in the range of 1000~1300 cm −1 were C−O−C stretching vibrations [33]. The FT−IR spectra of CMC−GE showed an obvious characteristic absorption of epoxy groups at 894 cm −1 [32]. After AQ was functionalized, the characteristic absorption of the epoxy group almost disappeared, and the absorptions at 1384 and 1625 cm −1 originated from C−N stretching vibration and N−H stretching vibration, respectively [36]. The FT−IR spectra data strongly elucidated that the AQ was introduced onto CMC−GE by the epoxy groups. The FT-IR spectra of CMC−GE−AQ, CMC−GE, CMC−Na and AQ are compared in Figure 1. The FT-IR spectra of AQ showed that absorptions at 3450 and 3350 cm −1 were primary amine stretching [35]. Absorptions at 3430 and 2925 cm −1 were found in CMC−GE−AQ, CMC−GE and CMC−Na. The former was O−H stretching vibration, and the latter was −CH 2 − stretching vibration. The absorptions of CMC in the range of 1000~1300 cm −1 were C−O−C stretching vibrations [33]. The FT-IR spectra of CMC−GE showed an obvious characteristic absorption of epoxy groups at 894 cm −1 [32]. After AQ was functionalized, the characteristic absorption of the epoxy group almost disappeared, and the absorptions at 1384 and 1625 cm −1 originated from C−N stretching vibration and   Table 1. The N content (1.44%) appeared in CMC−GE−AQ after the introduction of AQ, and C content changed from 56.51% to 57.39%. Thus, the conclusion of XPS confirmed that AQ was successfully introduced to CMC−GE. The fluorescence spectra and fluorescence photographs of AQ, CMC−Na, CMC−GE and CMC−GE−AQ are shown in Figure 3, and the concentrations of all the sample solutions were 1.5 × 10 −4 g mL −1 . The CMC and CMC−GE solution had no fluorescence. AQ solution showed weak green fluorescence at 565 nm, while CMC−GE−AQ solution showed strong orange fluorescence at 579 nm. When AQ was attached to CMC−GE, the fluorescence intensity of CMC−GE−AQ was about two times that of AQ, and the emission wavelength redshifted and the color changed significantly. According to the structural characteristics of CMC−GE−AQ, the enhanced fluorescence intensity could be related to the CMC skeleton. CMC−GE−AQ improved the weakness of the weak fluorescence emission intensity of AQ. Therefore, AQ was successfully introduced to CMC−GE.  Table 1. The N content (1.44%) appeared in CMC−GE−AQ after the introduction of AQ, and C content changed from 56.51% to 57.39%. Thus, the conclusion of XPS confirmed that AQ was successfully introduced to CMC−GE. The fluorescence spectra and fluorescence photographs of AQ, CMC−Na, CMC−GE and CMC−GE−AQ are shown in Figure 3, and the concentrations of all the sample solutions were 1.5 × 10 −4 g mL −1 . The CMC and CMC−GE solution had no fluorescence. AQ solution showed weak green fluorescence at 565 nm, while CMC−GE−AQ solution showed strong orange fluorescence at 579 nm. When AQ was attached to CMC−GE, the fluorescence intensity of CMC−GE−AQ was about two times that of AQ, and the emission wavelength redshifted and the color changed significantly. According to the structural characteristics of CMC−GE−AQ, the enhanced fluorescence intensity could be related to the CMC skeleton. CMC−GE−AQ improved the weakness of the weak fluorescence emission intensity of AQ. Therefore, AQ was successfully introduced to CMC−GE. Polymers 2022, 14, x FOR PEER REVIEW

Effect of Detection Conditions on Fluorescence Intensity of CMC−GE−AQ
The fluorescence intensity of CMC−GE−AQ was greatly affected by the enviro tal medium. This study focused on the effect of solvent polarity and pH value on fl cence intensity of CMC−GE−AQ.

Effect of Detection Conditions on Fluorescence Intensity of CMC−GE−AQ
The fluorescence intensity of CMC−GE−AQ was greatly affected by the environmental medium. This study focused on the effect of solvent polarity and pH value on fluorescence intensity of CMC−GE−AQ.

Solvents Polarity
The highly polar solvent ensured the fluorescence intensity of fluorescent materials, such as water, DMF, DMSO, DMAc, etc. [37]. Therefore, the mixture of DMF and water was selected as the detection solvent to study the effect of solvent on the fluorescence intensity of CMC−GE−AQ (Figure 4). With the increase in DMF amount, the fluorescence intensity of CMC−GE−AQ at the emission peak gradually increased, which was because water has a high dielectric constant (εr = 80.1) and more hydrogen bonds, while DMF has a low dielectric constant (εr = 36.7) and no hydrogen bonds [31]. With the increase in DMF ratio, the polarity of the solvent decreased and the dielectric constant decreased. Therefore, the fluorescence intensity of CMC−GE−AQ increased with the decrease in solvent polarity, and the increase trend was consistent with the decrease in the average dielectric constant [38]. The maximum fluorescence emission was obtained at a volume ratio DMF/H 2 O of 8/2. Therefore, the mixed solvent of DMF/H 2 O (v/v = 8/2) was selected as the detection solvent for subsequent experiments.

Solvents Polarity
The highly polar solvent ensured the fluorescence intensity of fluorescent materials, such as water, DMF, DMSO, DMAc, etc. [37]. Therefore, the mixture of DMF and water was selected as the detection solvent to study the effect of solvent on the fluorescence intensity of CMC−GE−AQ (Figure 4). With the increase in DMF amount, the fluorescence intensity of CMC−GE−AQ at the emission peak gradually increased, which was because water has a high dielectric constant (εr = 80.1) and more hydrogen bonds, while DMF has a low dielectric constant (εr = 36.7) and no hydrogen bonds [31]. With the increase in DMF ratio, the polarity of the solvent decreased and the dielectric constant decreased. Therefore, the fluorescence intensity of CMC−GE−AQ increased with the decrease in solvent polarity, and the increase trend was consistent with the decrease in the average dielectric constant [38]. The maximum fluorescence emission was obtained at a volume ratio DMF/H2O of 8/2. Therefore, the mixed solvent of DMF/H2O (v/v = 8/2) was selected as the detection solvent for subsequent experiments.

Solvents pH Value
As shown in Figure 5a, when the solvent of pH < 5, the CMC−GE−AQ solution showed weak fluorescence intensity. When the solvent of pH ranged from 5 to 7, the fluorescence intensity of CMC−GE−AQ solution increased significantly. When the solvent of pH > 7, the fluorescence intensity of CMC−GE−AQ solution remained at a high level. The suitable pH detection range of CMC−GE−AQ was 5~12. Different concentrations of Cu 2+ were added to the CMC−GE−AQ solution at the pH values 5 and 7, respectively. The relationship between fluorescence intensity and Cu 2+ concentration is shown in Figure 5b. The fluorescence intensity of CMC−GE−AQ solution (pH = 7) decreased with the increase in the concentration of Cu 2+ , which was consistent with the trend of CMC−GE−AQ solution (pH = 5). Since Cu 2+ did not hydrolyze at pH = 5, it also did not hydrolyze at pH = 7. Therefore, the pH value 7 of the CMC−GE−AQ solvent system was selected for subsequent metal ions detection.
Based on the above factors, the suitable detection conditions of CMC−GE−AQ for metal ions was as follows: Volume ratio of DMF/H2O was 8/2, and solvent pH was 7.

Solvents pH Value
As shown in Figure 5a, when the solvent of pH < 5, the CMC−GE−AQ solution showed weak fluorescence intensity. When the solvent of pH ranged from 5 to 7, the fluorescence intensity of CMC−GE−AQ solution increased significantly. When the solvent of pH > 7, the fluorescence intensity of CMC−GE−AQ solution remained at a high level. The suitable pH detection range of CMC−GE−AQ was 5~12. Different concentrations of Cu 2+ were added to the CMC−GE−AQ solution at the pH values 5 and 7, respectively. The relationship between fluorescence intensity and Cu 2+ concentration is shown in Figure 5b. The fluorescence intensity of CMC−GE−AQ solution (pH = 7) decreased with the increase in the concentration of Cu 2+ , which was consistent with the trend of CMC−GE−AQ solution (pH = 5). Since Cu 2+ did not hydrolyze at pH = 5, it also did not hydrolyze at pH = 7. Therefore, the pH value 7 of the CMC−GE−AQ solvent system was selected for subsequent metal ions detection.
Based on the above factors, the suitable detection conditions of CMC−GE−AQ for metal ions was as follows: Volume ratio of DMF/H 2 O was 8/2, and solvent pH was 7.

Fluorescence Responses to Various Metal Ions
The fluorescence selectivity of CMC−GE−AQ solution (1.5 × 10 −4 g mL −1 ) with ent metal ions (10 −4 mol L −1 ) in shown in Figure 6. Among the 10 metal ions, most o had no significant effects on the fluorescent phenomenon of CMC−GE−AQ, whil Cu 2+ significantly influenced the fluorescence emission, causing fluorescence quen (Figure 6a). Thus, the results showed that the CMC−GE−AQ could be used as a florescence sensor for identifying Cu 2+ . When CMC−GE−AQ reacted with Cu 2+ , it w affected by inner−filter effects. In order to study the anti−interference abil CMC−GE−AQ to Cu 2+ detection, the fluorescence emission spectra of CMC−GE−AQ recorded when other metal ions coexisted with Cu 2+ (Figure 6b). As seen in Figu when other metal ions coexist with Cu 2+ , the fluorescence intensity of CMC−GE−AQ tion changed slightly. In Figure 6d, among the 10 metal ions, fluorescence quenchin performed after the interaction between Cu 2+ and CMC−GE−AQ solution. Hence, results indicated that CMC−GE−AQ could be a fluorescence−quenching probe fo with good selectivity and anti−interference.

Fluorescence Responses to Various Metal Ions
The fluorescence selectivity of CMC−GE−AQ solution (1.5 × 10 −4 g mL −1 ) with different metal ions (10 −4 mol L −1 ) in shown in Figure 6. Among the 10 metal ions, most of them had no significant effects on the fluorescent phenomenon of CMC−GE−AQ, while only Cu 2+ significantly influenced the fluorescence emission, causing fluorescence quenching (Figure 6a). Thus, the results showed that the CMC−GE−AQ could be used as a novel florescence sensor for identifying Cu 2+ . When CMC−GE−AQ reacted with Cu 2+ , it was not affected by inner−filter effects. In order to study the anti−interference ability of CMC−GE−AQ to Cu 2+ detection, the fluorescence emission spectra of CMC−GE−AQ were recorded when other metal ions coexisted with Cu 2+ (Figure 6b). As seen in Figure 6c, when other metal ions coexist with Cu 2+ , the fluorescence intensity of CMC−GE−AQ solution changed slightly. In Figure 6d, among the 10 metal ions, fluorescence quenching was performed after the interaction between Cu 2+ and CMC−GE−AQ solution. Hence, those results indicated that CMC−GE−AQ could be a fluorescence−quenching probe for Cu 2+ with good selectivity and anti-interference.

Fluorescence and UV-Vis Responses of CMC−GE−AQ for Cu 2+
The concentration dependence of CMC−GE−AQ on Cu 2+ under fluorescence and UV conditions was studied. Figure 7a presents the fluorescence intensity of CMC−GE−AQ solution (1.5 × 10 −4 g mL −1 ) with different Cu 2+ concentrations (0~10 −4 mol L −1 ), and the fluorescence intensity of CMC−GE−AQ was reduced with the increase in Cu 2+ concentration. In Figure 7b, with the increase in Cu 2+ concentrations (0~7 × 10 −6 mol L −1 ), the maximum fluorescence intensity of CMC−GE−AQ fell quickly, and when the concentration of Cu 2+ was higher than 7 × 10 −6 mol L −1 , this decline became slow. In Figure 7c, the maximum fluorescence intensity of CMC−GE−AQ showed a good linear relationship (R 2 = 0.9903) with concentrations of Cu 2+ in the range of 0~7 × 10 −6 mol L −1 . According to Equation (1), the LOD of CMC−GE−AQ for Cu 2+ was as low as 6.4 × 10 −8 mol L −1 , which is lower than other probes that have been reported for the detection of Cu 2+ (Table 2). Therefore, CMC−GE−AQ could be used as a sensitive quenching probe to detect Cu 2+ at the micromolar level. Figure 7d shows the fluorescence intensity of CMC−GE−AQ solution (1.5 × 10 −4 g mL −1 ) after 3 cycles of Cu 2+ (10 −4 mol L −1 ) and EDTA (1.5 × 10 −4 mol L −1 ). The fluorescence intensity of CMC−GE−AQ recovered to 82% of the initial intensity after three cycles, indicating that CMC−GE−AQ is a reversible probe for Cu 2+ . In general, the fluorescence quenching process could be divided into static quenching and dynamic quenching [39]. In order to distinguish them, the UV-Vis absorption spectra of  (Figure 7f), indicating that the quenching effect of Cu 2+ on CMC−GE−AQ was attributed to a static quenching mechanism, and these absorption peak changes should be caused by the complexation of CMC−GE−AQ with Cu 2+ [39].

Fluorescence and UV-Vis Responses of CMC−GE−AQ for Cu 2+
The concentration dependence of CMC−GE−AQ on Cu 2+ under fluorescence an conditions was studied. Figure 7a presents the fluorescence intensity of CMC−GE−A lution (1.5 × 10 −4 g mL −1 ) with different Cu 2+ concentrations (0~10 −4 mol L −1 ), and the rescence intensity of CMC−GE−AQ was reduced with the increase in Cu 2+ concentr In Figure 7b, with the increase in Cu 2+ concentrations (0~7 × 10 −6 mol L −1 ), the max fluorescence intensity of CMC−GE−AQ fell quickly, and when the concentration o was higher than 7 × 10 −6 mol L −1 , this decline became slow. In Figure 7c, the max fluorescence intensity of CMC−GE−AQ showed a good linear relationship (R 2 = 0 with concentrations of Cu 2+ in the range of 0~7 × 10 −6 mol L −1 . According to Equatio the LOD of CMC−GE−AQ for Cu 2+ was as low as 6.4 × 10 −8 mol L −1 , which is lowe other probes that have been reported for the detection of Cu 2+ (Table 2). Ther CMC−GE−AQ could be used as a sensitive quenching probe to detect Cu 2+ at the mic lar level. Figure 7d shows the fluorescence intensity of CMC−GE−AQ solution (1.5 g mL −1 ) after 3 cycles of Cu 2+ (10 −4 mol L −1 ) and EDTA (1.5 × 10 −4 mol L −1 ). The fluores intensity of CMC−GE−AQ recovered to 82% of the initial intensity after three cycles cating that CMC−GE−AQ is a reversible probe for Cu 2+ . In general, the fluores quenching process could be divided into static quenching and dynamic quenching In order to distinguish them, the UV-Vis absorption spectra of CMC−GE−AQ (1.5 × mL −1 ) with different amounts of Cu 2+ (0~10 −4 mol L −1 ) were measured and illustra Figure 7e. With the increase in Cu 2+ concentrations, the UV-Vis absorption pe

Detection Mechanism Study
The static quenching was studied in terms of the Stern-Volmer equation shown in Equation (2) [39]. In addition, the binding constants and number of binding points could be used to determine whether the interaction between CMC−GE−AQ and Cu 2+ formed a complex. It is assumed that Cu 2+ has an independent number of binding sites (n) on CMC−GE−AQ. The apparent binding constant (K a ) and the number of binding sites (n) was determined based on Equation (3) [48]:  Figure 8b. Based on Equation (3), K a and n were obtained to be 1.7 × 10 6 mol −1 L and 1.2, respectively. The high correlation coefficient (R 2 = 0.998) indicated that the assumption proposed was reasonable. Moreover, K a was greater than 10 4 mol −1 L, indicating that CMC−GE−AQ had a strong binding ability with Cu 2+ , and n was close to 1, suggesting that there was one binding site for CMC−GE−AQ toward Cu 2+ .
Polymers 2022, 14, x FOR PEER REVIEW Cu 2+ on CMC−GE−AQ was attributed to a static quenching mechanism, and these a tion peak changes should be caused by the complexation of CMC−GE−AQ with Cu

Detection Mechanism Study
The static quenching was studied in terms of the Stern-Volmer equation sho Equation (2) [39]. In addition, the binding constants and number of binding points be used to determine whether the interaction between CMC−GE−AQ and Cu 2+ for complex. It is assumed that Cu 2+ has an independent number of binding sites CMC−GE−AQ. The apparent binding constant (Ka) and the number of binding si was determined based on Equation (3) Figure 8a. Based on Equation (2), Ks was calculated to 10 5 mol −1 L. The linear relation of lg((F0 − F)/F) versus lg[Cu 2+ ] is displayed in Fig  Based on Equation (3), Ka and n were obtained to be 1.7 × 10 6 mol −1 L and 1.2, respe The high correlation coefficient (R 2 = 0.998) indicated that the assumption propo reasonable. Moreover, Ka was greater than 10 4 mol −1 L, indicating that CMC−GE− a strong binding ability with Cu 2+ , and n was close to 1, suggesting that there w binding site for CMC−GE−AQ toward Cu 2+ . The recognition mechanism of CMC−GE−AQ for Cu 2+ was investigated by XP ysis and DFT calculations. As shown in Figure 9, after the reaction of CMC−GE−A Cu 2+ , the absorption of O 1s decreased significantly, and the absorption of CMC− at 497 eV vanished after the reaction with Cu 2+ . In addition, the CMC−GE−AQ + Cu plex showed a weak absorption at 933.34 eV, which originated from the character sorption of Cu 2+ [49]. Therefore, the O atom on CMC−GE−AQ was involved in t plexation of Cu 2+ . The recognition mechanism of CMC−GE−AQ for Cu 2+ was investigated by XPS analysis and DFT calculations. As shown in Figure 9, after the reaction of CMC−GE−AQ with Cu 2+ , the absorption of O 1s decreased significantly, and the absorption of CMC−GE−AQ at 497 eV vanished after the reaction with Cu 2+ . In addition, the CMC−GE−AQ + Cu 2+ complex showed a weak absorption at 933.34 eV, which originated from the characteristic absorption of Cu 2+ [49]. Therefore, the O atom on CMC−GE−AQ was involved in the complexation of Cu 2+ .  According to the high−resolution XPS spectra of O 1s (Figure 10a,b). The three absorptions at 532.  According to the High-resolution XPS spectra of O 1s (Figure 10a Density functional theory (DFT) calculations of the CMC−GE−AQ and CMC−G + Cu 2+ complexes were performed by the Gaussian 09 program. The optimal struct the CMC−GE−AQ and CMC−GE−AQ + Cu 2+ complexes along with their highest oc molecular orbital (HOMO) and lowest unoccupied molecular orbital (LUMO) a picted in Figure 11. The HOMOs of CMC−GE−AQ were mainly distributed on the thalene ring and hydroxyl, and the LUMOs were distributed on the whole napht ring. The HOMOs of the CMC−GE−AQ + Cu 2+ complex were mainly distributed naphthalene ring and oxygen−containing functional groups (carboxyl and hyd while the LUMOs were distributed on the whole naphthalene ring. Therefore CMC−GE−AQ interacted with Cu 2+ , the electrons were transferred from the carb the naphthalene ring, which would lead to the change in fluorescence, and the s mechanism of the interaction between CMC−GE−AQ and Cu 2+ was a photoinduce tron transfer (PET) [14,16]. After the interaction between CMC−GE−AQ and Cu HOMO−LUMO energy gaps changed from 3.82 to 3.00 eV, becoming more stabl may be due to the fact that the coordination with metal made the struct CMC−GE−AQ more planar, thus increasing the stiffness of the CMC−GE−AQ [50]. Density functional theory (DFT) calculations of the CMC−GE−AQ and CMC−GE−AQ + Cu 2+ complexes were performed by the Gaussian 09 program. The optimal structures of the CMC−GE−AQ and CMC−GE−AQ + Cu 2+ complexes along with their highest occupied molecular orbital (HOMO) and lowest unoccupied molecular orbital (LUMO) are depicted in Figure 11. The HOMOs of CMC−GE−AQ were mainly distributed on the naphthalene ring and hydroxyl, and the LUMOs were distributed on the whole naphthalene ring. The HOMOs of the CMC−GE−AQ + Cu 2+ complex were mainly distributed on the naphthalene ring and oxygen−containing functional groups (carboxyl and hydroxyl), while the LUMOs were distributed on the whole naphthalene ring. Therefore, after CMC−GE−AQ interacted with Cu 2+ , the electrons were transferred from the carboxyl to the naphthalene ring, which would lead to the change in fluorescence, and the sensing mechanism of the interaction between CMC−GE−AQ and Cu 2+ was a photoinduced electron transfer (PET) [14,16]. After the interaction between CMC−GE−AQ and Cu 2+ , the HOMO-LUMO energy gaps changed from 3.82 to 3.00 eV, becoming more stable. This may be due to the fact that the coordination with metal made the structure of CMC−GE−AQ more planar, thus increasing the stiffness of the CMC−GE−AQ [50].

Application of CMC−GE−AQ
This study focused on the environmental and materialized application of CMC−GE−AQ. The probe CMC−GE−AQ could be used to detect Cu 2+ in real water samples. Based on the good processing performance of carboxymethyl cellulose, CMC−GE−AQ prepared into portable microspheres also had the ability to detect Cu 2+ .

Application in Real Water Samples
With the development of modern industry, the discharge of a large number of industrial wastewater containing Cu 2+ has caused serious water pollution, which not only has seeped into the groundwater, but has also flowed into rivers and lakes. Therefore, three real water samples (Xuanwu Lake water, Yangtze River water and tap water) were selected to detect Cu 2+ . As shown in Figure 13, the fluorescence intensity of CMC−GE−AQ solution at 579 nm showed a good linear relationship with the concentration of Cu 2+ (0, 1, 3, 5 and 7 × 10 −6 mol L −1 ). The concentrations of Cu 2+ were calculated according to the fitting equation of CMC−GE−AQ solution and were measured by atomic absorption spectrometry, as shown in Table 3. Compared with the recovery of Cu 2+ measured by atomic absorption spectrometry (112.0%~183.0%), the recovery of Cu 2+ measured by CMC−GE−AQ (82.0%~118.7%) was closer to 100%. These results indicate that the probe CMC could detect Cu 2+ in environmental real water samples.

Application of CMC−GE−AQ
This study focused on the environmental and materialized applicat CMC−GE−AQ. The probe CMC−GE−AQ could be used to detect Cu 2+ in real wate ples. Based on the good processing performance of carboxymethyl ce CMC−GE−AQ prepared into portable microspheres also had the ability to detect C

Application in Real Water Samples
With the development of modern industry, the discharge of a large number of trial wastewater containing Cu 2+ has caused serious water pollution, which not o seeped into the groundwater, but has also flowed into rivers and lakes. Therefore real water samples (Xuanwu Lake water, Yangtze River water and tap water) w lected to detect Cu 2+ . As shown in Figure 13, the fluorescence intensity of CMC−G solution at 579 nm showed a good linear relationship with the concentration of Cu 3, 5 and 7 × 10 −6 mol L −1 ). The concentrations of Cu 2+ were calculated according to the equation of CMC−GE−AQ solution and were measured by atomic absorption spec try, as shown in Table 3. Compared with the recovery of Cu 2+ measured by atomic a tion spectrometry (112.0%~183.0%), the recovery of Cu 2+ measured by CMC−G (82.0%~118.7%) was closer to 100%. These results indicate that the probe CMC co tect Cu 2+ in environmental real water samples.  In Figure 14a, CMC−GE−AQ microspheres all aggregated and sank to the bottom, turning black in color and decreasing in size. In Figure 14b, under a 365 nm UV lamp, CMC−GE−AQ microspheres suspension showed bright orange fluorescence and fluorescence quenching after interacting with Cu 2+ . In Figure 14c, CMC−GE−AQ microspheres had obvious aggregation phenomena after the interaction with Cu 2+ , and the size of the microspheres decreased from 400 to 100 µm. Therefore, CMC−GE−AQ microspheres could be used as a fluorescence sensor of Cu 2+ and could recognize Cu 2+ in aqueous solution with the naked eye.   CMC−GE−AQ microspheres were added into DMF/H 2 O (v/v = 8/2) solution (1.5 × 10 −4 g mL −1 , pH = 7) with several typical metal ions and different concentrations (0~60 × 10 −6 mol L −1 ) of Cu 2+ . Afterward, the microspheres were employed for imaging by confocal laser scanning microscope (CLSM). As shown in Figure 15a, after interacting with several typical metal ions, only Cu 2+ could quench the fluorescence of CMC−GE−AQ suspension, indicating that the CMC−GE−AQ-based microspheres also have high selectivity for detecting Cu 2+ . As shown in Figure 15b,c the fluorescence of CMC−GE−AQ microspheres decreased continuously after adding different concentrations of Cu 2+ , which was consistent with the conclusion of fluorescence spectra. Therefore, CMC−GE−AQbased microspheres could be used as an effective and convenient tool for Cu 2+ sensing.

Conclusions
In summary, a simple fluorescent probe CMC−GE−AQ for the detection of Cu 2+ was successfully prepared. Upon coordination with Cu 2+ , the fluorescent color of CMC−GE−AQ changed significantly from orange to colorless. CMC−GEj−AQ exhibited a

Conclusions
In summary, a simple fluorescent probe CMC−GE−AQ for the detection of Cu 2+ was successfully prepared. Upon coordination with Cu 2+ , the fluorescent color of CMC−GE−AQ changed significantly from orange to colorless. CMC−GEj−AQ exhibited a good sensitivity, selectivity and reversibility for Cu 2+ . Its LOD for Cu 2+ was computed to be 6.4 × 10 −8 mol L −1 , and K a and n were obtained to be 1.7 × 10 6 mol −1 L and 1.2, respectively. The detection mechanism was confirmed by XPS and DFT calculations. In addition, probe CMC−GE−AQ could monitor Cu 2+ in real water samples. Furthermore, CMC−GE−AQ-based fluorescent microspheres could serve as an effective tool for detecting Cu 2+ . This work promoted the development of CMC in the field of fluorescent sensing.

Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.

Data Availability Statement:
The data presented in this study are available on request from the corresponding author.