A Nitro Functionalized MOF with Multi-Enzyme Mimetic Activities for the Colorimetric Sensing of Glucose at Neutral pH

Benefiting from the advantages like large surface area, flexible constitution, and diverse structure, metal-organic frameworks (MOFs) have been one of the most ideal candidates for nanozymes. In this study, a nitro-functionalized MOF, namely NO2-MIL-53(Cu), was synthesized. Multi-enzyme mimetic activities were discovered on this MOF, including peroxidase-like, oxidase-like, and laccase-like activity. Compared to the non-functional counterpart (MIL-53(Cu)), NO2-MIL-53(Cu) displayed superior enzyme mimetic activities, indicating a positive role of the nitro group in the MOF. Subsequently, the effects of reaction conditions on enzyme mimetic activities were investigated. Remarkably, NO2-MIL-53(Cu) exhibited excellent peroxidase-like activity even at neutral pH. Based on this finding, a simple colorimetric sensing platform was developed for the detection of H2O2 and glucose, respectively. The detection liner range for H2O2 is 1–800 μM with a detection limit of 0.69 μM. The detection liner range for glucose is linear range 0.5–300 μM with a detection limit of 2.6 μM. Therefore, this work not only provides an applicable colorimetric platform for glucose detection in a physiological environment, but also offers guidance for the rational design of efficient nanozymes with multi-enzyme mimetic activities.


Introduction
Nanozymes are a class of synthetic nanomaterials that exhibit enzyme-like catalytic activity. Compared with natural enzymes, nanozymes offer numerous advantages, including diverse structures, facile synthesis, high stability, and easy regulation and storage [1,2]. These characteristics have led to their widespread use in various fields, such as disease diagnosis, biosensing, environmental remediation, and so on [3][4][5]. Previous research has demonstrated that various nanomaterials can mimic different natural enzymes, including peroxidase [1][2][3], oxidase [4][5][6], superoxide dismutase [7], catalase, laccase, and others [8][9][10]. However, most of these studies have focused on the activity of single enzymes [6,7]. Recently, there has been a growing interest in multi-enzyme nanozymes, which can exhibit multi-enzyme mimetic activities alone or in combination, leading to synergistic effects, cascade reactions, and environmental response selectivity [8], as well as even broader application prospects. Despite the potential benefits, the development of highly efficient nanozymes with multiple activities is still in demand due to the limited number of nanozymes with well-defined structures, and multi-enzyme mimetic activities [9][10][11][12].
Metal-organic frameworks (MOFs) are porous crystalline materials composed of metal junctions and organic linkers [2]. Due to their large surface area, flexible constitution, and diverse structure, MOFs have emerged as ideal candidates for nanozymes [13,14]. Furthermore, the highly tunable and ordered structure of MOFs makes them attractive microreactors for dual-enzyme or multi-enzyme tandem-catalyzed reactions [15]. These metal junctions and organic linkers [2]. Due to their large surface area, flexible constitution, and diverse structure, MOFs have emerged as ideal candidates for nanozymes [13,14]. Furthermore, the highly tunable and ordered structure of MOFs makes them attractive microreactors for dual−enzyme or multi−enzyme tandem−catalyzed reactions [15]. These characteristics suggest a great potential to construct nanozymes with multi−enzyme mimetic activities, which warrants further investigation. Glucose, which is the crucial substance that produces energy in organisms, is very important for keeping the body functioning in good order [16,17]. Therefore, measuring blood glucose levels is essential for maintaining physiological health and treating diseases. Although there has been well−established research on glucose sensors, colorimetric techniques have gained high attention because of the development of the miniaturization concept, like paper−based analytical devices and image capture instruments [18]. Among the various colorimetric sensing modes, glucose oxidase (GOx)−based enzyme−catalyzed oxidation has proved to be a simple and reliable method for glucose detection. During the detection, glucose is hydrolyzed into gluconic acid, and H2O2 by GOx, and the in situ generated H2O2 is then catalyzed by peroxidase to oxidize the substrate for a visible color change. However, limited by the low catalytic efficiency, many previously reported peroxidase−like nanozyme to suffer from a low sensitivity for glucose detection. On the other hand, the instability of H2O2 and the difficulty of substrate oxidization under neutral pH have significantly hampered the development of this cascade reaction [19][20][21]. That means, most previous reports conducted the GOx incubation under neutral pH, and then, they had to adjust the pH into acid for peroxidase incubation. To overcome these shortcomings, it is still in demand to fabricate a novel peroxidase−like nanozyme with high catalytic efficiency under neutral pH, thus, constructing a simple and sensitive colorimetric platform for glucose detection.
In this work, we synthesized NO2−MIL−53(Cu) via a simple hydrothermal method. The regular complex structure centered on Cu allowed this material to mimic the activity of multiple enzymes, including peroxidase−like, oxidase−like, and laccase−like activity (as shown in Figure 1. To figure out the role of the functional group, MIL−53(Cu) was synthesized as a counterpart, and the activities were compared between NO2−MIL−53(Cu) and MIL−53(Cu). Then, the effects of different reaction conditions on enzyme mimetic activities were explored. Based on the excellent peroxidase−like activity of NO2−MIL−53(Cu) at neutral pH, a colorimetric sensing platform to detect H2O2 and glucose under a physiological environment was constructed.
The detection limits are calculated as LOD = 3σ/S, where σ was the standard deviation from 20 blank probe sample measurements, and s was the slope of the calibration plot from UV absorbance of solutions with different concentrations of targets (H 2 O 2 or glucose).

Synthesis and Characterization of NO 2 -MIL-53(Cu)
The synthesis of NO 2 -MIL-53(Cu) was carried out using a modified version of a previously reported method [22]. A total of 0.6334 g of NO 2 -BDC and 0.7986 g of Cu(CH 3 COO) 2 ·H 2 O were dissolved in 30 mL of H 2 O and stirred vigorously for 30 min. A total of 0.3903 g of urea was added to the mixed solution and stirred for another 30 min. The mixture was then transferred to a hydrothermal reactor with 50 mL of Teflon and subjected to solvothermal treatment at 150 • C for 5 h. After cooling to room temperature, the resulting precipitate was separated by using a 0.22 µm filter membrane and washed thoroughly with deionized water. Then, the collected product was washed with DMF and methanol several times. Finally, the obtained solid was dried at 60 • C for further characterization and application.
MIL-53(Cu) was synthesized by using 0.4984 g of H 2 BDC while keeping other steps unchanged.

Synthesis and Characterization of NO 2 -MIL-53(Cu)
Previous research has demonstrated that the enzyme mimetic activity of nanozymes can be influenced by their functional groups [23]. In order to obtain nanozymes with superior activity, a nitro-functionalized MOF was synthesized according to the previous method with some modifications. As shown in Figure 2, the resulting NO 2 -MIL-53(Cu) exhibited a leaf-like structure with plan view sizes ranging from 2 to 4 µm ( Figure 2a). In contrast, MIL-53(Cu), which was synthesized using the same method, displayed a stick-shaped structure with plan view sizes ranging from 0.2 to 1.5 µm (Figure 2b). The difference in morphology may be attributed to the addition of urea, a weak base used during the preparation process to deprotonate the groups in the organic linker and facilitate the coordination between metal ions and the organic linker. The protonation differences between BDC and NO 2 -BDC likely altered the kinetics of MOF formation [24,25].
M, pH 7.4) were consecutively added to obtain a total volume of 1000 μL. The resulting mixture was incubated for another 30 min at 37 °C, and then measured by UV-vis spectrometer.

Oxidase−like Catalytic Feature Evaluation of NO2−MIL−53(Cu)
To explore the oxidase−mimicking behavior, 200 μL of NO2−MIL−53(Cu) suspension (0.2 mg mL −1 ) was added to an OPD solution (1 mM) in 0.01 M Tris−HCl buffer (pH 7.4) with a total volume 1 mL. The solution was also incubated at 37 °C for 30 min and finally monitored via UV-vis spectrometer.

Synthesis and Characterization of NO2−MIL−53(Cu)
Previous research has demonstrated that the enzyme mimetic activity of nanozymes can be influenced by their functional groups [23]. In order to obtain nanozymes with superior activity, a nitro−functionalized MOF was synthesized according to the previous method with some modifications. As shown in Figure 2, the resulting NO2−MIL−53(Cu) exhibited a leaf−like structure with plan view sizes ranging from 2 to 4 μm ( Figure 2a). In contrast, MIL−53(Cu), which was synthesized using the same method, displayed a stick−shaped structure with plan view sizes ranging from 0.2 to 1.5 μm (Figure 2b). The difference in morphology may be attributed to the addition of urea, a weak base used during the preparation process to deprotonate the groups in the organic linker and facilitate the coordination between metal ions and the organic linker. The protonation differences between BDC and NO2−BDC likely altered the kinetics of MOF formation [24,25].       (Figure 4b). These changes in concentration would promote the oxidase-like activity of NO 2 -MIL-53(Cu) via offered more active sites to initiate the redox reaction between oxygen and OPD. As expected, the solution presented a clear OPD oxidation peak at 420 nm, accompanied by a distinguishable color change from colorless to yellow. No peak was observed in the solution of NO 2 -MIL-53(Cu) and OPD alone, confirming the oxidase-like activity of NO 2 -MIL-53(Cu). Laccases were a family of copper-containing oxidases. To test the feasibility of NO 2 -MIL-53(Cu) as a laccase mimic, we incubated 2,4-dichlorophenol (2,4-DP) and 4-aminoantipyrine (4-AP) with NO 2 -MIL-53(Cu). As shown in Figure 4c, an absorption peak at 510 nm was observed after incubation, which can be attributed to the oxidation product of 2,4-DP. No absorption was observed with each compound alone. Similar results were observed in the image, in which only the solution with NO 2 -MIL-53(Cu) turned pink. The above results distinctly indicated that the prepared NO 2 -MIL-53(Cu) had laccase-like activity.
To uncover the role of the nitro group in the organic linker, MIL-53(Cu) was used as a non-functional counterpart to NO 2 -MIL-53(Cu). As shown in Figure S1, all of the incubation systems containing NO 2 -MIL-53(Cu) catalyzed substrates to produce high characteristic peaks, accompanied by obvious color change. In contrast, the systems containing MIL-53(Cu) only produced weak characteristic peaks and color changes. These results indicated that NO 2 -MIL-53(Cu) exhibited overwhelming enzyme mimetic activities, and the nitro group appears to play a positive role in the multi-enzyme mimetic catalysis. In previous reports by Wei et al., the nitro group was proposed as an electron-absorbing group that made the metal nodes in MOFs more electron-deficient, thereby promoting electron transfer between substrates and active sites [23]. This explanation could also be used to account for the improved activity of NO 2 -MIL-53(Cu).  To uncover the role of the nitro group in the organic linker, MIL−53(Cu) was used as a non−functional counterpart to NO2−MIL−53(Cu). As shown in Figure S1, all of the incubation systems containing NO2−MIL−53(Cu) catalyzed substrates to produce high characteristic peaks, accompanied by obvious color change. In contrast, the systems containing MIL−53(Cu) only produced weak characteristic peaks and color changes. These results indicated that NO2−MIL−53(Cu) exhibited overwhelming enzyme mimetic activities, and the nitro group appears to play a positive role in the multi−enzyme mimetic catalysis. In previous reports by Wei et al., the nitro group was proposed as an electron−absorbing group that made the metal nodes in MOFs more electron−deficient, thereby promoting electron transfer between substrates and active sites [23]. This explanation could also be used to account for the improved activity of NO2−MIL−53(Cu).

Optimization of the Peroxidase−like Activity for NO2−MIL−53(Cu)
As it is known, the peroxidase−like activity of nanozyme is always dependent on reaction conditions. Therefore, the effects of typical conditions for NO2−MIL−53(Cu) were investigated. For solution pH, the peroxidase−like activity increased before pH 7.4 and decreased when pH exceeded 7.4 (as shown in Figure 5a), suggesting that the optimal pH for this reaction was around 7.4, which was consistent with the physiological environment. This applicability was further confirmed by testing different substrates. As shown in Figure S2 To study the effect of catalyst concentrations, seven concentrations, including 5, 10, 15, 20, 25, 30, and 35 mg/L of NO2−MIL−53(Cu), were selected. As shown in Figure 5b, a growth trend was observed as the catalyst concentration increased, but the trend slowed down when the catalyst concentration exceeded 20 mg/L.
We also explored the effects of OPD concentrations and reaction time, as shown in Figure 5c

Optimization of the Peroxidase-like Activity for NO 2 -MIL-53(Cu)
As it is known, the peroxidase-like activity of nanozyme is always dependent on reaction conditions. Therefore, the effects of typical conditions for NO 2 -MIL-53(Cu) were investigated. For solution pH, the peroxidase-like activity increased before pH 7.4 and decreased when pH exceeded 7.4 (as shown in Figure 5a), suggesting that the optimal pH for this reaction was around 7.4, which was consistent with the physiological environment. This applicability was further confirmed by testing different substrates. As shown in Figure S2, when we replaced OPD with other typical enzyme-linked chromogenic substrates, such as 3,3 ,5,5 -tetramethylbenzidine (TMB), and 2,2 -azino-bis(3-ethylbenzothiazoline-6-sulfonic acid (ABTS), NO 2 -MIL-53(Cu) was able to catalyze the oxidation of different substrates under neutral conditions, demonstrating the wide applicability of peroxidase-like NO 2 -MIL-53(Cu).
To study the effect of catalyst concentrations, seven concentrations, including 5, 10, 15, 20, 25, 30, and 35 mg/L of NO 2 -MIL-53(Cu), were selected. As shown in Figure 5b, a growth trend was observed as the catalyst concentration increased, but the trend slowed down when the catalyst concentration exceeded 20 mg/L.
We also explored the effects of OPD concentrations and reaction time, as shown in Figure 5c,d, respectively. When the OPD concentration increased to 0.3 mM, further increases in reaction concentration had little effect. A similar phenomenon was observed when the reaction time reached 30 min.
The stability of the peroxidase-like activity for NO 2 -MIL-53(Cu) was evaluated by storing NO 2 -MIL-53(Cu) in aqueous solution at 2-8 • C. Its peroxidase-like activity was measured for seven consecutive days. As shown in Figure S3, it was found that the activity of NO 2 -MIL-53(Cu) remained above 90% on the seventh day, indicating good stability.

Optimization of the Oxidase-like Activity and Laccase-like Activity for NO 2 -MIL-53(Cu)
Factors affecting the oxidase-like activity of NO 2 -MIL-53(Cu) were also investigated, including pH (ranging from 4 to 9), catalyst concentration (0, 5,10,15,20,25,30,35,40,45, 50 mg/L) and reaction time (5, 10, 15, 20, 25, 30, 35, 40, 45, 50 min). As shown in Figure S4a, NO 2 -MIL-53(Cu) exhibited high catalytic efficiency at pH values ranging from 7 to 8.5. With respect to reaction time, the absorbance of oxidized OPD increased with increasing reaction time and reached an appropriate UV measure range at 30 min (as shown in Figure S4b). Regarding the effect of catalyst concentration, the background absorbance from the catalyst should be taken into account. As shown in Figure S4c, the oxidase-like activity of NO 2 -MIL-53(Cu) reached stable when the catalyst concentration was up to 40 mg/L, since the differential value of absorbance tended to be constant at this concentration.
Sensors 2023, 23, x FOR PEER REVIEW 7 of 11 measured for seven consecutive days. As shown in Figure S3, it was found that the activity of NO2−MIL−53(Cu) remained above 90% on the seventh day, indicating good stability.

Optimization of the Oxidase−like Activity and Laccase−like Activity for NO2−MIL−53(Cu)
Factors affecting the oxidase−like activity of NO2−MIL−53(Cu) were also investigated, including pH (ranging from 4 to 9), catalyst concentration (0, 5,10,15,20,25,30,35,40,45, 50 mg/L) and reaction time (5, 10, 15, 20, 25, 30, 35, 40, 45, 50 min). As shown in Figure S4a, NO2−MIL−53(Cu) exhibited high catalytic efficiency at pH values ranging from 7 to 8.5. With respect to reaction time, the absorbance of oxidized OPD increased with increasing reaction time and reached an appropriate UV measure range at 30 min (as shown in Figure S4b). Regarding the effect of catalyst concentration, the background absorbance from the catalyst should be taken into account. As shown in Figure S4c, the oxidase−like activity of NO2−MIL−53(Cu) reached stable when the catalyst concentration was up to 40 mg/L, since the differential value of absorbance tended to be constant at this concentration.
In addition, we studied the effect of pH on the laccase−like activity of NO2−MIL−53(Cu). As shown in Figure S4d, NO2−MIL−53(Cu) exhibited the highest catalytic activity at pH 6.8, which was similar to natural laccase. Nanomaterials with oxidase−like activity have been widely used for biosensors (such as the detection of metal ions and reducing substances), antibacterial and environmental protection [29][30][31]. As a green catalyst, nanomaterials with laccase−like activity have broad application prospects in environmental pollutant removal and environmental remediation [32,33]. In addition, laccase can also be used in biosensors (especially the detection of epinephrine), the food industry, and the paper industry [34][35][36]. Benefit from the synergistic effect of nanomaterials with multi−enzyme activity; they are also widely used in disease treatments. For example, Fe3O4/Ag/Bi2MoO6 nanoparticles with multiple−enzyme mimic activities are designed for multimodal imaging−guided chemi- In addition, we studied the effect of pH on the laccase-like activity of NO 2 -MIL-53(Cu). As shown in Figure S4d, NO 2 -MIL-53(Cu) exhibited the highest catalytic activity at pH 6.8, which was similar to natural laccase. Nanomaterials with oxidase-like activity have been widely used for biosensors (such as the detection of metal ions and reducing substances), antibacterial and environmental protection [29][30][31]. As a green catalyst, nanomaterials with laccase-like activity have broad application prospects in environmental pollutant removal and environmental remediation [32,33]. In addition, laccase can also be used in biosensors (especially the detection of epinephrine), the food industry, and the paper industry [34][35][36]. Benefit from the synergistic effect of nanomaterials with multi-enzyme activity; they are also widely used in disease treatments. For example, Fe 3 O 4 /Ag/Bi 2 MoO 6 nanoparticles with multiple-enzyme mimic activities are designed for multimodal imaging-guided chemical/photodynamic/photothermal therapy of tumors [37]. All of these examples have promised the great potential of NO2-MIL-53(Cu) in various fields.

Colorimetric Detection of H 2 O 2 and Glucose
Based on the peroxidase-like activity of the NO 2 -MIL-53(Cu), we established a NO 2 -MIL-53(Cu)/OPD sensing system for colorimetric detection of H 2 O 2 . As shown in Figure 6, the color of the reaction solution gradually turned yellow upon the addition of H 2 O 2 to MIL-53(Cu)/OPD. It was shown that the absorption intensity at 420 nm of the oxidized OPD increased sharply with increasing concentration of H 2 O 2 . A good linear relationship was found between the absorbance and H 2 O 2 concentrations ranging from 1 to 800 µM (R 2 = 0.9964). The limit of detection(LOD) for H 2 O 2 was calculated as 0.69 µM (S/N = 3). NO2−MIL−53(Cu)/OPD sensing system for colorimetric detection of H2O2. As shown in Figure 6, the color of the reaction solution gradually turned yellow upon the addition of H2O2 to MIL−53(Cu)/OPD. It was shown that the absorption intensity at 420 nm of the oxidized OPD increased sharply with increasing concentration of H2O2. A good linear relationship was found between the absorbance and H2O2 concentrations ranging from 1 to 800 μM (R 2 = 0.9964). The limit of detection(LOD) for H2O2 was calculated as 0.69 μM (S/N = 3). As well known, glucose can be hydrolyzed by glucose oxidase(GOx) to produce H2O2 [38]. Therefore, a colorimetric platform can be achieved for glucose detection via the cascading reactions of NO2−MIL−53(Cu) and GOx. As shown in Figure S5, even in the incubation system of OPD/NO2−MIL−53(Cu), there was a weak absorption around 420 nm due to the inherent oxidase−like activity of NO2−MIL−53(Cu). This result is consistent with Figure 4a. When GOx or glucose was added separately, little change in this peak was observed. For the system containing GOx and glucose together, a significant increase in this peak can be observed. These results confirm that H2O2 is only produced when GOx is co−incubated with glucose, which in turn causes a peroxidase−like catalytic reaction.
Based on this, the absorbance at 420 nm increased with increasing glucose concentration (as shown in Figure 7a). Additionally, the inset image showed that the yellow color of the reaction system became darker with increasing glucose concentration. Figure  7b showed a good linear relationship between the glucose concentrations and the absorbance in the range of 0.5 μM to 300 μM (R 2 = 0.9928), with a LOD of 2.6 μM (S/N = 3). This result is sufficient for the routine health screening of glucose in body fluids. Compared with previous reports, the performance of NO2−MIL−53(Cu) is also considerable (Table S1). As well known, glucose can be hydrolyzed by glucose oxidase(GOx) to produce H 2 O 2 [38]. Therefore, a colorimetric platform can be achieved for glucose detection via the cascading reactions of NO 2 -MIL-53(Cu) and GOx. As shown in Figure S5, even in the incubation system of OPD/NO 2 -MIL-53(Cu), there was a weak absorption around 420 nm due to the inherent oxidase-like activity of NO 2 -MIL-53(Cu). This result is consistent with Figure 4a. When GOx or glucose was added separately, little change in this peak was observed. For the system containing GOx and glucose together, a significant increase in this peak can be observed. These results confirm that H 2 O 2 is only produced when GOx is co-incubated with glucose, which in turn causes a peroxidase-like catalytic reaction.
Based on this, the absorbance at 420 nm increased with increasing glucose concentration (as shown in Figure 7a). Additionally, the inset image showed that the yellow color of the reaction system became darker with increasing glucose concentration. Figure 7b showed a good linear relationship between the glucose concentrations and the absorbance in the range of 0.5 µM to 300 µM (R 2 = 0.9928), with a LOD of 2.6 µM (S/N = 3). This result is sufficient for the routine health screening of glucose in body fluids. Compared with previous reports, the performance of NO 2 -MIL-53(Cu) is also considerable (Table S1).  In order to evaluate the feasibility of the established platform for glucose detection, the effects of other sugars (sucrose, maltose, lactose, etc.) and common biochemical species in body fluids (amino acids, metal ions, ascorbic acid, etc.) were studied on the detection system. As shown in Figure S6, only the addition of the glucose triggered a significant chromogenic reaction, while the effect of other targets was negligible. These results demonstrated that the method has good selectivity for the detection of glucose.

Conclusions
In conclusion, we synthesized a nitro−functionalized MOF using hydrothermal In order to evaluate the feasibility of the established platform for glucose detection, the effects of other sugars (sucrose, maltose, lactose, etc.) and common biochemical species in body fluids (amino acids, metal ions, ascorbic acid, etc.) were studied on the detection system. As shown in Figure S6, only the addition of the glucose triggered a significant chromogenic reaction, while the effect of other targets was negligible. These results demonstrated that the method has good selectivity for the detection of glucose.

Conclusions
In conclusion, we synthesized a nitro-functionalized MOF using hydrothermal methods. The synthesized NO 2 -MIL-53(Cu) demonstrated peroxidase-like, oxidase-like, and laccase-like catalytic activity. The effects of reaction conditions were discussed, and it was found that NO 2 -MIL-53(Cu) exhibited excellent peroxidase-like activity under neutral conditions. Based on this, a colorimetric sensing platform for hydrogen peroxide and glucose was constructed. Wide linear ranges could be achieved for H 2 O 2 (1-800 µM) and glucose (0.5-300 µM), accompanied by low LODs of 0.69 µM for H 2 O 2 and 2.6 µM for glucose, respectively. The platform exhibited good selectivity and promising potential feasibility for glucose detection in physiological environments. This work not only offered a more applicable colorimetric platform for glucose detection, but also provided guidelines for the rational design of nanozymes, especially for the materials with multi-enzyme mimetic activities.