Dual-Enzyme Cascade Composed of Chitosan Coated FeS2 Nanozyme and Glucose Oxidase for Sensitive Glucose Detection

Immobilizing enzymes with nanozymes to catalyze cascade reactions overcomes many of the shortcomings of biological enzymes in industrial manufacturing. In the study, glucose oxidases were covalently bound to FeS2 nanozymes as immobilization carriers while chitosan encapsulation increased the activity and stability of the immobilized enzymes. The immobilized enzymes exhibited a 10% greater increase in catalytic efficiency than the free enzymes while also being more stable and catalytically active in environments with an alkaline pH of 9.0 and a high temperature of 100 °C. Additionally, the FeS2 nanozyme-driven double-enzyme cascade reaction showed high glucose selectivity, even in the presence of lactose, dopamine, and uric acid, with a limit of detection (LOD) (S/N = 3) as low as 1.9 × 10−6 M. This research demonstrates that nanozymes may be employed as ideal carriers for biological enzymes and that the nanozymes can catalyze cascade reactions together with natural enzymes, offering new insights into interactions between natural and synthetic biosystems.


Introduction
As with mostly protein-based biological catalysts, enzymes accelerate chemical reactions by reducing activation energy [1]. Due to their great substrate specificity and high catalytic efficiency, natural enzymes have significant uses in biochemical analysis, medicine, chemical synthesis, and food preparation [2][3][4][5]. However, the usage of enzymes is costly, their recycling is challenging, and their tertiary protein structure is easily damaged under harsh process conditions, resulting in the loss of catalytic activity [6][7][8]. Additionally, the stability of enzymes must meet strict standards in the industrial manufacturing environment, greatly restricting the broader industrial application of enzymes to date [9][10][11].
Accordingly, enhancing enzyme stability in harsh environments is a good strategy to promote the use of enzymes [12,13]. Enzyme immobilization by encasing them in solid substances or restricting them to certain regions using physical or chemical methods enables their reuse while also greatly increasing their stability and catalytic efficiency [14][15][16][17]. Additionally, the immobilization of enzymes makes it easier to separate enzymes from products, culture medium, and substrates, which improves process economics [18,19]. Conventional enzyme-immobilization carriers are chemically inert and have no catalytic effect themselves [14,20,21]. Some carrier materials even limit the contact between the substrate and the enzyme, thereby affecting the catalytic efficiency. Additionally, certain carriers will interact with the enzyme to reduce its ability to catalyze reactions [22][23][24]. Therefore, the most important factor for successful enzyme immobilization is the selection of an appropriate carrier material [25].
The creation of new and highly effective nanozymes is another strategy for addressing the shortcomings of enzymes [26,27]. Since in 2007 it was discovered that Fe 3 O 4 has enzyme-like capabilities, nanozymes have gained significant attention [28]. Nanozymes

Characterization of FeS 2 /CS@Gox
In order to assess the state of the immobilized enzyme, the morphology of the FeS 2 enzyme particles was first studied. The FeS 2 nanomaterial exhibited a homogeneous hexagonal nanosheet shape ( Figure S1), whereas the FeS 2 with immobilized enzymes had a hexagonal nanosheet shape with compound surface attachments ( Figure 1A). When Gox was loaded onto the surface of FeS 2 nanosheets, polymeric attachments were clearly visible ( Figure 1B). The enzyme is physically and chemically bonded to the surface of FeS 2 , preserving the FeS 2 's original form in the composite material. The establishment of the link between FeS 2 and Gox was then confirmed by FTIR ( Figure 1C and Figure S2), and the typical absorption peak of cysteine-rich FeS 2 nanosheets was observed at 3300-3400 cm −1 which was attributed to the NH stretching vibration. The symmetric and asymmetric stretching vibrations of CH and CH 2 were reflected in the peaks at 2750 and 3200 cm −1 [39]. The presence of S-H groups in FeS 2 was confirmed by the weak signal at 2550 cm −1 . Stretching vibrations of the C=O molecule resulted in both asymmetric and symmetric peaks at 1600-1700 cm −1 . The broad peak at 900-1300 cm −1 was attributed to C=S double bonds, while the broad peak at 1600-1700 cm −1 can be attributed to the newly formed C=O of NH 2 in FeS 2 and COOH in Gox after conjugation with CS and Gox. The N-H bending vibration caused a peak at 1538 cm −1 , while the vibration peaks at 1295 and 1059 were related to the C-O and C-N stretching vibrations, respectively. The distinctive infrared functional group peaks demonstrate the chemical interaction between FeS 2 and Gox. The successful conjugation was further confirmed by the elemental distribution in Fe/CS@Gox ( Figure S3). Finally, pure FeS 2 displayed distinct crystal characteristic peaks, which also demonstrated that FeS 2 had been successfully prepared. The same crystal characteristic peaks also developed in FeS 2 when the enzyme was immobilized. The consistency of the XRD pattern also demonstrated that Gox and CS did not alter the structure of FeS 2 after immobilization ( Figure 1D). FeS 2 can efficiently exhibit its catalytic activity provided that its crystal structure remains unaffected, which is advantageous for the creation of immobilized enzyme systems. These results confirmed that Gox was successfully grafted onto the FeS 2 nanosheets.
Molecules 2023, 28, 1357 3 of 12 strated that FeS2 had been successfully prepared. The same crystal characteristic peaks also developed in FeS2 when the enzyme was immobilized. The consistency of the XRD pattern also demonstrated that Gox and CS did not alter the structure of FeS2 after immobilization ( Figure 1D). FeS2 can efficiently exhibit its catalytic activity provided that its crystal structure remains unaffected, which is advantageous for the creation of immobilized enzyme systems. These results confirmed that Gox was successfully grafted onto the FeS2 nanosheets.

Immobilization Ratio and Immobilized Enzyme Activity
The conjugation of Gox to the FeS2 nanosheets was confirmed by green fluorescent labeling. As shown in Figure S4, there was a clearly visible green fluorescence under ultraviolet light which confirmed the presence of Gox in the FeS2/CS particles. Furthermore, fluorescence microscopy of the FeS2/CS@Gox particles showed that FeS2 exhibited an aggregated morphology in the field of view, and bright green fluorescence coincided with the distribution of FeS2 (Figure 2A,B). Subsequently, we investigated the effect of adding different amounts of Gox to the enzyme immobilization ratio. The enzyme immobilization did not exceed 30% when only FeS2 and Gox were linked covalently, but the FeS2/CS had higher immobilization efficiency. FeS2/CS had the highest efficiency and loading capacity when Gox was added at 0.5 mg, which was the most suitable concentration for enzyme immobilization in this system (Table S1 and Figure 2C). In an aqueous solution, Gox readily self-dissociates when it is directly chemically bound to FeS2, but CS creates a confined space for FeS2@Gox, making Gox dissociation challenging. The effects of pH and temperature on immobilized enzymes were then further investigated. As shown in Figure 2D,E,

Immobilization Ratio and Immobilized Enzyme Activity
The conjugation of Gox to the FeS 2 nanosheets was confirmed by green fluorescent labeling. As shown in Figure S4, there was a clearly visible green fluorescence under ultraviolet light which confirmed the presence of Gox in the FeS 2 /CS particles. Furthermore, fluorescence microscopy of the FeS 2 /CS@Gox particles showed that FeS 2 exhibited an aggregated morphology in the field of view, and bright green fluorescence coincided with the distribution of FeS 2 (Figure 2A,B). Subsequently, we investigated the effect of adding different amounts of Gox to the enzyme immobilization ratio. The enzyme immobilization did not exceed 30% when only FeS 2 and Gox were linked covalently, but the FeS 2 /CS had higher immobilization efficiency. FeS 2 /CS had the highest efficiency and loading capacity when Gox was added at 0.5 mg, which was the most suitable concentration for enzyme immobilization in this system (Table S1 and Figure 2C). In an aqueous solution, Gox readily self-dissociates when it is directly chemically bound to FeS 2 , but CS creates a confined space for FeS 2 @Gox, making Gox dissociation challenging. The effects of pH and temperature on immobilized enzymes were then further investigated. As shown in Figure 2D,E, the immobilized enzyme exhibited higher catalytic activity than the free enzyme, which may be due to the attachment to the support changing the functional groups of the enzyme. In addition, it has been reported that the sulfide helps to enhance the activity of the enzyme, and the catalytic activity of Gox can be significantly improved by the numerous thiol groups in FeS 2 [40]. Additionally, the immobilized enzymes demonstrated improved catalytic activity in extreme circumstances. The immobilized enzyme may retain more than 80% of its catalytic activity in acidic and alkaline environments, whereas the catalytic activity of the free enzyme is less than 60%. The catalytic activity of enzymes is also impacted by temperature. Free enzymes barely exhibit any catalytic activity at 100 • C, but immobilized enzymes exhibit more than 60% catalytic activity. The results demonstrated that the stability and catalytic activity of the enzyme were significantly improved by immobilizing it with FeS 2 and CS. Reusability is a crucial aspect of immobilized enzymes, and Figure 2F shows the reuse of the FeS 2 /CS@Gox. The catalytic efficiency of FeS 2 /CS@Gox remained above 80% over the first eight reuse cycles, but further usage eventually resulted in structural degradation and a loss of mass transfer, which would lower catalytic performance [41]. The use of enzyme engineering in actual manufacturing will be encouraged by its excellent reusability, which will lower costs and boost yield.
thiol groups in FeS2 [40]. Additionally, the immobilized enzymes demonstrated improved catalytic activity in extreme circumstances. The immobilized enzyme may retain more than 80% of its catalytic activity in acidic and alkaline environments, whereas the catalytic activity of the free enzyme is less than 60%. The catalytic activity of enzymes is also impacted by temperature. Free enzymes barely exhibit any catalytic activity at 100 °C, but immobilized enzymes exhibit more than 60% catalytic activity. The results demonstrated that the stability and catalytic activity of the enzyme were significantly improved by immobilizing it with FeS2 and CS. Reusability is a crucial aspect of immobilized enzymes, and Figure 2F shows the reuse of the FeS2/CS@Gox. The catalytic efficiency of FeS2/CS@Gox remained above 80% over the first eight reuse cycles, but further usage eventually resulted in structural degradation and a loss of mass transfer, which would lower catalytic performance [41]. The use of enzyme engineering in actual manufacturing will be encouraged by its excellent reusability, which will lower costs and boost yield.

The POD-like Activity of FeS2
Subsequently, we investigated the peroxidase-like catalytic capacity of the composite through the oxidation of 3,3′,5,5′-tetramethylbenzidine (TMB) in the presence of H2O2. We tested the effects of nFeS concentration, H2O2 concentration, pH, and temperature on the

The POD-like Activity of FeS 2
Subsequently, we investigated the peroxidase-like catalytic capacity of the composite through the oxidation of 3,3 ,5,5 -tetramethylbenzidine (TMB) in the presence of H 2 O 2 . We tested the effects of nFeS concentration, H 2 O 2 concentration, pH, and temperature on the peroxidase-like activity. With the increase of FeS 2 concentration, the oxidation rate of TMB also increased ( Figure 3B). Moreover, with the increase of H 2 O 2 concentration, the rate of TMB oxidation also gradually increased ( Figure 3C). After optimizing the temperature and pH for the peroxidase-like activity of nFeS, it was found that the peroxidase-like activity of FeS 2 decreased with the increase in temperature, and the optimum pH was 4.0 ( Figure 3D,E). The above results show that the peroxidase-like catalytic performance of FeS 2 nanoparticles is dependent on catalyst concentration, substrate concentration, temperature, and pH, similar to natural enzymes. The mechanism of peroxidase-like activity of FeS 2 nanoparticles was then further explored. Terephthalic acid was used as a fluorescent probe to detect the production of hydroxyl radicals, which can be produced from H 2 O 2 by peroxidase [42]. The generation of hydroxyl radicals from H 2 O 2 catalyzed by FeS 2 was detected. In the presence of H 2 O 2 , FeS 2 can convert TA into TA-OH, which has increased fluorescence intensity. The production of hydroxyl radicals was detected under acidic or neutral conditions, indicating that the peroxidase-like activity of FeS 2 is due to its ability to decompose H 2 O 2 to produce hydroxyl radicals ( Figure S5). To determine the catalytic efficiency of FeS 2 nanosheets, we performed enzyme kinetic analysis with testing at optimal pH and room temperature. Unexpectedly, FeS 2 nanosheets showed a very high affinity for H 2 O 2 , with a K m of 0.01743 mM and a V max of 110.9848 nM/s ( Figure 3F). The results demonstrate that FeS 2 mimics natural peroxidase catalytically and has the benefit of stability over natural enzymes. The excellent peroxidase-like activity of FeS 2 nanosheets encouraged us to further apply them for two-enzyme coupled catalysis.
FeS2 nanoparticles is dependent on catalyst concentration, substrate concentration, temperature, and pH, similar to natural enzymes. The mechanism of peroxidase-like activity of FeS2 nanoparticles was then further explored. Terephthalic acid was used as a fluorescent probe to detect the production of hydroxyl radicals, which can be produced from H2O2 by peroxidase [42]. The generation of hydroxyl radicals from H2O2 catalyzed by FeS2 was detected. In the presence of H2O2, FeS2 can convert TA into TA-OH, which has increased fluorescence intensity. The production of hydroxyl radicals was detected under acidic or neutral conditions, indicating that the peroxidase-like activity of FeS2 is due to its ability to decompose H2O2 to produce hydroxyl radicals ( Figure S5). To determine the catalytic efficiency of FeS2 nanosheets, we performed enzyme kinetic analysis with testing at optimal pH and room temperature. Unexpectedly, FeS2 nanosheets showed a very high affinity for H2O2, with a Km of 0.01743 mM and a Vmax of 110.9848 nM/s ( Figure 3F). The results demonstrate that FeS2 mimics natural peroxidase catalytically and has the benefit of stability over natural enzymes. The excellent peroxidase-like activity of FeS2 nanosheets encouraged us to further apply them for two-enzyme coupled catalysis.

Sensitivity and Selectivity of Glucose Detection
Based on the exceptional peroxidase-mimicking activity of FeS 2 , we initially employed the immobilized Gox-TMB system to detect glucose in a solution. With the increase of glucose content, the absorbance at 652 nm of the detecting system also increased ( Figure 4A). The color response of the immobilized Gox-TMB system showed a good linear relationship with the glucose concentration ( Figure 4B). Moreover, the detection limit was as low as 1.9 × 10 −6 M, which was sufficiently sensitive for the actual glucose sample. We created a standard color card with the colors representing the various glucose concentrations in order to more easily determine the concentration of glucose in the solution ( Figure 4C). The selectivity was assessed by examining the reactions to the typical interfering substances, including the selectivity of FeS 2 for H 2 O 2 and the selectivity of the system for glucose. At the same substrate concentration, only H 2 O 2 produced the response of FeS 2 , while there was no reaction with common metal ions and TMB, demonstrating its strong selectivity for H 2 O 2 ( Figure S6). Similarly, only glucose caused a discernible change in solution color, while the solutions containing interfering compounds remained colorless and behaved exactly like the blank, clearly demonstrating the great selectivity for glucose ( Figure 4D and Figure S7). The FeS 2 /CS@Gox system's strong selectivity and sensitivity for glucose make it suited for the identification of samples that contain the substance. In addition, the catalytic efficiency of the glucose detection system based on FeS 2 /CS@Gox was further evaluated over time. As shown in Figure 4E, the catalytic activity of the glucose detection system decreased during one month of storage, but the overall value was higher than 80%, demonstrating the good storage stability of the glucose detection system. The good shelf life of the FeS 2 /CS@Gox system will also further improve its application in the detection of real samples. The nanozyme-based glucose-sensing system offers a good detection limit [43], and our system offers obvious benefits over other systems (Table 1). In conclusion, the FeS 2 /CS@Gox system constructed by the immobilized enzyme strategy has excellent sensitivity, selectivity, and shelf life and is a potential product for glucose detection. created a standard color card with the colors representing the various glucose concentrations in order to more easily determine the concentration of glucose in the solution ( Figure  4C). The selectivity was assessed by examining the reactions to the typical interfering substances, including the selectivity of FeS2 for H2O2 and the selectivity of the system for glucose. At the same substrate concentration, only H2O2 produced the response of FeS2, while there was no reaction with common metal ions and TMB, demonstrating its strong selectivity for H2O2 ( Figure S6). Similarly, only glucose caused a discernible change in solution color, while the solutions containing interfering compounds remained colorless and behaved exactly like the blank, clearly demonstrating the great selectivity for glucose (Figures 4D and S7). The FeS2/CS@Gox system's strong selectivity and sensitivity for glucose make it suited for the identification of samples that contain the substance. In addition, the catalytic efficiency of the glucose detection system based on FeS2/CS@Gox was further evaluated over time. As shown in Figure 4E, the catalytic activity of the glucose detection system decreased during one month of storage, but the overall value was higher than 80%, demonstrating the good storage stability of the glucose detection system. The good shelf life of the FeS2/CS@Gox system will also further improve its application in the detection of real samples. The nanozyme-based glucose-sensing system offers a good detection limit [43], and our system offers obvious benefits over other systems (Table 1). In conclusion, the FeS2/CS@Gox system constructed by the immobilized enzyme strategy has excellent sensitivity, selectivity, and shelf life and is a potential product for glucose detection.

Glucose Detection in Actual Samples
Patients suffering from kidney disease or diabetes have high levels of glucose in their urine, and their physical condition can be judged by detecting the glucose content in urine or blood [48,49]. To assess the viability of FeS 2 /CS@Gox, we created a mixed solution that contained glucose to mimic actual samples. The standard color card provides a rough estimate of the concentration of glucose when the test sample reacts with the detection system ( Figure 5A). The glucose concentration in the sample to be tested was further judged by measuring the absorbance, and the glucose concentration that was calculated using the standard curve is shown in Table 2 and Figure 5B. When we compared the detected concentration to the actual concentration of the material to be tested, there was only a slight discrepancy, and the error was less than 3%. This finding demonstrates that the glucose detection system based on FeS 2 /CS@Gox can determine the concentration of glucose present in different complex samples, such as blood and urine. In addition, we can also detect glucose in blood and sweat using the FeS 2 /CS@Gox system, and the stability based on the FeS 2 /CS@Gox system can be applied to the development and production of glucose detection medical devices.

Glucose Detection in Actual Samples
Patients suffering from kidney disease or diabetes have high levels of glucose in their urine, and their physical condition can be judged by detecting the glucose content in urine or blood [48,49]. To assess the viability of FeS2/CS@Gox, we created a mixed solution that contained glucose to mimic actual samples. The standard color card provides a rough estimate of the concentration of glucose when the test sample reacts with the detection system ( Figure 5A). The glucose concentration in the sample to be tested was further judged by measuring the absorbance, and the glucose concentration that was calculated using the standard curve is shown in Table 2 and Figure 5B. When we compared the detected concentration to the actual concentration of the material to be tested, there was only a slight discrepancy, and the error was less than 3%. This finding demonstrates that the glucose detection system based on FeS2/CS@Gox can determine the concentration of glucose present in different complex samples, such as blood and urine. In addition, we can also detect glucose in blood and sweat using the FeS2/CS@Gox system, and the stability based on the FeS2/CS@Gox system can be applied to the development and production of glucose detection medical devices.

Synthesis and Characterization of FeS 2
FeS 2 was synthesized as described previously, with minor modifications as follow [38]. Briefly, 0.5 g of FeCl 3 and 1.0 g of cysteine were dissolved in 40 mL of ethylene glycol, and then 3.6 g sodium acetate was added. The mixture was then allowed to react for 12 h at 200 • C in an autoclave. The produced black powder was collected, washed with ethanol three times, and dried at 60 • C.

Immobilization of Gox
In order to promote covalent conjugation, Gox and FeS 2 were mixed in 0.1 mM phosphate buffer with pH = 9 and reacted at 37 • C for 24 h under constant stirring at 12,000 rpm. The solids were then separated, added to the CS solution without washing, and left for 10 min to allow chitosan to coat the alkaline FeS 2 surface. FeS 2 particles containing Gox and CS were centrifuged, washed three times with distilled water, and lyophilized. The loading of the FeS 2 particles with the enzyme was confirmed by the FITC fluorescent labeling and confocal laser scanning microscopy (Nikon, Tokyo, Japan) [50].

Materials Characterization
The morphology of the prepared FeS 2 and FeS 2 /CS@Gox was observed using a scanning electron microscope (SEM, S4800). The solid sample was dispersed in absolute ethanol, dried on a silicon wafer, scanned, and photographed using SEM. A transmission electron microscope (TEM) was used to further analyze the morphology of FeS 2 /CS@Gox. FeS 2 /CS@Gox was dispersed in an ethanol solution, dried on a copper grid, and then examined and photographed under the TEM. The dried product was positioned on a quartz glass slide for X-ray diffraction examination (XRD) (Rigaku Miniflex 600, Tokyo, Japan). With 0.008 θ steps and a 3-degree-per-minute scanning speed, the scanning range was 5 to 60 degrees. In order to prepare the FeS 2 and FeS 2 /CS@Gox powders for Fourier transform infrared spectroscopy, they were combined with dry KBr powder in an agate mortar, crushed to a fine powder, and then heated to remove any leftover moisture. After being formed into a pellet, the combined powder was scanned using the device. For the Fourier transform infrared spectrum of the Gox solution, KBr was first made into thin slices, and then a small amount of Gox solution was dropped on the KBr thin slices and scanned by the instrument.

The Effect of pH on the Activity of Immobilized Enzyme
To study the effect of pH on free enzymes and immobilized enzymes, the free enzymes and immobilized enzymes were put in different pH reaction systems to catalyze glucose, the enzyme activities under different conditions were recorded, and the highest activity of the free enzymes was defined as 100% relative enzyme activity.

The Effect of Temperature on the Activity of Immobilized Enzyme
To study the effect of temperature on free enzymes and immobilized enzymes, the free enzymes and immobilized enzymes were put in different temperature reaction systems to catalyze glucose, the enzyme activities under different conditions were recorded, and the highest activity of the free enzymes was defined as 100% relative enzyme activity.

POD-Like Activity and Kinetic Assay of FeS 2 Nanozyme
TMB was used as the substrate for the measurement of peroxidase-like (POD-like) activity with H 2 O 2 . Different concentrations of H 2 O 2 and FeS 2 were mixed in a phosphate buffer with pH = 4.5, and 10 µL of 80 mM TMB solution was then added. The absorbance of the mixture at 652 nm was assessed using a UV-vis spectrophotometer (Perkin Elmer, Waltham, MA, USA) after 5 min of reaction at 37 • C [42].
The nanozyme's affinity for its substrate was assessed by measuring the Michaelis constant (K M ) defined as the substrate concentration at which the nanozyme exhibits half the maximum reaction rate. Maximum reaction velocity (V max ) was defined as the maximum reaction rate measured at a saturated substrate concentration. The kinetic constants K M and V max were determined using Origin software, by fitting the substrate concentrations and initial reaction velocities (v) to the Michaelis-Menten equation: where A is the value of the change in absorbance before and after the reaction; t (s) is the reaction time; ε (M −1 cm −1 ) = 39000 is the molar absorption coefficient of the colorimetric substrate, and L (cm) is the width of the quartz cuvette [32].

Enzyme Activity Assay of Gox
The reducing sugar assay was used to measure the amount of glucose consumed. Briefly, 100 µL of the Gox reaction mixture containing residual glucose was mixed with 200 µL of DNS reagent and heated at 100 • C for 10 min. Then 900 µL of distilled water was added to the mixture, and the absorbance at 540 nm was measured. The consumption of glucose was used to calculate the enzyme activity titer of Gox.

Immobilized Enzyme Cycle Assay
In short, the enzyme activity of Gox in the FeS 2 /CS/Gox system was determined first, and the FeS 2 /CS/Gox system was filtered and dried after the reaction was complete. For subsequent enzymatic activity assays, the FeS 2 /CS/Gox system obtained above was used. Enzyme activity was recorded for each reaction.

Construction of the System for Glucose Detection
The system for glucose detection consisted of 1mg of FeS 2 /CS@Gox, 890 µL of 0.1 M phosphate buffer (pH = 4.5), and 10 µL of TMB (80 mM). The amount of 100 µL of the sample to be tested was then added to the glucose detection system. After 5 min of reaction at room temperature, the absorbance at 652 nm was measured, and the glucose concentration was determined using a standard curve. The LOD was calculated using the equation: where σ is the standard deviation of 20 measurements, and s is the slope of the standard curve.

The Selectivity of FeS 2 for H 2 O 2 Detection and Glucose Detection
The selectivity of FeS 2 for H 2 O 2 was detected in solutions containing H 2 O 2 (1 mM) or disrupters (1 mM). These assays were tested in the solution which contained TMB (1 mM) and FeS 2 (100 µg mL −1 ). Color change of the above solutions was observed at 2 min, and their absorbance was recorded at 652 nm. Pure HAc-NaAc buffer was used as a control.
The selectivity of the FeS 2 /CS/Gox system for glucose was detected in a solution containing glucose (50 µM) or disrupters (50 µM). The amount of 100 µL of glucose solution (100 µM) or other interfering chemicals (100 µM) was mixed with 100 µL of 1 mg mL −1 FeS 2 /CS/Gox system. Finally, a color change of the above solutions was observed, and their absorbance was recorded at 652 nm.

Preparation of Urine Simulant
For the preparation of the urine-simulated solution, a certain amount of glucose was dissolved in water first, and then the same amount of amino acids and urea as the glucose was added. After thorough mixing, impurities were filtered out and stored at 4 • C for future use. For the urine-simulated solution, the glucose content is known, and the FeS 2 /CS/Gox system is used to measure the glucose content in the solution and compare it with the actual content.

Shelf Life of the Glucose Detection System Assay
The effectively created FeS 2 /CS@Gox system was dispersed in an aqueous solution initially, and then it was kept in an aqueous solution for various amounts of time to assess its stability. Glucose was detected by the Gox system, and the stability of the FeS 2 /CS@Gox system was assessed using absorbance at 652 nm.

Conclusions
In this study, we used FeS 2 nanozymes to encapsulate Gox, and the resulting immobilized enzyme system demonstrated increased catalytic efficiency and resistance to harsh conditions. FeS 2 /CS@Gox was further used to construct a glucose detection system with great selectivity, sensitivity, and accuracy which has the potential to be applied to the actual detection of glucose in urine and blood. This system demonstrates that nanozymes may be efficient carriers of natural enzymes and that the cascade interaction between nanozymes and natural enzymes is a powerful strategy for advancing the use of natural enzymes. It is anticipated that the biocatalytic catalyst nanozyme will serve as a link between biological and abiotic catalysis.