Fe Doping Enhances the Peroxidase-Like Activity of CuO for Ascorbic Acid Sensing

: Although signiﬁcant advances have been witnessed in the application of nanozymes in recent years, exploring new strategies to enhance the enzyme-like activity of nanozymes is of urgent importance. Herein, we investigate the feasibility of accelerating the peroxidase-like reaction rate of CuO nanostructures through Fe doping. The coprecipitation method was used to synthesize Fe-doped CuO (Fe-CuO) nanozymes, and the results indicate that the diversiﬁed valence of Fe beneﬁts the redox reaction driven by CuO-based nanozymes. With the improved peroxidase-like activity, the Fe-CuO nanozyme enables the signiﬁcant chromogenic oxidation reaction of 3,3 (cid:48) ,5,5 (cid:48) - tetramethylbenzidine (TMB), facilitating the construction of a visual sensing platform for the sensitive and selective determination of ascorbic acid. Under optimal conditions, the absorbance at 652 nm decreases linearly with the concentration of ascorbic acid in the range of 5–50 µ M, with a limit of detection as low as 4.66 µ M. This work exempliﬁes the activity enhancement for peroxidase-mimicking nanozymes with a metal-doping strategy and provides a broad prospect for the design of more high-performance nanozymes for biosensing applications.


Introduction
The biosensing of biomarkers and small molecules is of great significance in biomedicines, the environment, and the food industry [1][2][3]. A variety of nanomaterials with unique optical, electrical, and catalytic properties and recognition functions play important roles [4][5][6][7][8]. Among them, nanozymes, as a collection of nanomaterials with natural enzyme-like activities, have been used in the field of biosensors to replace natural enzymes because of their high stability, low cost, and ease of mass production [9][10][11][12][13][14][15]. Although massive kinds of nanozymes have received tremendous attention over the past years, the catalytic activity of some nanozymes is still relatively low, which restricts their potential applications [16,17]. Notably, as an essential metal element to life, Cu is an important part of many natural enzymes [15]. However, the catalytic activity of various existing Cu-based nanozymes, for example CuO nanozyme, is still lower than that of natural PODs, making the development of new methods to enhance the activity of the CuO nanozyme still challenging.
Recently, various strategies have been developed to enhance the activity of nanozymes [18][19][20]. For example, higher peroxidase (POD)-like activity can be obtained by fabricating hybrid nanomaterials, tuning the morphology of nanomaterials, or modifying with functional molecules on the surface of nanozymes [14,15]. Another effective route was reported in which doping with suitable heteroatoms could enhance the enzyme-like catalytic activity of nanomaterials. For instance, Mo-doped Co 3 O 4 nanotubes endow an enhanced POD-like activity compared with pristine Co 3 O 4 nanotubes, which was Attributed to the additional reaction of oxygen species by the doping elements [21]. Therefore, it was a great opportunity to improve the POD-like activity by preparing novel types of heteroatom-doped CuO nanomaterials. Fe 3 O 4 is considered to be the first reported metal-based nanozymes [9]. The diversified valence of Fe benefits the redox reaction driven by the nanozyme which acts similarly to the natural oxidoreductase. Thus, we are inspired to introduce Fe into CuO nanostructures to improve the POD-like activity of CuO nanozyme. In this contribution, Fe-doped CuO (Fe-CuO) nanostructures are first fabricated using a coprecipitation method. After verifying that the Fe-CuO nanozyme only possesses POD-like activity, the steady-state kinetics are systematically investigated and the roles of radicals in the Fe-doping-enhanced nanozymatic catalysis are studied. Moreover, we figure out the linear relationship between the concentration of H 2 O 2 and the absorbance of the colorimetric reaction. Thus, a fast colorimetric sensing strategy based on the Fe-CuO nanozyme is proposed for the highly sensitive and selective detection of ascorbic acid (AA). Additionally, on the basis of the superior POD-like activity of the Fe-CuO nanozyme, we constructed a total antioxidant capacity (TAC) biosensor with AA as an antioxidant model by expressing TAC as an AA-equivalent antioxidant capacity.

Apparatus and Characterization
The morphologies of CuO and Fe-CuO were characterized using a JEM-2100 Plus (JEOL, Tokyo, Japan) transmission electron microscope (TEM) operating at 300 kV. X-ray diffraction (XRD) patterns were carried out on a D8 (Bruker, Karlsruhe, Germany) using Ni-filtered Cu Ka radiation (l = 1.5406 Å). The UV-Vis spectra were recorded at room temperature on a UV-2600 (Shimadzu, Kyoto, Japan) using a quartz cuvette with an optical path of 10 mm. Fluorometric data were obtained on a spectrofluorophotometer (F-7000, Hitachi, Tokyo, Japan).

Preparation of CuO and Fe-CuO
The preparation of CuO and Fe-CuO was carried out using a coprecipitation method as described in previous work with little modification [22]. The doped and undoped CuO nanostructures were prepared in a 50 mL flask. First, 248.5 mg of FeCl 2 ·4H 2 O and 1917.9 mg of CuCl 2 ·2H 2 O were dissolved in 25.0 mL water and stirred for 30 min at room temperature. After the addition of 5.0 mL of NaOH (15.0 M), the solution was stirred for 2 h at 70 • C. Subsequently, the colloidal solution was cooled down to room temperature. The resulting product was centrifuged at 5000 rpm for 10 min and washed with ultrapure water and acetone twice each to remove the unused salts. The precipitate was dried under a vacuum oven at 373 K for 2 h and ground in a mortar to make fine powders.

POD-like Activity Assay for Fe-CuO Nanozymes
The peroxidase activity of undoped CuO and Fe-CuO nanozymes was studied using the catalytic oxidation of TMB with H 2 O 2 . In a typical experiment, 36 µL of TMB solution (10 mg·mL −1 ) and 150 µL of H 2 O 2 (100 mM) were added to acetate buffer (pH 4.0) at 25 • C. Then, 270 µL of CuO or Fe-CuO nanozymes (1 mg·mL −1 ) were introduced to make the final acetate (0.2 M) buffered solution of 3 mL. After 5 min of reaction, the solution was filtered using an ultrafilter film to collect the filtrate. The UV-Vis spectrum and the absorbance at 652 nm of the filtrate were measured using a UV-Vis spectrophotometer.
To calculate kinetic parameters, the Michaelis-Menten equation and its Lineweaver-Burk double reciprocal representation were used: where V 0 and V max are the initial and maximum reaction velocities, respectively. [S] represents the substrate concentration. K m represents the Michaelis-Menten constant.
To determine the POD-like catalytic kinetic parameters, 90 µg·mL −1 of CuO and Fe-CuO were used in 0.2 M acetate buffer (pH 4.0) to measure the initial reaction rate at 25 • C. When evaluating the kinetic parameters obtained for TMB or H 2 O 2 as the substrate, a constant concentration of H 2 O 2 (5 mM) or TMB (0.12 mg·mL −1 ) was used, respectively. The V 0 value is calculated using the following equations: where A and t represent the absorbance of oxTMB at 652 nm and the reaction time, respectively. ε 652 = 39,000 M −1 ·cm −1 and b = 1 cm.

Detection of AA Using Fe-CuO Nanozymes
Colorimetric detection of AA was carried out as follows. First, 36 µL of TMB solution (10 mg·mL −1 ), 45 µL of H 2 O 2 (10 mM), and a series of AA solutions with various concentrations were added to acetate buffer (pH 4.0) at 25 • C. Then, 270 µL of Fe-CuO nanozyme (1 mg·mL −1 ) was introduced to make the final acetate (0.2 M) buffered solution of 3 mL. After 5 min of reaction, the solution was filtered using an ultrafilter film to collect the filtrate. The absorbance at 652 nm of the filtrate was measured using a UV-Vis spectrophotometer. The experiment was repeated three times under the same conditions. The limit of detection (LOD) was calculated as LOD = 3σ/k, where σ is the standard deviation of the experiment and k is the slope of the linear curve.

TAC Assay
A commercial beverage (Mizone) was used for the TAC assay. The above-mentioned protocol for AA detection was employed. The beverage was diluted appropriately for the sample preparation. After the addition of 30 µL sample, the reaction of 3 mL system containing 120 µg·mL −1 TMB, 150 µM H 2 O 2 , and 90 µg·mL −1 Fe-CuO nanozyme in 0.2 M acetate buffer (pH 4.0) was conducted at 25 • C for 5 min. In the spiked recovery experiments, the standard AA solution with the concentration of 1 mM was introduced to calculate the recovery.

Characterization of CuO and Fe-CuO Nanozymes
To prepared CuO-based POD-mimicking nanozymes, CuO and Fe-doped CuO (Fe-CuO) nanostructures were synthesized using a classical coprecipitation method ( Figure 1). The expected molar ratio of Fe and Cu is 1:9 for Fe-CuO. Thus, for the preparation of doped nanostructures with a nominal composition of Fe x Cu 1-x O (x = 0.1), FeCl 2 is mixed with CuCl 2 in the stoichiometric ratio first. The addition of NaOH at 70 • C induces the reactions as follows.
After the resultant solution was filtered, washed, and dried, it was seen that the prepared samples were fine powders with dark colors (Figure 1).  For undoped CuO, CuCl 2 + 2 NaOH = Cu(OH) 2 + 2 NaCl → CuO + H 2 O. (1) For doped Fe-CuO, After the resultant solution was filtered, washed, and dried, it was seen that the prepared samples were fine powders with dark colors (Figure 1).
The morphologies of CuO and Fe-CuO were characterized using SEM. Figure   Interesting is noted that characteristic peaks remain unchanged after doping with Fe. This indi that the crystal structure of CuO is not distorted in Fe-CuO, which may result from similarity of the ionic radii of Fe and Cu. Therefore, the above investigations verify successful preparation of Fe-CuO with the uniform doping of Fe. Representative TEM images further reveal the flat nanostructures of CuO and the rough nanostructures of Fe-CuO (Figure 3a,b). These patterns are well corroborated with the surface morphology results obtained from SEM. In addition, we examined the XRD patterns of CuO and Fe-CuO for the crystal structure and phase analysis ( Figure 3c). The diffraction peaks could be indexed to the phase of CuO (JCPDS#48-1548). Interestingly, it is noted that characteristic peaks remain unchanged after doping with Fe. This indicates that the crystal structure of CuO is not distorted in Fe-CuO, which may result from the similarity of the ionic radii of Fe and Cu. Therefore, the above investigations verify the successful preparation of Fe-CuO with the uniform doping of Fe.

Fe-Doping-Enhanced Peroxidase-like Activity of CuO
Five different reaction systems were designed to study the influence of Fe doping on the peroxidase-like catalytic property of CuO ( Figure 4a). TMB is a commonly used substrate for the chromogenic oxidation reaction catalyzed by peroxidases with the presence of H 2 O 2 . During the reaction, the amino group of TMB loses an electron to become a cationic free radical and forms a charge-transfer complex which has maximum absorptions at 371 nm and 652 nm, gifting the oxTMB a characteristic blue color [23]. The oxidation of TMB by H 2 O 2 under different conditions is displayed in Figure 4b. Compared with the reaction catalyzed by the CuO nanozyme (curve #2), the doping of Fe results in a significant increase in the absorption at 652 nm for the Fe-CuO nanozyme (curve #1). As a comparison, the Fe-CuO nanozyme cannot catalyze the reaction between O 2 and TMB (curve #3), indicating that the Fe-CuO nanozyme does not possess an obvious oxidase-like activity. Moreover, it is not surprising to find that neither the reaction between H 2 O 2 and TMB without any catalysts (curve #4) nor the reaction system in the absence of TMB as a visible indicator (curve #5) exhibit a negligible absorbance. The above results substantiate that Fe doping enhances the POD-like activity of CuO in the proposed Fe-CuO nanozyme. diffraction peaks could be indexed to the phase of CuO (JCPDS#48-1548). Interestingly is noted that characteristic peaks remain unchanged after doping with Fe. This indica that the crystal structure of CuO is not distorted in Fe-CuO, which may result from t similarity of the ionic radii of Fe and Cu. Therefore, the above investigations verify t successful preparation of Fe-CuO with the uniform doping of Fe.

Fe-Doping-Enhanced Peroxidase-like Activity of CuO
Five different reaction systems were designed to study the influence of Fe doping the peroxidase-like catalytic property of CuO (Figure 4a). TMB is a commonly used su strate for the chromogenic oxidation reaction catalyzed by peroxidases with the presen of H2O2. During the reaction, the amino group of TMB loses an electron to become a ca onic free radical and forms a charge-transfer complex which has maximum absorptions 371 nm and 652 nm, gifting the oxTMB a characteristic blue color [23]. The oxidation TMB by H2O2 under different conditions is displayed in Figure 4b. Compared with reaction catalyzed by the CuO nanozyme (curve #2), the doping of Fe results in a sign cant increase in the absorption at 652 nm for the Fe-CuO nanozyme (curve #1). As a co parison, the Fe-CuO nanozyme cannot catalyze the reaction between O2 and TMB (cur #3), indicating that the Fe-CuO nanozyme does not possess an obvious oxidase-like act ity. Moreover, it is not surprising to find that neither the reaction between H2O2 and TM without any catalysts (curve #4) nor the reaction system in the absence of TMB as a visi indicator (curve #5) exhibit a negligible absorbance. The above results substantiate that doping enhances the POD-like activity of CuO in the proposed Fe-CuO nanozyme.

Steady-State Kinetic Assay
As is known, the catalytic behaviors of enzyme mimics follow the Michaelis−Men equation. To further understand the peroxidase-like catalytic activity of CuO enhanc by Fe doping, the kinetic parameters of CuO and Fe-CuO nanozymes were analyzed a

Steady-State Kinetic Assay
As is known, the catalytic behaviors of enzyme mimics follow the Michaelis−Menten equation. To further understand the peroxidase-like catalytic activity of CuO enhanced by Fe doping, the kinetic parameters of CuO and Fe-CuO nanozymes were analyzed and compared. By fixing the concentration of one substrate (TMB or H 2 O 2 ) and varying the concentration of the other substrate, the initial catalytic rates for both CuO and Fe-CuO were tested. Thus, we obtained the reaction velocity vs. substrate concentration curves, which follow the classic Michaelis-Menten equation (Figure 5, insets). Furthermore, the catalytic kinetic parameters (V max and K m ) of H 2 O 2 and TMB were calculated by using the double reciprocal Lineweaver-Burk diagram ( Figure 5). In enzymatic catalysis, the value of Km indicates the affinity of the enzyme and su strates. For both CuO and Fe-CuO nanozymes, the Km and Vmax for H2O2 and TMB a summarized in Table 1. It is seen that, when using H2O2 as the substrate, the Km of Fe-Cu is 15.9 mM, which is 5.5 times lower than that of CuO (Km = 87.9 mM). The results sugg that Fe-CuO possesses a stronger affinity to H2O2 in comparison with CuO. Interesting In enzymatic catalysis, the value of K m indicates the affinity of the enzyme and substrates. For both CuO and Fe-CuO nanozymes, the K m and V max for H 2 O 2 and TMB are summarized in Table 1. It is seen that, when using H 2 O 2 as the substrate, the K m of Fe-CuO is 15.9 mM, which is 5.5 times lower than that of CuO (K m = 87.9 mM). The results suggest that Fe-CuO possesses a stronger affinity to H 2 O 2 in comparison with CuO. Interestingly, we find that the affinity to TMB of Fe-CuO (K m = 0.986 mM) is slightly lower than that of CuO (K m = 0.497 mM). It is rationally speculated that a typical ping-pong mechanism occurs over the Fe-CuO nanozyme, which indicates that the Fe-CuO nanozyme reacts with the first substrate and then releases the first product before reacting with the second substrate. However, the V max of Fe-CuO for TMB is larger than that of CuO for TMB. The faster reaction rates for the colorimetric substrate make up for the deficiency of weak affinity between Fe-CuO and TMB to a certain extent, suggesting an accelerated rate of enzymatic conversion of the substrate by the CuO-based nanozyme after Fe is doped. Since Fe doping strengthens the POD-like activity of CuO, the kinetic parameters (including K m and V max ) of the Fe-CuO nanozyme are comparable to other state-of-the-art POD-mimicking nanozymes reported recently (Table 1).

Reactive Oxygen Species in the Nanozymatic Catalysis
To further confirm the catalytic mechanism of the Fe-CuO nanozyme, we investigated reactive oxygen species as they are usually known to be produced in nanozymatic catalysis. It is known that DMPO is a kind of free radical scavenger, which can specifically combine with short-lived hydroxyl (•OH) radicals to form longer-lived DMPO-OH•. Thus, by simply adding DMPO to the catalytic reaction, •OH radicals will be trapped to generate the aforementioned long-lived radical adducts. Therefore, it can be qualitatively judged whether •OH radicals are produced from the ESR measurement. Therefore, we performed ESR spectroscopy analysis for the determination of •OH radicals. A total of 20 µL of as-prepared Fe-CuO dispersion (4 mg·mL −1 ), 20 µL of H 2 O 2 (100 mM), and 20 µL of DMPO aqueous solution (1 M) were sequentially added into a final solution of 200 µL containing 200 mM acetate buffer (pH 4.0). The mixture was reacted for 10 min before recording the EPR spectra. Figure 6a clearly shows that the characteristic quartet peak of •OH radicals (1:2:2:1) exists in the Fe-CuO and H 2 O 2 mixture system, which suggests that a Fenton-like reaction occurs in TMB oxidation. In addition, •OH radicals in the system were also investigated using the oxidation of terephthalic acid (TA), which reacts with •OH radicals to produce 2-hydroxyterephthalic acid exhibiting a strong emission peak at 450 nm. Figure 6b indicates that the Fe-CuO nanozyme can catalyze H 2 O 2 into •OH radicals (black curve). While the other systems (including the system of Fe-CuO and TA, the system of H 2 O 2 and TA, and the system of TA only) do not exhibit notable fluorescence peaks, respectively. These results further confirm that the TMB oxidation originates from •OH radicals. Accordingly, we propose that the POD-like activity of the Fe-CuO nanozyme is ascribed to the Fenton-type mechanism. 450 nm. Figure 6b indicates that the Fe-CuO nanozyme can catalyze H cals (black curve). While the other systems (including the system of Fe system of H2O2 and TA, and the system of TA only) do not exhibit no peaks, respectively. These results further confirm that the TMB oxidati •OH radicals. Accordingly, we propose that the POD-like activi nanozyme is ascribed to the Fenton-type mechanism.

H 2 O 2 Concentration-Dependent Colorimetric Signals Using Fe-CuO Nanozyme
On the basis of the above results, we constructed a platform for H 2 O 2 detection by monitoring the absorption intensity changes of oxTMB at 652 nm [33]. To achieve higher sensitivity, the dependence of the reaction system on the concentration of the Fe-CuO nanozyme, pH of the reaction system, reaction temperature, and reaction time were systematically investigated to figure out the optimized experimental conditions of the sensing strategy (Figure 7).
The concentration of the catalyst will greatly affect the intensity of the colorimetric signal, so it needs to be optimized first. By comparing the absorbance of different Fe-CuO nanozyme concentrations, it is found that as the concentration increases from 0 to 210 µg·mL −1 ; the absorbance of oxTMB at 652 nm gradually increases (Figure 7a). The low catalyst concentration only causes a weak signal; however, too many catalyst nanoparticles affect the light transmittance of the resultant solution and cause a waste of the materials. Therefore, 90 µg·mL −1 of Fe-CuO nanozyme was used as the catalyst concentration for subsequent experiments.
For lots of nanozymes, the pH of the reaction system is a key factor that influences the POD-like activity. Thus, the effect of different pHs (3.0, 3.5, 4.0, 4.5, 5.0, and 5.5) was also investigated (Figure 7b). It is found that the nanozyme system has good POD-like activity in the range of pH from 3.0 to 5.0. As a result, pH 4.0 was selected as the optimal pH for subsequent experiments. signal, so it needs to be optimized first. By comparing the absorbance of different Fe-CuO nanozyme concentrations, it is found that as the concentration increases from 0 to 210 μg·mL −1 ; the absorbance of oxTMB at 652 nm gradually increases (Figure 7a). The low catalyst concentration only causes a weak signal; however, too many catalyst nanoparticles affect the light transmittance of the resultant solution and cause a waste of the materials. Therefore, 90 μg·mL −1 of Fe-CuO nanozyme was used as the catalyst concentration for subsequent experiments. For lots of nanozymes, the pH of the reaction system is a key factor that influences the POD-like activity. Thus, the effect of different pHs (3.0, 3.5, 4.0, 4.5, 5.0, and 5.5) was also investigated (Figure 7b). It is found that the nanozyme system has good POD-like activity in the range of pH from 3.0 to 5.0. As a result, pH 4.0 was selected as the optimal pH for subsequent experiments. Moreover, in addition to the catalyst concentration and pH of the reaction system, the effect of the reaction temperature is also very important. When the temperature varies from 20 to 35 • C, the absorbance value of oxidized TMB is continuously increased (Figure 7c). Considering the convenience of conducting the nanozymatic reaction procedure, 25 • C was chosen as the optimal temperature for subsequent experiments.
Lastly, the reaction time was explored based on the optimal conditions of 90 µg·mL −1 of Fe-CuO nanozyme, pH 4.0, and 25 • C ( Figure 7d). As the incubation time prolongs, the absorbance of oxTMB gradually increases during 45 min. Therefore, in order to save the detecting time for H 2 O 2 assay, 5 min was selected as the reaction time for subsequent experiments.
Encouraged by the optimization of experimental conditions, we constructed a colorimetric H 2 O 2 bioassay. It is seen that as the H 2 O 2 concentration rises, an obvious color change from colorless to blue occurs (Figure 8a). Therefore, we obtained a linear range of 5-150 µM with a limit of detection (LOD) of 7.07 µM (Figure 8b). detecting time for H2O2 assay, 5 min was selected as the reaction time for subsequent experiments.
Encouraged by the optimization of experimental conditions, we constructed a colorimetric H2O2 bioassay. It is seen that as the H2O2 concentration rises, an obvious color change from colorless to blue occurs (Figure 8a). Therefore, we obtained a linear range of 5-150 μM with a limit of detection (LOD) of 7.07 μM (Figure 8b).

Analytical Performance of Fe-CuO Nanozyme-Based AA Detection
Ascorbic acid (AA), is one of the important vitamins that can participate in many important biological reactions to adjust the normal physiological function of the body [34]. AA is also considered to be an important indicator of the freshness and nutritional quality of fruits [35]. Thus, it is vital to develop fast and sensitive methods for detecting AA. As a typical reducing agent, AA can effectively reduce oxTMB to its reduction form, leading to a discoloration of the solution. By introducing AA to the chromogenic TMB oxidation reaction catalyzed by Fe-CuO nanozymes, the shade of the blue color can reflect the amount of AA. Accordingly, we are inspired to develop a colorimetric AA sensing strategy by utilizing the improved POD-like activity of Fe-CuO nanozymes (Figure 9a).

Analytical Performance of Fe-CuO Nanozyme-Based AA Detection
Ascorbic acid (AA), is one of the important vitamins that can participate in many important biological reactions to adjust the normal physiological function of the body [34]. AA is also considered to be an important indicator of the freshness and nutritional quality of fruits [35]. Thus, it is vital to develop fast and sensitive methods for detecting AA. As a typical reducing agent, AA can effectively reduce oxTMB to its reduction form, leading to a discoloration of the solution. By introducing AA to the chromogenic TMB oxidation reaction catalyzed by Fe-CuO nanozymes, the shade of the blue color can reflect the amount of AA. Accordingly, we are inspired to develop a colorimetric AA sensing strategy by utilizing the improved POD-like activity of Fe-CuO nanozymes (Figure 9a). Figure 9b shows that the blue color of the resultant solution becomes lighter as the concentration of AA increases. Thus, a good linear relationship between the absorbance at 652 nm and AA concentration is established in the range from 5 to 50 µM with R 2 = 0.991 (Figure 9c). The results are averaged values obtained from three repetitive measurements under the same experimental conditions, which represent a good reproducibility of the method. At a signal-to-noise ratio (S/N) of 3, the limit detection for AA is calculated to be 4.66 µM, which is comparable to the AA detection methods proposed in recent reports ( Table 2). The results of this analysis indicate that the high sensitivity of AA detection with Fe-CuO nanozymes has a good usability.   Figure 9b shows that the blue color of the resultant solution becomes lighter as the concentration of AA increases. Thus, a good linear relationship between the absorbance at 652 nm and AA concentration is established in the range from 5 to 50 μM with R 2 = 0.991 (Figure 9c). The results are averaged values obtained from three repetitive measurements under the same experimental conditions, which represent a good reproducibility of the method. At a signal-to-noise ratio (S/N) of 3, the limit detection for AA is calculated to be 4.66 μM, which is comparable to the AA detection methods proposed in recent reports ( Table 2). The results of this analysis indicate that the high sensitivity of AA detection with Fe-CuO nanozymes has a good usability. To validate the selectivity of the proposed colorimetric AA sensing system, a variety of possible interferences, including amino acids, glucose, metal ions, and other vitamin molecules, were challenged ( Figure 9d). As expected, only the presence of AA results in a significant decrease in the absorbance at the same substrate concentration (Figure 9e). This selectivity test substantiates that the proposed detection strategy has excellent specificity for AA. Therefore, the above results indicate that highly sensitive and selective AA detection with Fe-CuO nanozymes has a good usability.

Practicability for TAC Assay in Real-World Scenarios
As a critical indicator for estimating antioxidants in antioxidant foods and medicines, the sensitive determination of TAC is attractive [36,37]. AA is usually selected to be the antioxidative model for a TAC assay. With the sensing strategy proposed in this work, we developed a TAC biosensor for real-world samples to verify the feasibility of the Fe-CuO nanozyme-based biosensor for practical usage [43]. For example, Mizone is one of the popular functional sports drinks produced by Danone. After appropriate dilution of the commercial beverage, the content of equivalent AA was detected using the proposed method, followed by the spiked recovery experiments using the standard addition method to verify the accuracy of our biosensor (Table 3). It is found that the concertation of equivalent AA in Mizone is 59.1 mM. Moreover, the recoveries of the standard AA are in the range of 98% to 104%, while the corresponding relative standard deviations (RSD) of three repetitive experiments are less than 10%. Thus, the competency of the Fe-CuO nanozyme-based biosensor for TAC assays in practical applications is proved.

Conclusions
In summary, we developed a facile strategy to synthesize Fe-CuO nanozymes, in which the doping of Fe can enhance the POD-like activity of CuO. Working as an effective nanocatalyst, Fe-CuO can promote the chromogenic oxidation reaction between TMB and H 2 O 2 . Based on the splendid POD-like activity, a proof-of-concept colorimetric sensing platform for AA detection was established. The proposed sensing strategy exhibits a LOD of 4.66 µM and excellent anti-interference ability. The current work provides a facile metal-doping method to prepare effective CuO-based nanozymes with improved catalytic activity, which also extends the toolkit for building facile biosensors.