Facile Synthesis of Iron and Nitrogen Co-Doped Carbon Dot Nanozyme as Highly Efficient Peroxidase Mimics for Visualized Detection of Metabolites

Visual detection based on nanozymes has great potential for the rapid detection of metabolites in clinical analysis or home-based health management. In this work, iron and nitrogen co-doped carbon dots (Fe,N-CDs) were conveniently synthesized as a nanozyme for the visual detection of glucose (Glu) or cholesterol (Chol). Using inexpensive and readily available precursors, Fe,N-CDs with peroxidase-like activity were conveniently prepared through a simple hydrothermal method. Co-doping of Fe and N atoms enhanced the catalytic activity of the nanozyme. The nanozyme had a low Michaelis constant (Km) of 0.23 mM when hydrogen peroxide (H2O2) was used as the substrate. Free radical trapping experiments revealed that the reactive oxygen species (ROS) generated in the nanozyme-catalyzed process were superoxide anion radicals (•O2−), which can oxidize colorless 3,3′,5,5′-tetramethylbenzidine (TMB) to generate blue oxidation product (ox-TMB) with characteristics absorbance at 652 nm. Based on this mechanism, a colorimetric sensor was constructed to detect H2O2 ranging from 0.1 μM to 200 μM with a detection limit (DL) of 75 nM. In the presence of glucose oxidase (Gox) or Chol oxidase (Chox), Glu or Chol was oxidized, respectively, and generated H2O2. Based on this, indirect detection of Glu and Chol was realized with linear detection ranges of 5–160 μM and 2–200 μM and DLs of 2.8 μM and 0.8 μM, respectively. A paper-based visual detection platform was fabricated using Fe,N-CDs as nanozyme ink to prepare testing paper by inkjet printing. Using a smartphone to record the RGB values of the testing paper after the reaction, visual detection of Glu and Chol can be achieved with linear detection ranges of 5–160 μM (DL of 3.3 μM) and 2–200 μM (DL of 1.0 μM), respectively.


Introduction
Metabolite detection provides vital health information, enabling early disease detection and guiding treatment, while also playing an important role in individual health management [1][2][3][4][5]. For example, Glu is a necessary nutrient for metabolism in organisms [6,7]. In the body, Glu is oxidized into carbon dioxide and water, while also providing energy and being stored in the form of glycogen. Abnormal blood Glu levels are an important factor in triggering serious health problems such as cardiovascular diseases and diabetes. On the other hand, Chol is primarily synthesized by the human body [8]. Under normal circumstances, the body synthesizes Chol in the liver and obtains it from food. Chol can be converted into steroid hormones or become a component of cell membranes, while also maintaining stable Chol levels in the blood. When the liver experiences severe disease, Chol levels can decrease. However, in patients with obstructive jaundice and nephrotic syndrome, Chol levels often increase. Therefore, effective and convenient monitoring of the

Strategy for One-Pot Synthesis of Fe,N-CDs and Colorimetric Detection of Metabolism
CDs possess several advantages, including adjustable size, excellent optical properties, biocompatibility, and chemical modifiability [38][39][40]. Figure 1a illustrates the synthesis of Fe,N-CDs using a one-pot hydrothermal method for colorimetric detection. Inexpensive and easily accessible histidine and ferric chloride were utilized as raw materials to synthesize Fe,N-CDs through a bottom-up approach. The resulting yield of Fe,N-CDs from this method is 7.4%. Fe,N-CDs exhibit peroxidase-like activity and can catalyze the oxidation of colorless TMB to blue oxides of TMB (ox-TMB) by H 2 O 2 . Thus, the detection of H 2 O 2 can be achieved by measuring the characteristic absorption of ox-TMB using UV-visible spectroscopy. As is known, H 2 O 2 is an intermediate product of metabolism. For example, the oxidation of Glu or Chol catalyzed by Gox or Chox can generate H 2 O 2 . Therefore, colorimetric detection of H 2 O 2 enables indirect detection of Glu or Chol.
28, x FOR PEER REVIEW 3 of 15

Strategy for One-Pot Synthesis of Fe,N-CDs and Colorimetric Detection of Metabolism
CDs possess several advantages, including adjustable size, excellent optical properties, biocompatibility, and chemical modifiability [38][39][40]. Figure 1a illustrates the synthesis of Fe,N-CDs using a one-pot hydrothermal method for colorimetric detection. Inexpensive and easily accessible histidine and ferric chloride were utilized as raw materials to synthesize Fe,N-CDs through a bottom-up approach. The resulting yield of Fe,N-CDs from this method is 7.4%. Fe,N-CDs exhibit peroxidase-like activity and can catalyze the oxidation of colorless TMB to blue oxides of TMB (ox-TMB) by H2O2. Thus, the detection of H2O2 can be achieved by measuring the characteristic absorption of ox-TMB using UVvisible spectroscopy. As is known, H2O2 is an intermediate product of metabolism. For example, the oxidation of Glu or Chol catalyzed by Gox or Chox can generate H2O2. Therefore, colorimetric detection of H2O2 enables indirect detection of Glu or Chol. Visual detection is a straightforward and user-friendly method that does not require specialized equipment or technical expertise. It yields immediate results, enabling fast and convenient testing, particularly for on-site or home monitoring purposes. Figure 1b illustrates the process of preparing testing paper using nanozyme ink for visually detecting H2O2. As shown, nanozymes can be directly used as ink with a commonly available inkjet printer to create the testing paper. After placing the testing paper containing nanozymes into a mixture solution of TMB substrate and different concentrations of H2O2 for reaction, a smartphone can capture images of the reacted testing paper. H2O2 detection can be achieved by extracting the RGB values using Image J software. As inkjet printing for the preparation of testing paper offers several advantages, including precise control, efficient production, easy integration of multiple functions, and cost-effectiveness, the fabricated paper-based sensor exhibits significant potential for rapid, large-scale, and customized testing paper production. Visual detection is a straightforward and user-friendly method that does not require specialized equipment or technical expertise. It yields immediate results, enabling fast and convenient testing, particularly for on-site or home monitoring purposes. Figure 1b illustrates the process of preparing testing paper using nanozyme ink for visually detecting H 2 O 2 . As shown, nanozymes can be directly used as ink with a commonly available inkjet printer to create the testing paper. After placing the testing paper containing nanozymes into a mixture solution of TMB substrate and different concentrations of H 2 O 2 for reaction, a smartphone can capture images of the reacted testing paper. H 2 O 2 detection can be achieved by extracting the RGB values using Image J software. As inkjet printing for the preparation of testing paper offers several advantages, including precise control, efficient production, easy integration of multiple functions, and cost-effectiveness, the fabricated paper-based sensor exhibits significant potential for rapid, large-scale, and customized testing paper production.

Characterization of Fe,N-CDs
The size of Fe,N-CDs was characterized by TEM. From the TEM image (Figure 2a), it can be observed that Fe,N-CDs are well dispersed without aggregation, and the size is relatively uniform. The particle size distribution shows that the size of Fe,N-CDs ranges from 2.5 nm to 4.5 nm, with an average size of 3.4 nm (Figure 2b).

Characterization of Fe,N-CDs
The size of Fe,N-CDs was characterized by TEM. From the TEM image (Figure 2a), it can be observed that Fe,N-CDs are well dispersed without aggregation, and the size is relatively uniform. The particle size distribution shows that the size of Fe,N-CDs ranges from 2.5 nm to 4.5 nm, with an average size of 3.4 nm (Figure 2b). The functional groups of Fe,N-CDs were characterized by FT-IR. As shown in Figure  2c, the broad absorption peak at 3425 cm −1 and the weaker absorption peak at 3132 cm −1 can be attributed to the stretching vibration of N-H/O-H and the characteristic absorption peak of C-H, respectively. The strong absorption peaks at 1634 cm −1 and 1384 cm −1 are attributed to the stretching vibration of C=O and C=C/C=N, respectively. These results indicate that Fe,N-CDs contain abundant oxygen and nitrogen functional groups, which contribute to their good dispersion in aqueous solutions. The peak observed at 680 cm −1 corresponds to the stretching vibration of Fe-N, indicating the successful doping of Fe atoms into the carbon framework of the CDs.
The element composition and chemical groups of Fe,N-CDs were further characterized by XPS. Figure 3a shows the XPS survey spectrum of Fe,N-CDs. As seen, Fe,N-CDs mainly consist of three elements, C, N, and O, with corresponding percentages of 78.7%, 3.2%, and 17.8%, respectively. In addition, a very low signal of Fe atoms can be observed, accounting for 0.3% of the total composition. Furthermore, the Fe content in Fe,N-CDs was determined by ICP-OES, and 5.6% of Fe is revealed. The difference in the Fe content measured by the two methods is attributed to the fact that XPS can only detect the surface Fe atoms of Fe,N-CDs. Figure 3b presents the high-resolution C 1s spectrum. The signals with binding energies at 284.6 eV, 286.0 eV, and 288.0 eV correspond to graphitic C (C-C=C, sp 2 carbon), C-N/C-O bond, and C=N/C=O bond, respectively. The high-resolution N 1s spectrum in Figure 3c shows peaks at binding energies of 399.5 eV, 400.8 eV, and 401.5 eV, corresponding to C-N-C, C=N-C, and N-H structures, respectively. The highresolution O 1s spectrum in Figure 3d exhibits peaks at binding energies of 531.7 eV and 533.0 eV, corresponding to C=O and C-OH, respectively. These results further confirm the presence of functional groups such as imidazole, carbonyl, and hydroxyl groups on the surface of Fe,N-CDs. The element composition and chemical groups of Fe,N-CDs were further characterized by XPS. Figure 3a shows the XPS survey spectrum of Fe,N-CDs. As seen, Fe,N-CDs mainly consist of three elements, C, N, and O, with corresponding percentages of 78.7%, 3.2%, and 17.8%, respectively. In addition, a very low signal of Fe atoms can be observed, accounting for 0.3% of the total composition. Furthermore, the Fe content in Fe,N-CDs was determined by ICP-OES, and 5.6% of Fe is revealed. The difference in the Fe content measured by the two methods is attributed to the fact that XPS can only detect the surface Fe atoms of Fe,N-CDs. Figure 3b presents the high-resolution C 1s spectrum. The signals with binding energies at 284.6 eV, 286.0 eV, and 288.0 eV correspond to graphitic C (C-C=C, sp 2 carbon), C-N/C-O bond, and C=N/C=O bond, respectively. The high-resolution N 1s spectrum in Figure 3c shows peaks at binding energies of 399.5 eV, 400.8 eV, and 401.5 eV, corresponding to C-N-C, C=N-C, and N-H structures, respectively. The high-resolution O 1s spectrum in Figure 3d exhibits peaks at binding energies of 531.7 eV and 533.0 eV, corresponding to C=O and C-OH, respectively. These results further confirm the presence of functional groups such as imidazole, carbonyl, and hydroxyl groups on the surface of Fe,N-CDs.

Peroxidase-like Activity of Fe,N-CDs
The peroxidase-like activity of Fe,N-CDs can be examined using TMB as a substrate, which is a commonly used substrate to evaluate the enzymatic activity of peroxidase-like nanozymes. Commonly, nanozymes can catalyze the oxidation of TMB by H2O2 to form blue ox-TMB. The higher the absorbance of ox-TMB, the higher the nanozyme activity. Figure 4a shows the time-dependent changes in absorbance at 652 nm (A652 nm) for different reaction solutions. For comparison, CDs with only N doping (N-CDs), synthesized with only L-histidine as a precursor, were applied as the control. As seen, the addition of N-CDs resulted in slight increases in absorbance. In the presence of Fe,N-CDs, a significantly higher increase in absorbance is observed, indicating higher nanozyme activity. Figure 4b shows the absorption spectra of different reaction solutions after a 10-min reaction. As seen, the absorbance of the Fe,N-CDs + TMB + H2O2 solution is significantly higher than that of the other groups. These results indicate that Fe,N-CDs exhibit the highest nanozyme activity, which can be attributed to the co-doping of Fe and N atoms [40][41][42][43]. Doping nitrogen and iron atoms into CDs might alter their band structure and energy level distribution. On the one hand, N atoms establish robust covalent bonds with carbon atoms, improving electrical conductivity and enhancing chemical stability by increasing electron density. On the other hand, Fe elements play an important role in the catalytical

Peroxidase-like Activity of Fe,N-CDs
The peroxidase-like activity of Fe,N-CDs can be examined using TMB as a substrate, which is a commonly used substrate to evaluate the enzymatic activity of peroxidase-like nanozymes. Commonly, nanozymes can catalyze the oxidation of TMB by H 2 O 2 to form blue ox-TMB. The higher the absorbance of ox-TMB, the higher the nanozyme activity. Figure 4a shows the time-dependent changes in absorbance at 652 nm (A 652 nm ) for different reaction solutions. For comparison, CDs with only N doping (N-CDs), synthesized with only L-histidine as a precursor, were applied as the control. As seen, the addition of N-CDs resulted in slight increases in absorbance. In the presence of Fe,N-CDs, a significantly higher increase in absorbance is observed, indicating higher nanozyme activity. Figure 4b shows the absorption spectra of different reaction solutions after a 10-min reaction. As seen, the absorbance of the Fe,N-CDs + TMB + H 2 O 2 solution is significantly higher than that of the other groups. These results indicate that Fe,N-CDs exhibit the highest nanozyme activity, which can be attributed to the co-doping of Fe and N atoms [40][41][42][43]. Doping nitrogen and iron atoms into CDs might alter their band structure and energy level distribution. On the one hand, N atoms establish robust covalent bonds with carbon atoms, improving electrical conductivity and enhancing chemical stability by increasing electron density. On the other hand, Fe elements play an important role in the catalytical property of natural peroxidase.
The introduction of Fe atoms might increase active sites on the surface of CDs and facilitate electron transfer during catalysis, thereby enhancing substrate adsorption capacity and improving catalytic reaction rate and efficiency. property of natural peroxidase. The introduction of Fe atoms might increase active sites on the surface of CDs and facilitate electron transfer during catalysis, thereby enhancing substrate adsorption capacity and improving catalytic reaction rate and efficiency.  The enzymatic performance of Fe,N-CDs as a peroxidase mimic was evaluated by determining the steady-state kinetic constants. The Michaelis-Menten curves were obtained by using TMB and H 2 O 2 as substrates, and the Michaelis constant (K m ) and maximum initial reaction rate (V max ) values were calculated using the Lineweaver-Burk plot. When TMB is used as the substrate, the K m value is determined to be 0.23 mM, and the V max is 5.510 × 10 −8 M/s (Figure 4c,d). When H 2 O 2 is used as the substrate, the K m value is 1.10 mM, and the V max is 5.598 × 10 −8 M/s (Figure 4e,f).

Catalytic Mechanism of Fe,N-CD Nanozyme
The catalytic mechanism of Fe,N-CDs was investigated by adding different scavengers to capture ROS generated during the Fe,N-CDs catalytic process. The scavengers used for capturing different free radicals were benzoquinone (BQ) for •O 2− , tryptophan (Trp) for singlet oxygen ( 1 O 2 ), and tert-butanol (TBA) for hydroxyl radical (•OH). When free radicals are captured by scavengers, the production of ox-TMB might significantly decrease, leading to a decrease in absorbance. As shown in Figure 5a,  The enzymatic performance of Fe,N-CDs as a peroxidase mimic was evaluated by determining the steady-state kinetic constants. The Michaelis-Menten curves were obtained by using TMB and H2O2 as substrates, and the Michaelis constant (Km) and maximum initial reaction rate (Vmax) values were calculated using the Lineweaver-Burk plot. When TMB is used as the substrate, the Km value is determined to be 0.23 mM, and the Vmax is 5.510 × 10 −8 M/s (Figure 4c,d). When H2O2 is used as the substrate, the Km value is 1.10 mM, and the Vmax is 5.598 × 10 −8 M/s (Figure 4e,f).

Catalytic Mechanism of Fe,N-CD Nanozyme
The catalytic mechanism of Fe,N-CDs was investigated by adding different scavengers to capture ROS generated during the Fe,N-CDs catalytic process. The scavengers used for capturing different free radicals were benzoquinone (BQ) for •O 2− , tryptophan (Trp) for singlet oxygen ( 1 O2), and tert-butanol (TBA) for hydroxyl radical (•OH). When free radicals are captured by scavengers, the production of ox-TMB might significantly decrease, leading to a decrease in absorbance. As shown in Figure 5a, the addition of BQ results in a sharp decrease in absorbance, indicating that Fe,N-CDs mainly generate •O 2− as the ROS during nanozyme-catalyzed H2O2 decomposition.

Colorimetric Detection of H2O2
Fe,N-CDs exhibit nanozyme activity and can catalyze the decomposition of H2O2 to generate ROS, which further oxidizes colorless TMB to produce blue-colored ox-TMB. Thus, the detection of H2O2 can be achieved by the quantification of ox-TMB. To obtain the optimal conditions for detecting H2O2, the effects of reaction pH, temperature, and reaction time on the catalytic reaction were investigated. As shown in Figure 5b, Fe,N-CDs exhibit the highest peroxidase-like activity at pH 4.0. At a reaction temperature of 40 °C, Fe,N-CDs show the highest catalytic activity, which is attributed to the fact that both the Fe,N-CD nanozyme and natural enzymes have the highest activity near physiological temperatures (Figure 5c). Therefore, the subsequent experiments were conducted at physiological temperatures for detection. When TMB, H2O2, and Fe,N-CDs reacted for 10 min, the reaction reached equilibrium (Figure 5d). The optimized conditions mentioned above were used for subsequent experiments. Figure 5e shows the absorption spectra of the solution after adding different concentrations of H2O2 to TMB and Fe,N-CDs, followed by a 10-minute reaction. It can be observed that the absorbance values of the solution increase with increasing H2O2 concentration. As shown in Figure 5f, the absorbance values at 652 nm (A652) exhibit a good linear relationship with H2O2 concentration (C) in the range of 0.1-200 µM (A652 = 0.00377C + 0.0200, R 2 = 0.998). The detection limit (DL) calculated based on a signal-to-noise ratio of three (S/N = 3) is 75 nM.

Colorimetric Detection of Glu and Chol
The detection of metabolites such as Glu and Chol is of significant importance in the diagnosis and treatment of chronic diseases such as diabetes and dyslipidemia. In the presence of Gox and Chox, Chol and Glu can be converted to H2O2. Therefore, indirect detection of Glu or Chol can be achieved through quantitatively measuring H2O2. Figure  6a shows the absorption curves of the mixed solution containing Fe,N-CDs, TMB, and Gox after adding different concentrations of Glu. As seen, the absorbance of the solution increases with increasing Glu concentration. As shown in Figure 6b, there is a linear relationship between A652 and Glu concentration within the ranges of 5-40 µM (A652 = 0.0023CGlu + 0.097, R 2 = 0.996) and 40-160 µM (A652 = 0.0015CGlu + 0.13, R 2 = 0.999). The DL

Colorimetric Detection of H 2 O 2
Fe,N-CDs exhibit nanozyme activity and can catalyze the decomposition of H 2 O 2 to generate ROS, which further oxidizes colorless TMB to produce blue-colored ox-TMB. Thus, the detection of H 2 O 2 can be achieved by the quantification of ox-TMB. To obtain the optimal conditions for detecting H 2 O 2 , the effects of reaction pH, temperature, and reaction time on the catalytic reaction were investigated. As shown in Figure 5b, Fe,N-CDs exhibit the highest peroxidase-like activity at pH 4.0. At a reaction temperature of 40 • C, Fe,N-CDs show the highest catalytic activity, which is attributed to the fact that both the Fe,N-CD nanozyme and natural enzymes have the highest activity near physiological temperatures ( Figure 5c). Therefore, the subsequent experiments were conducted at physiological temperatures for detection. When TMB, H 2 O 2 , and Fe,N-CDs reacted for 10 min, the reaction reached equilibrium (Figure 5d). The optimized conditions mentioned above were used for subsequent experiments. Figure 5e shows the absorption spectra of the solution after adding different concentrations of H 2 O 2 to TMB and Fe,N-CDs, followed by a 10-minute reaction. It can be observed that the absorbance values of the solution increase with increasing H 2 O 2 concentration. As shown in Figure 5f

Colorimetric Detection of Glu and Chol
The detection of metabolites such as Glu and Chol is of significant importance in the diagnosis and treatment of chronic diseases such as diabetes and dyslipidemia. In the presence of Gox and Chox, Chol and Glu can be converted to H 2 O 2 . Therefore, indirect detection of Glu or Chol can be achieved through quantitatively measuring H 2 O 2 . Figure 6a shows the absorption curves of the mixed solution containing Fe,N-CDs, TMB, and Gox after adding different concentrations of Glu. As seen, the absorbance of the solution increases with increasing Glu concentration. As shown in Figure 6b, there is a linear relationship between A 652 and Glu concentration within the ranges of 5-40 µM (A 652 = 0.0023C Glu + 0.097, R 2 = 0.996) and 40-160 µM (A 652 = 0.0015C Glu + 0.13, R 2 = 0.999). The DL is 2.8 µM (S/N = 3). Similar to the mechanism described above, Chol is also converted into H 2 O 2 in the presence of Chox. As shown in Figure 6c, the absorbance of the mixed solution increases with increasing Chol concentration. In Figure 6d, A 652 exhibits a linear relationship with Chol concentration within the ranges of 2-10 µM (A 652 = 0.004C Chol + 0.023, R 2 = 0.996) and 10-200 µM (A 652 = 0.001C Chol + 0.059, R 2 = 0.998). The DL is 0.8 µM (S/N = 3). As seen, the sensitivity for the Glu or Chol detection decreases at a high concentration range. Such a phenomenon is commonly observed in many sensors, attributable to decreased active binding sites and mass transfer.
Molecules 2023, 28, x FOR PEER REVIEW 9 of 15 is 2.8 µM (S/N = 3). Similar to the mechanism described above, Chol is also converted into H2O2 in the presence of Chox. As shown in Figure 6c, the absorbance of the mixed solution increases with increasing Chol concentration. In Figure 6d, A652 exhibits a linear relationship with Chol concentration within the ranges of 2-10 µM (A652 = 0.004CChol + 0.023, R 2 = 0.996) and 10-200 µM (A652 = 0.001CChol + 0.059, R 2 = 0.998). The DL is 0.8 µM (S/N = 3). As seen, the sensitivity for the Glu or Chol detection decreases at a high concentration range. Such a phenomenon is commonly observed in many sensors, attributable to decreased active binding sites and mass transfer. (e) (f) Selectivity is an important parameter for evaluating a sensor. To investigate the selectivity of the constructed colorimetric sensor for Glu detection, several common sugars such as sucrose, lactose, fructose (Fru), and maltose were selected as possible interfering substances. As shown in Figure 6e, even when five times the concentration of other sugars was added, there was no significant increase in absorbance, indicating that the sensor has good selectivity for Glu. The selectivity of the colorimetric sensor for Chol detection was also evaluated by adding common interferents in serum, such as uric acid (UA), urea, cysteine (Cys), glycine (Gly), MgCl2, glutamic acid (Glc), Glu, ascorbic acid (AA), and dopamine (DA). As shown in Figure 6f, a significant change in the signal is observed even with a five times concentration of interferents, demonstrating the excellent selectivity of the sensor.
The content of Glu and Chol in serum was detected using the standard addition method. As shown in Table 1, satisfactory recovery rates were obtained for Glu or Chol detection (96.0-98.8% for Glu detection, 97.5-104% for Chol detection). The relative standard deviation (RSD) of the measurements is no higher than 2.2%, indicating good accuracy of the detection.

Visual Detection of Glu and Chol Using Nanozyme Paper and Smartphone
Smartphone-based visual detection offers advantages such as convenience, simplicity, real-time results, remote monitoring, and multi-functionality. Nanozyme testing paper can be conveniently prepared using inkjet printing, serving as a paper-based sensor Selectivity is an important parameter for evaluating a sensor. To investigate the selectivity of the constructed colorimetric sensor for Glu detection, several common sugars such as sucrose, lactose, fructose (Fru), and maltose were selected as possible interfering substances. As shown in Figure 6e, even when five times the concentration of other sugars was added, there was no significant increase in absorbance, indicating that the sensor has good selectivity for Glu. The selectivity of the colorimetric sensor for Chol detection was also evaluated by adding common interferents in serum, such as uric acid (UA), urea, cysteine (Cys), glycine (Gly), MgCl 2 , glutamic acid (Glc), Glu, ascorbic acid (AA), and dopamine (DA). As shown in Figure 6f, a significant change in the signal is observed even with a five times concentration of interferents, demonstrating the excellent selectivity of tthe sensor.
The content of Glu and Chol in serum was detected using the standard addition method. As shown in Table 1, satisfactory recovery rates were obtained for Glu or Chol detection (96.0-98.8% for Glu detection, 97.5-104% for Chol detection). The relative standard deviation (RSD) of the measurements is no higher than 2.2%, indicating good accuracy of the detection.

Visual Detection of Glu and Chol Using Nanozyme Paper and Smartphone
Smartphone-based visual detection offers advantages such as convenience, simplicity, real-time results, remote monitoring, and multi-functionality. Nanozyme test-ing paper can be conveniently prepared using inkjet printing, serving as a paper-based sensor and enabling the visual detection of Glu or Chol. As shown in the insets of Figure 7a, the blue color of the testing paper intensifies as the concentration of the analyte increases. The sum of green and blue chromaticity values divided by twice the red chromaticity value [(G + B)/2R, designated as y] exhibits good linearity with Glu concentration within the ranges of 5-40 µM (y = 0.0013 C Glu + 1.04, R 2 = 0.996) and 40-160 µM (y = 0.0010 C Glu + 0.98, R 2 = 0.998). The detection limit is 3.3 µM (S/N = 3). Similarly, as shown in Figure 7b, [(G + B)/2R] shows good linearity with Chol concentration within the ranges of 2-80 µM (y = 0.0029 C Chol + 1.04, R 2 = 0.999) and 80-200 µM (y = 0.0020 C Chol + 1.12, R 2 = 0.999). The detection limit is 1.0 µM.

Characterizations and Instrumentations
The size and morphology of CDs were examined using a transmission electron microscope (TEM, JEM-2100, JEOL Ltd., Tokyo, Japan) with a testing voltage of 200 kV, using ultrathin carbon film as the substrate. X-ray photoelectron spectroscopy (XPS, PHI5300, Physical Electronics, Boston, MA, USA) and Fourier-transform infrared spectroscopy (FT-IR, Vertex 70, Bruker Corporation, Karlsruhe, Germany) were employed to investigate the functional groups of CDs. XPS measurements were performed under the conditions of 250 W, 14 kV, and with Mg Kα radiation. The UV-Vis absorption spectrum of Fe,N-CDs was recorded using a UV-2450 UV-Visible Spectrophotometer (UV-Vis, Shimadzu Corporation, Tokyo, Japan). The particle size distribution of CDs was determined using a particle

Characterizations and Instrumentations
The size and morphology of CDs were examined using a transmission electron microscope (TEM, JEM-2100, JEOL Ltd., Tokyo, Japan) with a testing voltage of 200 kV, using ultrathin carbon film as the substrate. X-ray photoelectron spectroscopy (XPS, PHI5300, Physical Electronics, Boston, MA, USA) and Fourier-transform infrared spectroscopy (FT-IR, Vertex 70, Bruker Corporation, Karlsruhe, Germany) were employed to investigate the functional groups of CDs. XPS measurements were performed under the conditions of 250 W, 14 kV, and with Mg Kα radiation. The UV-Vis absorption spectrum of Fe,N-CDs was recorded using a UV-2450 UV-Visible Spectrophotometer (UV-Vis, Shimadzu Corporation, Tokyo, Japan). The particle size distribution of CDs was determined using a particle size analyzer (SZ-100V2, Horiba Corporation, Tokyo, Japan). The iron content in CDs was determined using an inductively coupled plasma optical emission spectrometer (ICP-OES, Agilent 730, Agilent Technologies, Palo Alto, CA, USA).

One-Step Synthesis of Fe,N-CD Nanozyme
Fe,N-CDs were synthesized using a one-pot hydrothermal method. Briefly, 0.1 g of L-histidine and 0.1 g of anhydrous ferric chloride were added to 20 mL of ultrapure water. The resulting mixture was stirred at room temperature for 30 min before being transferred to a reaction vessel lined with polytetrafluoroethylene (PTFE). The reaction was carried out at 180 • C for 10 h. The resulting solution was first filtered through a 0.22 µm membrane to remove large particles, and then dialyzed for 48 h using a dialysis bag with a cut-off molecular weight of 500 Da. The liquid inside the dialysis bag was freeze-dried to obtain solid Fe,N-CDs.

Enzyme Activity Testing of Fe,N-CD Pseudo-Peroxidase
The catalytic behavior of the Fe,N-CD nanozyme was studied using 3,3 ,5,5tetramethylbenzidine (TMB) and H 2 O 2 as substrates. The experiment was carried out in NaAc-HAc buffer solution (0.1 M, pH 4.0), with the addition of Fe,N-CDs (60 µg/mL) for the test group and non-doped CDs (60 µg/mL) for the control group. After incubation in a constant temperature water bath at 40 • C for 10 min, the UV absorption spectra of the reaction system were recorded. Subsequently, the kinetics parameters of Fe,N-CDs were studied using the Michaelis-Menten model. When H 2 O 2 was applied as substrate, the H 2 O 2 concentration was varied from 0.1 mM to 3 mM, and the TMB concentration was fixed at 0.5 mM under optimal pH conditions. In the case of the TMB substrate, the concentration of H 2 O 2 was fixed at 0.2 mM, while the TMB concentration was varied from 0.01 mM to 0.6 mM. The changes in absorbance at 652 nm over time were recorded for 10 min. Finally, the K m and Vmax of Fe,N-CDs were calculated using the reciprocal of the double reciprocal plot according to the Lineweaver-Burk equation.

Detection of Free Radicals Generated during the Catalytic Process of Fe,N-CDs
The scavengers used for capturing different free radicals were benzoquinone for •O 2− , tryptophan for singlet oxygen ( 1 O 2 ), and tert-butanol for hydroxyl radical (•OH). Using TMB as the substrate, the absorbance values were measured after 10 min of reaction in the TMB + Fe,N-CDs + H 2 O 2 system with the addition of the three free radical scavengers mentioned above. The final concentrations of TMB, Fe,N-CDs, and H 2 O 2 were 0.5 mM, 60 µg/mL, and 6.6 mM, respectively. The concentrations of benzoquinone, tryptophan, and tert-butanol were 100 µM and 100 µg/mL, respectively. Further validation of the experimental results was conducted through ESR testing, using DMPO + H 2 O 2 or DMPO + H 2 O 2 + Fe,N-CD solutions. The final concentrations of DMPO, H 2 O 2 , and Fe,N-CDs were 20 mM, 20 mM, and 100 µg/mL, respectively.

Colorimetric Detection of H 2 O 2
A single-factor optimization method was employed to optimize the temperature, pH value, reaction time, and Fe,N-CD concentration in the colorimetric detection of H 2 O 2 . Under optimal conditions, the detection of H 2 O 2 was performed. Briefly, different concentrations of H 2 O 2 were added to a solution containing NaAc-HAc buffer (0.1 M, pH 4.0), TMB (0.5 mM), and Fe,N-CDs (60 µg/mL). After incubation at 37 • C for 10 min, the UV absorption spectra of the solution were recorded.

Colorimetric Detection of Glu
Colorimetric detection of Glu was performed in the presence of Fe,N-CDs and glucose oxidase. In simple terms, different concentrations of Glu were added to a mixed solution containing NaAc-HAc buffer (0.1 M, pH 4), TMB (0.5 mM), Fe,N-CDs (60 µg/mL), and Gox (0.1 mg/mL). After incubating at 37 • C for 10 min, the UV absorption spectra of the solution were recorded. To investigate the selectivity of the constructed colorimetric sensor for detecting Glu, several common sugars such as sucrose, maltose, fructose, and lactose were chosen as interfering substances. The changes in absorbance of the above detection solution were measured when the concentration of each sugar substance (1 mM) was 5 times the concentration of Glu. To further explore the potential application of the constructed colorimetric sensor in real sample analysis, Glu content in bovine serum (diluted 50 times) was determined using the standard addition method.

Colorimetric Detection of Chol
Colorimetric detection of Chol was achieved using Fe,N-CDs and cholesterol oxidase. Briefly, different concentrations of Chol were added to a mixed solution containing NaAc-HAc buffer (0.1 M, pH 4), TMB (0.5 mM), Fe,N-CDs (60 µg/mL), and Chox (0.05 mg/mL). After incubating at 37 • C for 10 min, the UV absorption spectra of the solution were recorded. To explore the selectivity of the sensor for Chol detection, uric acid, urea, cysteine, glycine, MgSO 4 , glutamic acid, Glu, ascorbic acid, and dopamine were used as the possible interfering substances, and the changes in absorbance of the detection solution were measured when each substance had a concentration of 1 mM. The determination of Chol in bovine serum (diluted 50 times) was also investigated using the standard addition method.
3.9. Visual Detection of H 2 O 2 , Glu, or Chol Based on Nanozyme Testing Paper Nanozyme testing paper was prepared using an inkjet printer (HP Deskjet 1112, HP Inc., Palo Alto, CA, USA). Fe,N-CDs were used as ink (0.5 mg/mL) and filter paper as the printing medium. The printed filter paper was cut into 2 cm × 2 cm pieces for further investigation. For the detection of H 2 O 2 , different concentrations of H 2 O 2 were mixed with NaAc-HAc buffer (0.1 M, pH 4.0) and TMB (0.5 mM). Then, 200 µL of the mixture was dropped onto the paper-based sensor, followed by incubation at 37 • C for 30 min. The color of the testing paper was recorded by taking a photo using a smartphone. For detecting Glu, glucose oxidase was used. Briefly, different concentrations of Glu were mixed with NaAc-HAc buffer (0.1 M, pH 4.0), TMB (0.5 mM), and Gox (0.1 mg/mL) Similarly, 200 µL of the mixture was dropped onto the paper-based sensor, followed by incubation at 37 • C for 30 min. The color of the testing paper was then recorded using a smartphone. Subsequently, the red-green-blue (RGB) values of the testing paper were analyzed using Image J software. The visual detection of Chol was performed using the same procurement when Gox was replaced with Chox (0.05 mg/mL).

Conclusions
In this work, Fe,N-CDs were synthesized through a one-step hydrothermal method. The steady-state kinetic constants and catalytic mechanism of Fe,N-CDs were investigated. The results showed that Fe,N-CDs exhibited high peroxidase-like activity and substrate affinity. Based on the ability of Fe,N-CDs to catalyze the decomposition of H 2 O 2 and generate ·O 2-, colorimetric detection of H 2 O 2 was achieved by oxidizing TMB to ox-TMB with characteristic absorption at 652 nm. Indirect detection of Glu or Chol was accomplished by incorporating ChOx or GOx. Nanozyme-based ink was used to prepare testing paper as a paper-based sensor using inkjet printing. A simple visual detection method for Glu and Chol was developed using a smartphone. The synthesized nanozyme and developed visual detection platform exhibit great potential for the rapid and convenient detection of metabolite.
Author Contributions: Investigation, S.X. and S.Z.; data curation, S.X., S.Z., and Y.L.; writing-original draft preparation, S.X. and S.Z.; conceptualization and supervision, J.L.; writing-review and editing, J.L. All authors have read and agreed to the published version of the manuscript.

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

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