Fabrication of a Ratiometric Fluorescence Sensor Based on Carbon Dots as Both Luminophores and Nanozymes for the Sensitive Detection of Hydrogen Peroxide

The construction of novel fluorescent nanozymes is highly desirable for providing new strategies for nanozyme-based sensing systems. Herein, a novel ratiometric fluorescence sensing platform was constructed based on carbon dots (CDs) as both luminophores and nanozymes, which could realize the sensitive detection of hydrogen peroxide (H2O2). CDs with peroxidase-mimicking activity were prepared with a one-step hydrothermal method using L-histidine as an inexpensive precursor. CDs had bright blue fluorescence. Due to the pseudo-peroxidase activity, CDs catalyzed the oxidation of o-phenylenediamine (OPD) with H2O2 to generate 2,3-diaminophenolazine (DAP). The fluorescence resonance energy transfer (FRET) between CDs and DAP resulted in a decrease in the fluorescence of CDs and an increase in the fluorescence of DAP, leading to a ratiometric fluorescence system. The free radical trapping experiment was used to investigate the reactive oxygen radicals (ROS) in the catalytic process of CD nanozymes. The enzymatic parameters of CD nanozymes, including the Michaelis constant (Km) and the maximum initial reaction velocities (Vmax), were investigated. A good affinity for both OPD and H2O2 substrates was proven. Based on the FRET between CDs and OPD, a ratiometric fluorescence analysis of H2O2 was achieved and results ranged from 1 to 20 μM and 20 to 200 μM with a low limit of detection (LOD, 0.42 μM). The detection of H2O2 in milk was also achieved.


Introduction
Natural enzymes are efficient biocatalysts and participate in almost all metabolic processes of living organisms. Due to their high catalytic efficiency and substrate selectivity, natural enzymes have broad application prospects in the fields of industrial production, medical research, bioanalysis, etc. [1]. However, natural enzymes have disadvantages such as high preparation and purification costs, easy denaturation under conditions such as high temperature or extreme pH, and degradation by proteases. In recent years, researchers have been working to develop "artificial enzymes" with enzymatic activity as substitutes for natural enzymes. Amongst researches, nanozymes with the catalytic function of natural enzymes have attracted much attention [2][3][4]. Nanozymes are nanomaterials that have the catalytic ability of natural enzymes and follow the enzymatic characteristics (e.g., catalytic kinetics) of natural enzymes. Due to their excellent properties such as good stability, high catalytic activity, highly tunable structure, and unique properties of nanomaterials, nanozymes are expected to serve as excellent substitutes for natural enzymes in the fields of chemical and biosensing, biomedicine, etc. [5][6][7]. The development of nanozymes the enhancement of the DAP's fluorescence, leading to a ratiometric fluorescence analysis system. The fabricated ratiometric fluorescence sensor could achieve the sensitive detection of H 2 O 2 in real food samples.

Synthesis and Characterization of CDs
Nanomaterials usually exhibit novel optical/electrical/magnetic properties and have a high specific surface area due to their unique microstructure and nanoscale size [33][34][35]. In recent years, the use of various functional nanomaterials to construct novel sensing systems has attracted much attention [36][37][38][39]. As 0D fluorescent carbon nanomaterials, carbon dots (CDs) have the advantages of a wide range of synthetic raw materials and good biocompatibility and have been widely applied in the development of advanced fluorescence sensors. Through changing the precursors and the synthesis conditions, the structure, carbonization degree, conjugated structure, lattice, and energy level structure of CDs can be regulated, leading to highly adjustable fluorescence properties. Among the developed methods for the synthesis of CDs, hydrothermal synthesis usually involves the carbonization or molecular fusion of organic small molecules (precursors) in a hydrothermal reactor at high temperature [40][41][42]. This synthetic method has the advantages of low-cost synthesis and environmental friendliness. It has been proven that the pseudo-peroxidase properties of carbon nanomaterials can be generated through nitrogen doping or the introduction of functional groups (e.g., imidazole) [7,[43][44][45]. Inspired by these research works, the amino acid histidine, which was inexpensive and contained an imidazole group, was chosen in this work as the precursor for the synthesis of CDs. As illustrated in Figure 1, CD nanozymes were synthesized with a one-step hydrothermal method. The production yield for the one-step synthesis of CDs was 37.2%.
of CDs overlapped with the absorption spectrum of DAP, a fluorescence resonance energy transfer (FRET) occurred with the quenching of the CDs' fluorescence and the enhancement of the DAP's fluorescence, leading to a ratiometric fluorescence analysis system. The fabricated ratiometric fluorescence sensor could achieve the sensitive detection of H2O2 in real food samples.

Synthesis and Characterization of CDs
Nanomaterials usually exhibit novel optical/electrical/magnetic properties and have a high specific surface area due to their unique microstructure and nanoscale size [33][34][35]. In recent years, the use of various functional nanomaterials to construct novel sensing systems has attracted much attention [36][37][38][39]. As 0D fluorescent carbon nanomaterials, carbon dots (CDs) have the advantages of a wide range of synthetic raw materials and good biocompatibility and have been widely applied in the development of advanced fluorescence sensors. Through changing the precursors and the synthesis conditions, the structure, carbonization degree, conjugated structure, lattice, and energy level structure of CDs can be regulated, leading to highly adjustable fluorescence properties. Among the developed methods for the synthesis of CDs, hydrothermal synthesis usually involves the carbonization or molecular fusion of organic small molecules (precursors) in a hydrothermal reactor at high temperature [40][41][42]. This synthetic method has the advantages of lowcost synthesis and environmental friendliness. It has been proven that the pseudo-peroxidase properties of carbon nanomaterials can be generated through nitrogen doping or the introduction of functional groups (e.g., imidazole) [7,[43][44][45]. Inspired by these research works, the amino acid histidine, which was inexpensive and contained an imidazole group, was chosen in this work as the precursor for the synthesis of CDs. As illustrated in Figure 1, CD nanozymes were synthesized with a one-step hydrothermal method. The production yield for the one-step synthesis of CDs was 37.2%. The size of CDs was investigated with transmission electron microscopy (TEM). As shown in Figure 2a, CDs had good dispersibility and the particle size varied. The diameter distribution plot of CDs measured with dynamic light scattering (DLS) revealed the average size of 3.2 nm (Figure 2b). Figure 2c shows the UV absorption spectrum of CDs. The absorption peaks at 210 nm and 300 nm corresponded to the π→π * and n→π * transitions. The size of CDs was investigated with transmission electron microscopy (TEM). As shown in Figure 2a, CDs had good dispersibility and the particle size varied. The diameter distribution plot of CDs measured with dynamic light scattering (DLS) revealed the average size of 3.2 nm (Figure 2b). Figure 2c shows the UV absorption spectrum of CDs. The absorption peaks at 210 nm and 300 nm corresponded to the π→π * and n→π * transitions. The fluorescence characteristics of CDs were also investigated. The CD solution was light yellow under sunlight, and emitted blue fluorescence under ultraviolet light (365 nm, inset in Figure 2c). As shown in the inset in Figure 2d, the maximum excitation wavelength of CDs was 370 nm. When the excitation wavelength varied from 350 nm to 410 nm, the maximum emission wavelength of CDs slightly changed, indicating an excitationdependent fluorescence emission. The absolute fluorescence quantum yield was 4.7%.
Molecules 2022, 27, x FOR PEER REVIEW 4 of 17 The fluorescence characteristics of CDs were also investigated. The CD solution was light yellow under sunlight, and emitted blue fluorescence under ultraviolet light (365 nm, inset in Figure 2c). As shown in the inset in Figure 2d, the maximum excitation wavelength of CDs was 370 nm. When the excitation wavelength varied from 350 nm to 410 nm, the maximum emission wavelength of CDs slightly changed, indicating an excitation-dependent fluorescence emission. The absolute fluorescence quantum yield was 4.7%. The elemental and chemical compositions of CDs were characterized with X-ray photoelectron spectroscopy (XPS). Figure 3a-d shows the XPS survey spectrum and high-resolution C 1s, N 1s, and O 1s spectra of the prepared CDs. As revealed in the XPS survey spectrum, CDs were composed of three elements including C, N, and O, corresponding to the atomic percentages of 68.5%, 3.3%, and 28.2%, respectively (Figure 3a). This result The elemental and chemical compositions of CDs were characterized with X-ray photoelectron spectroscopy (XPS). Figure 3a-d shows the XPS survey spectrum and highresolution C 1s, N 1s, and O 1s spectra of the prepared CDs. As revealed in the XPS survey spectrum, CDs were composed of three elements including C, N, and O, corresponding to the atomic percentages of 68.5%, 3.3%, and 28.2%, respectively (Figure 3a). This result proved that CDs contained abundant oxygen-and nitrogen-containing groups in addition to the carbon backbone. In the high-resolution C 1s spectrum (Figure 3b), three peaks corresponding to the graphitic C structure (C-C=C, sp 2 carbon domain), C-N/C-O bond, and C=O/C=N bond were observed. As shown in the high-resolution N 1s spectrum (Figure 3c), the peaks at the binding energies of 399.5 eV, 400.8 eV, and 401.5 eV were related to the C-N-C, C=N-C, and N-H structures, respectively. In addition, the C-OH and C=O structures were proven with the peaks at the binding energies of 531.7 eV and 533.0 eV in the high-resolution O 1s spectrum (Figure 3d). The above results showed that the CDs had imidazole groups and oxygen-containing functional groups such as hydroxyl groups, which were beneficial to the water solubility and nanozyme properties of the CDs. proved that CDs contained abundant oxygen-and nitrogen-containing groups in addition to the carbon backbone. In the high-resolution C 1s spectrum (Figure 3b), three peaks corresponding to the graphitic C structure (C-C=C, sp 2 carbon domain), C-N/C-O bond, and C=O/C=N bond were observed. As shown in the high-resolution N 1s spectrum ( Figure  3c), the peaks at the binding energies of 399.5 eV, 400.8 eV, and 401.5 eV were related to the C-N-C, C=N-C, and N-H structures, respectively. In addition, the C-OH and C=O structures were proven with the peaks at the binding energies of 531.7 eV and 533.0 eV in the high-resolution O 1s spectrum ( Figure 3d). The above results showed that the CDs had imidazole groups and oxygen-containing functional groups such as hydroxyl groups, which were beneficial to the water solubility and nanozyme properties of the CDs. Fourier transform infrared spectroscopy (FT-IR) was used to further characterize the surface functional groups of CDs. As shown in Figure 4, peaks at 3406 cm −1 , 3128 cm −1 , and 2860 cm −1 were attributed to the stretching vibration of N-H/O-H, -OH, and C-H groups, respectively. In addition, the peaks at 1628 cm −1 , 1534 cm −1 , 1357 cm −1 , and 1093 cm −1 were related to the stretching vibrations of C=O, C=C/C=N, C-N, and C-O, respectively. These

Peroxidase-Mimicking Activity of CDs
Peroxidase is a class of oxidoreductases that use hydrogen p acceptor to catalyze the oxidation of substrates. Generally, perox dation of phenolic, amine, or hydrocarbon compounds with hydro chosen as the model substrate to investigate the pseudo-peroxida Generally, CDs catalyze the oxidation of OPD to DAP with H2O2. D with a characteristic absorption peak at 420 nm. Thus, the enzymat be investigated by measuring the characteristic absorption of the nm (A420). Figure 5a shows the change curve of A420 of different m action time. As shown, the solution which only contained CDs + absorption. When OPD was mixed with H2O2, the absorbance of because H2O2 could catalyze the oxidation of OPD. However, only was produced. When CDs were added to the mixture of OPD an value of the CDs + OPD + H2O2 ternary mixture increased rapid reaction time. Thus, CDs catalyzed the oxidation of OPD with H duced more DAP. Similar results were also observed from the abs ure 5b. As demonstrated in the digital images (inset of Figure 5b), solution had a deep yellow color, proving that the presence of C DAP. These phenomena demonstrated the peroxidase-mimickin which catalyzed the oxidation of OPD with H2O2.

Peroxidase-Mimicking Activity of CDs
Peroxidase is a class of oxidoreductases that use hydrogen peroxide as an electron acceptor to catalyze the oxidation of substrates. Generally, peroxidase catalyzes the oxidation of phenolic, amine, or hydrocarbon compounds with hydrogen peroxide. OPD was chosen as the model substrate to investigate the pseudo-peroxidase activity of the CDs. Generally, CDs catalyze the oxidation of OPD to DAP with H 2 O 2 . DAP is a yellow solution with a characteristic absorption peak at 420 nm. Thus, the enzymatic activity of CDs could be investigated by measuring the characteristic absorption of the reaction system at 420 nm (A 420 ). Figure 5a shows the change curve of A 420 of different mixed solutions with reaction time. As shown, the solution which only contained CDs + OPD basically had no absorption. When OPD was mixed with H 2 O 2 , the absorbance of the solution was lower because H 2 O 2 could catalyze the oxidation of OPD. However, only a small amount of DAP was produced. When CDs were added to the mixture of OPD and H 2 O 2 , the absorbance value of the CDs + OPD + H 2 O 2 ternary mixture increased rapidly with the increase in reaction time. Thus, CDs catalyzed the oxidation of OPD with H 2 O 2 , which in turn produced more DAP. Similar results were also observed from the absorbance spectra in Figure 5b. As demonstrated in the digital images (inset of Figure 5b), the OPD + CDs + H 2 O 2 solution had a deep yellow color, proving that the presence of CDs produced the most DAP. These phenomena demonstrated the peroxidase-mimicking activity of the CDs, which catalyzed the oxidation of OPD with H 2 O 2 .

Steady-State Dynamics of CD Nanozymes
As the peroxidase mimicked, the steady-state kinetic constants of CD nanozymes were investigated to evaluate their enzymatic properties. The double-reciprocal curve of the Michaelis equation (Lineweaver-Burk plots, 1/V 0 = K m /V max C + 1/Vmax) was used to analyze the Michaelis constant (K m ) and the maximum reaction rate (V max ). The Michaelis-Menten curve was measured with H 2 O 2 and OPD as substrates, respectively (Figure 5c,e). According to the Lineweaver-Burk curve, K m was calculated to be 0.7468 mM and the V max was 1.086 × 10 −8 m/s with H 2 O 2 as the substrate (Figure 5d). When OPD was applied as the substrate, the calculated K m and V max were 6.81 mM and 7.60 × 10 −9 m/s, respectively (Figure 5f). The small K m proved high affinity towards the substrates. The Km with OPD as the substrate was lower than that obtained on Fe-doped carbon dots (Fe-CDs) [46] and mesoporous MnCo 2 O 4 [47]. The K m with H 2 O 2 as the substrate was lower than that obtained on Co-and N co-doped carbon dots (Co, N/CDs) [48], ruthenium ion-complexed carbon nitride (Ru-C 3 N 4 ) [49], and horseradish peroxidase (HRP) [50].

Catalytic Mechanism of CD Nanozymes
To identify the types of reactive oxygen species (ROS) produced in the CD-catalyzed process, experiments to capture the ROS were carried out [9]. TMB was applied as the substrate and the oxidation of TMB with H 2 O 2 catalyzed with CDs was studied. 1,4benzoquinone (BQ) was used as the capture agent of the superoxide anion radical (•O 2− ), tryptophan (Trp) was employed to capture singlet oxygen ( 1 O 2 ), and tert-butanol (TBA) was used as the capture agent of •OH. When free radicals were captured, the oxidation process of TMB was inhibited, resulting in a lower amount of oxidized TMB and a low characteristic absorption at 652 nm. As shown in Figure 6a, the absorbance of the system with BQ decreased significantly, proving that the ROS produced in the catalytic process of CDs was mainly •O 2− . These conclusions were further verified with electron spin resonance (ESR) spectra in Figure 6b of CD activity obtained using Michaelis-Menten model at different concentrations of H2O2 (d) or OPD (f).

Steady-State Dynamics of CD Nanozymes
As the peroxidase mimicked, the steady-state kinetic constants of CD nanozymes were investigated to evaluate their enzymatic properties. The double-reciprocal curve of the Michaelis equation (Lineweaver-Burk plots, 1/V0 = Km/Vmax C + 1/Vmax) was used to analyze the Michaelis constant (Km) and the maximum reaction rate (Vmax). The Michaelis-Menten curve was measured with H2O2 and OPD as substrates, respectively (Figure 5c,e). According to the Lineweaver-Burk curve, Km was calculated to be 0.7468 mM and the Vmax was 1.086 × 10 −8 M/s with H2O2 as the substrate (Figure 5d). When OPD was applied as the substrate, the calculated Km and Vmax were 6.81 mM and 7.60×10 −9 M/s, respectively ( Figure  5f). The small Km proved high affinity towards the substrates. The Km with OPD as the substrate was lower than that obtained on Fe-doped carbon dots (Fe-CDs) [46] and mesoporous MnCo2O4 [47]. The Km with H2O2 as the substrate was lower than that obtained on Co-and N co-doped carbon dots (Co, N/CDs) [48], ruthenium ion-complexed carbon nitride (Ru-C3N4) [49], and horseradish peroxidase (HRP) [50].

Catalytic Mechanism of CD Nanozymes
To identify the types of reactive oxygen species (ROS) produced in the CD-catalyzed process, experiments to capture the ROS were carried out [9]. TMB was applied as the substrate and the oxidation of TMB with H2O2 catalyzed with CDs was studied. 1,4-benzoquinone (BQ) was used as the capture agent of the superoxide anion radical (•O 2− ), tryptophan (Trp) was employed to capture singlet oxygen ( 1 O2), and tert-butanol (TBA) was used as the capture agent of •OH. When free radicals were captured, the oxidation process of TMB was inhibited, resulting in a lower amount of oxidized TMB and a low characteristic absorption at 652 nm. As shown in Figure 6a, the absorbance of the system with BQ decreased significantly, proving that the ROS produced in the catalytic process of CDs was mainly •O 2− . These conclusions were further verified with electron spin resonance (ESR) spectra in Figure 6b

FRET Phenomenon between CDs and DAP
A fluorescence resonance energy transfer (FRET) is a special phenomenon that occurs directly between two fluorescent substances. The occurrence of this phenomenon requires two prerequisites. One is that the emission spectrum of one fluorescent material has a certain overlap with the absorption spectrum of another material. Second, the distance between the two fluorophores has to be relatively close. When these two conditions are satisfied at the same time, the phenomenon of fluorescence energy transfer from the donor to the acceptor can be observed. As shown in Figure 6c, CDs had blue fluorescence and DAP emitted yellow fluorescence. In the mixture of CDs and DAP, a decrease in the CDs' fluorescence was observed, indicating the potential of the FRET phenomenon. As shown in Figure 6d, the absorption spectrum of DAP and the fluorescence emission spectrum of CDs had obvious overlap. The fluorescence lifetime of CDs in the presence or absence of DAP was further investigated. As shown, after the addition of DAP, the fluorescence lifetime of CDs decreased from the original 3.1 ns (Figure 6e) to 2.8 ns (Figure 6f), proving that the presence of DAP led to the dynamic quenching of the CDs' fluorescence. These results indicated the FRET between the CDs and DAP.

FRET Phenomenon between CDs and DAP
A fluorescence resonance energy transfer (FRET) is a special phenomenon that occurs directly between two fluorescent substances. The occurrence of this phenomenon requires two prerequisites. One is that the emission spectrum of one fluorescent material has a certain overlap with the absorption spectrum of another material. Second, the distance between the two fluorophores has to be relatively close. When these two conditions are satisfied at the same time, the phenomenon of fluorescence energy transfer from the donor to the acceptor can be observed. As shown in Figure 6c, CDs had blue fluorescence and DAP emitted yellow fluorescence. In the mixture of CDs and DAP, a decrease in the CDs' fluorescence was observed, indicating the potential of the FRET phenomenon. As shown in Figure 6d, the absorption spectrum of DAP and the fluorescence emission spectrum of CDs had obvious overlap. The fluorescence lifetime of CDs in the presence or absence of DAP was further investigated. As shown, after the addition of DAP, the fluorescence lifetime of CDs decreased from the original 3.1 ns (Figure 6e) to 2.8 ns (Figure 6f), proving that the presence of DAP led to the dynamic quenching of the CDs' fluorescence. These results indicated the FRET between the CDs and DAP.

Ratiometric Fluorescence Detection of H 2 O 2
H 2 O 2 is an important metabolite in cells and a signaling molecule related to oxidative stress and physiological activities. The accumulation of H 2 O 2 can damage DNA, protein, and lipid chains in cells and lead to accelerated apoptosis of cells. An abnormal H 2 O 2 concentration is considered to be a direct trigger of various physiological diseases, including autoimmune diseases, Alzheimer's disease, and cancer. Therefore, the detection of H 2 O 2 is of great significance in biomedical research, clinical diagnosis, food safety, etc. In this work, a ratiometric fluorescence assay was constructed for the simple, rapid, and highly sensitive detection of H 2 O 2 . As illustrated in Figure 1, a ratiometric fluorescence system to detect H 2 O 2 was constructed since CDs and DAP could directly undergo the FRET. On the one hand, CD nanozymes catalyzed the decomposition of H 2 O 2 to generate •O 2− , and •O 2− oxidized OPD to reduce DAP. On the other hand, the FRET occurred between DAP and CDs, resulting in a decrease in the fluorescence intensity of CDs at 450 nm and an increase in the fluorescence intensity of DAP at 560 nm. Thus, a dual signal change in the fluorescence emission was observed. Based on this ratiometric fluorescence signal, the detection of H 2 O 2 could be achieved.
To obtain the best conditions for the detection of H 2 O 2 , the detection parameters including the concentration of OPD and the reaction conditions (pH, temperature, and reaction time) were optimized (Figure 7). The effects of the above parameters were investigated using the ratio of the fluorescence intensity at 560 nm to the fluorescence intensity at 450 nm (I 560 /I 450 ) as the signal. The increase in the substrate of OPD firstly led to the increase in I 560 /I 450 resulting from the formation of DAP (Figure 7a). Then, a reaction plateau was achieved. It was found that I 560 /I 450 had a maximum value at pH = 4.0, which proved that the CD nanozymes had the highest reactivity (Figure 7b). This was possibly due to the high reactivity of H 2 O 2 at this pH. When the reaction temperature was 40 • C, I 560 /I 450 had a maximum value, proving that the CD nanozymes had the highest reactivity close to the physiological temperature like natural enzymes (Figure 7c). In addition, the reaction reached equilibrium at 35 min (Figure 7d). These optimized conditions were used for further experiments. Figure 7e shows the fluorescence spectra after adding different concentrations of H 2 O 2 to the CDs + OPD system. As can be seen, with the increase in H 2 O 2 concentration, the fluorescence intensity of CDs at 450 nm decreased, and the fluorescence intensity of DAP at 560 nm increased. A linear relationship was observed between I 560 /I 450 and the logarithmic value of the hydrogen peroxide concentration (logC H2O2 ) at 1-20 µM (I 560 /I 450 = 0.326logC H2O2 + 0.300, R 2 = 0.991) and 20-200 µM (I 560 /I 450 = 0.707logC H2O2 -0.204, R 2 = 0.994) (Figure 7f). The detection limit calculated based on the signal-to-noise ratio of three (S/N = 3) was 0.42 µM. The comparison between the determination of H 2 O 2 using different nanomaterials and methods is demonstrated in Table 1 [47,49,[51][52][53][54]. The LOD was lower than that obtained using iron-doped CuSn(OH) 6 microspheres (Fe/CuSn(OH) 6 ) [51], FePt-Au ternary metallic hybrid nanoparticles (Fe Pt/AuHNPs) [52], mesoporous MnCo 2 O 4 [47], Ru/C 3 N 4 [49], Fe-doped carbon dots immobilized on cellulose nanofibrils (CNF/FeCDs) [53], and zinc and nitrogen co-doped multifunctional carbon dots (N,Zn/CDs) [54]. eau was achieved. It was found that I560/I450 had a maximum value at pH = 4.0, which proved that the CD nanozymes had the highest reactivity (Figure 7b). This was possibly due to the high reactivity of H2O2 at this pH. When the reaction temperature was 40 °C, I560/I450 had a maximum value, proving that the CD nanozymes had the highest reactivity close to the physiological temperature like natural enzymes (Figure 7c). In addition, the reaction reached equilibrium at 35 min (Figure 7d). These optimized conditions were used for further experiments.  Figure 7e shows the fluorescence spectra after adding different concentrations of H2O2 to the CDs + OPD system. As can be seen, with the increase in H2O2 concentration, the fluorescence intensity of CDs at 450 nm decreased, and the fluorescence intensity of DAP at 560 nm increased. A linear relationship was observed between I560/I450 and the log-

The Selectivity of the Detection and Real Sample Analysis
Selectivity is important for the application of the sensor. To evaluate the detection selectivity of the constructed ratiometric fluorescence sensor using CDs as luminophores and nanozymes, the effect of ascorbic acid (AA), L-cysteine, D-fructose (D-fru), glucose (Glu), lactose, Na + , Mg 2+ , Ca 2+ , D,L-tryptophan (D,L-try), D-sorbitol, PO 4 3− , Zn 2+ , Mn 2+ , vitamin B1, melamine, and Fe 3+ was investigated. Figure 8 shows the fluorescence intensity ratioof the OPD + CDs solution in the absence (F 0 ) or presence (F) of different substances, where F or F 0 were calculated using the fluorescence intensity (I) at 560 nm and 450 nm (F = I 560 /I 450 ). As shown, in the presence of H 2 O 2 , the signal increased significantly. In contrast, when the above substances were added, there was no obvious change in I 560 /I 450 , indicating that the ratiometric fluorescence sensor had good selectivity.

The Selectivity of the Detection and Real Sample Analysis
Selectivity is important for the application of the sensor. To evaluate the detection selectivity of the constructed ratiometric fluorescence sensor using CDs as luminophores and nanozymes, the effect of ascorbic acid (AA), L-cysteine, D-fructose (D-fru), glucose (Glu), lactose, Na + , Mg 2+ , Ca 2+ , D,L-tryptophan (D,L-try), D-sorbitol, PO4 3− , Zn 2+ , Mn 2+ , vitamin B1, melamine, and Fe 3+ was investigated. Figure 8 shows the fluorescence intensity ratioof the OPD + CDs solution in the absence (F0) or presence (F) of different substances, where F or F0 were calculated using the fluorescence intensity (I) at 560 nm and 450 nm (F = I560/I450). As shown, in the presence of H2O2, the signal increased significantly. In contrast, when the above substances were added, there was no obvious change in I560/I450, indicating that the ratiometric fluorescence sensor had good selectivity. The H2O2 in milk was detected with a standard addition method. As shown in Table  2, a good recovery, ranging from 97.0% to 102%, was obtained when the constructed ratiometric fluorescence sensor was applied to detect H2O2. The relative standard deviation (RSD) of the determination was less than 2.9%, proving good reliability. The H 2 O 2 in milk was detected with a standard addition method. As shown in Table 2, a good recovery, ranging from 97.0% to 102%, was obtained when the constructed ratiometric fluorescence sensor was applied to detect H 2 O 2 . The relative standard deviation (RSD) of the determination was less than 2.9%, proving good reliability.

Experiments and Instrumentations
The size of the CDs was characterized using a JEM-2100 transmission electron microscopy (TEM) with an ultrathin carbon film as the supporting substrate. The applied voltage was 200 kV. The dynamic light scattering (DLS) was measured using a SZ-100V2 nanoparticle analyzer (HORIBA, Kyoto, Japan) equipped with a 633 nm He-Ne laser. The scattering light at a 90 angle was detected and used to analyze the size and size distribution. The chemical composition of CDs was analyzed with X-ray photoelectron spectroscopy (XPS, PHI5300, PE, Waltham, MA, USA) using Mg Kα radiation (250 W, 14 kV). UV-Vis absorption spectra of the CDs were recorded using a UV-2450 UV-Vis spectrophotometer (Shimadzu, Japan). The fluorescence properties of CDs were investigated using a Fluoromax-4 spectrophotometer (Horiba, Montpellier, France). Electron paramagnetic resonance (EPR) spectra were obtained using an EMX-10/12 spectroscopic instrument (Bruker, Karlsruhe, Germany). Fourier transform infrared spectroscopy (FT-IR) was measured using a Vertex 70 spectrometer (Bruker, Karlsruhe, Germany) at room temperature on power-pressed KBr pellets.

Synthesis of CDs
CDs were synthesized with a one-step hydrothermal method. Specifically, 0.31 g of L-histidine was added into 20 mL of deionized water. Then, the mixture was added into a Teflon-lined autoclave and reacted at 180 • C for 10 h. The obtained solution was filtered with a filter film (0.22 µm) to remove large-sized particles, and the filtered solution was dialyzed at room temperature in a dialysis bag with a cut-off molecular weight of 500 Da for 72 h to remove unreacted small molecules or salts generated by the reaction. The dialysate was freeze-dried to obtain CD solids.

Peroxidase-Mimicking Activity of CDs
OPD was selected as the substrate to investigate the peroxidase-mimicking activity of CDs. The absorbance spectrum of the oxidized product, DAP, generated by the CDcatalyzed oxidation of OPD with H 2 O 2 was determined. Briefly, a Tris-HCl buffer solution (Tris-HCl, 50 mM, pH = 4.0) was used as the medium. Three samples including the control groups (OPD + H 2 O 2 and OPD + CDs) and the experimental group (OPD + H 2 O 2 + CDs) were investigated. The absorbance spectra were measured after 35 min of reaction at 40 • C. The used concentrations of OPD, H 2 O 2 , and CDs were 7.5 mM, 0.2 mM, and 15 µg/mL, respectively.
To investigate the catalytic mechanism of CD nanozymes, the trapping of free radicals generated during the catalytic process was investigated. TMB was applied as the substrate of peroxidase-mimicking nanozymes and CDs (15 µg/mL), and in the catalyzed oxidation of TMB, hydrogen peroxide was used as the model. P-benzoquinone, tryptophan, and tert-butanol were chosen as the scavengers for the superoxide anion radical (•O 2− ), singlet oxygen ( 1 O 2 ), and hydroxyl radical (•OH). Specifically, one of the above three capture agents (100 µM for p-benzoquinone and 100 µg/mL for both tryptophan and tert-butanol) was added to the TMB (0.5 mM) + CDs (15 µg/mL) + H 2 O 2 (3.3 mM) system and the absorbance value of the solution at 652 nm (A 652 ) was measured after 10 min of reaction.
The ESR test was further carried out using 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) as a trapping agent to determine the amount of •O 2− generated by the catalytic reaction. Experiments were performed in methanol solution (pH = 4.0). DMPO + H 2 O 2 and DMPO + H 2 O 2 + CDs solutions were respectively prepared and then exposed to UV light for 2 min. The concentrations of DMPO, H 2 O 2 , and CDs were 20 mM, 200 mM, and 30 µg/mL, respectively.

Ratiometric Fluorescence Detection of H 2 O 2 and Real Sample Analysis
For the detection of H 2 O 2 , OPD (7.5 mM), and CDs (15 µg/mL), a Tris-HCL (50 mM, pH = 4.0) was used as the detection medium. When different concentrations of H 2 O 2 were added, the mixture was incubated in the dark at 40 • C for 35 min. Then, the emission spectrum of the solution was recorded with an excitation wavelength of 390 nm. To investigate the selectivity of the constructed ratiometric fluorescence sensor for detecting H 2 O 2 , ascorbic acid (AA), D-sorbitol, melamine, vitamin B1, L-cysteine, D-fructose, glucose, lactose, Mn 2+ , PO 4 3− , Zn 2+ , Na + , Mg 2+ , Ca 2+ , and D,L-tryptophan, which are commonly found in milk, were selected as the possible interfering substances. The influence of the above substances on the detection of hydrogen peroxide (0.2 mM) was investigated. The concentration of Fe 3+ was 0.6 µg/mL and the concentration of the other substances was 4 mM. For real sample analysis, H 2 O 2 in milk was detected using a standard addition method.

Conclusions
In summary, a novel ratiometric fluorescence sensing platform was constructed based on carbon dots as both luminophores and nanozymes, which realized the sensitive detection of hydrogen peroxide. CDs with peroxidase activity were prepared with a one-step hydrothermal method using L-histidine as an inexpensive precursor. The synthesis method was simple, green, and of a low cost. CDs had bright blue fluorescence and peroxidasemimicking activity. The catalytic mechanism and enzymatic parameters of CD nanozymes were investigated. It was proven that CDs had good affinity for both OPD and H 2 O 2 substrates. From the fluorescence resonance energy transfer (FRET) phenomenon between CDs and DAP, the oxidation product of OPD was confirmed. CDs catalyzed H 2 O 2 to generate •O 2− , which oxidized OPD to generate DAP. The FRET between CDs and DAP resulted in a decrease in the fluorescence of CDs and an increase in the fluorescence of DAP, leading to a ratiometric fluorescence analysis system. The fabricated ratiometric fluorescence system had high detection and good selectivity in the detection of H 2 O 2 . The strategy using CDs as both luminophores and nanozymes could provide new ideas for nanozyme-based sensing systems and the construction of ratiometric fluorescence sensors.