FeCoP-NC diatomic nano-enzyme as well as preparation method and application thereof

Abstract

The invention provides a FeCoP-NC diatomic nano enzyme as well as a preparation method and application thereof. The used FeCoP-NC nano-enzyme has active sites of FeN3P-CoN3P, the mass fraction of doped P is 0.08%-0.12%, the mass fraction of Fe is 0.25%-0.32%, the mass fraction of Co is 0.5%-0.58%, the FeCoP-NC nano-enzyme is of a dodecahedron structure, the surface area of the FeCoP-NC nano-enzyme is 810-830 m < 2 > / g, the FeCoP-NC nano-enzyme is of a porous structure, and the pore diameter of the FeCoP-NC nano-enzyme is 3-3.5 nm. The FeCoP-NC nano-enzyme disclosed by the invention has excellent oxide-like enzyme activity, and can be used as a catalyst for catalyzing a chromogenic reaction of a chromogenic substrate in a colorimetric sensor.

Description

Technical Field

[0001] This invention relates to the field of nanozyme technology, specifically to a FeCoP-NC diatomic nanozyme, its preparation method, and its applications. Background Technology

[0002] Nanozymes are a class of artificial nanomaterials possessing the catalytic activity of natural enzymes. They combine the physicochemical properties of nanomaterials with the catalytic function of biological enzymes, mimicking the functions of natural enzymes such as peroxidase, oxidase, and superoxide dismutase through surface atomic arrangement or specific structures to catalyze substrate transformation. For example, iron(III) oxide nanoparticles can mimic peroxidase, decomposing hydrogen peroxide to generate reactive oxygen species. Currently, common applications of nanozymes include their use in colorimetric sensors to catalyze the colorimetric reactions of chromogenic substrates.

[0003] Single-atom nanozymes (SAzymes) and diatomic nanozymes (DAzymes) are two types of artificial enzymes designed based on atomically dispersed active sites. The active site of a single, isolated metal atom (such as Fe or Cu) forms an active center similar to that of a natural enzyme through nitrogen or carbon coordination, mimicking various natural enzymes such as oxidase (OXD), peroxidase (POD), catalase (CAT), and superoxide dismutase (SOD). The active site of a diatomic nanozyme contains two adjacent atomically dispersed metal atoms (homonuclear or heteronuclear, such as Fe-Co or Mo-Cu), forming a diatomic synergistic catalytic center. Single-atom nanozymes achieve ultra-high catalytic efficiency by maximizing atom utilization, while the dual-single-atom system can further enhance performance through metal synergistic effects. Compared to single-atom nanozymes, the synergistic effect between the two atomic sites in diatomic nanozymes provides additional adsorption sites and modulates the local electronic structure, thereby achieving superior enzyme activity. For example, Fe-Co diatomic nanozymes, in which the Co atomic sites synergistically influence the d-band center position of the Fe atomic sites and serve as a second reaction center, exhibit excellent oxidase-like (OXD) activity.

[0004] Currently, research on diatomic nanozymes is still in its early stages, and there are few reports on further enhancing their catalytic activity. Summary of the Invention

[0005] The purpose of this invention is to provide FeCo-NC diatomic nanozymes with excellent oxidase-like activity.

[0006] Another object of the present invention is to provide a method for preparing FeCo-NC diatomic nanozymes.

[0007] Another object of the present invention is to provide applications of FeCo-NC diatomic nanozymes.

[0008] To achieve the above objectives, the present invention adopts the following technical solution: A FeCoP-NC diatomic nanozyme, wherein the FeCoP-NC diatomic nanozyme has FeN3P-CoN3P active sites and exhibits oxidase-like activity.

[0009] In some embodiments, the FeCoP-NC diatomic nanozyme contains 0.08-0.12% P, 0.25%-0.32% Fe, and 0.5%-0.58% Co by mass.

[0010] Furthermore, the FeCoP-NC diatomic nanozyme contains 0.09-0.11% P, 0.28%-0.3% Fe, and 0.5%-0.55% Co by mass.

[0011] Most preferably, the FeCoP-NC diatomic nanozyme contains 0.1% P, 0.29% Fe, and 0.53% Co by mass.

[0012] Furthermore, the FeCoP-NC diatomic nanozyme exhibits a dodecahedral structure with a surface area of ​​810~830 m². 2 / g, and has a porous structure with an average pore size of 3~3.5nm.

[0013] More preferably, the FeCoP-NC diatomic nanozyme has a dodecahedral structure with a surface area of ​​820~825 m². 2 It has a porous structure with an average pore size of 3.2~3.5nm.

[0014] A second aspect of this invention provides a method for preparing the above-mentioned FeCoP-NC diatomic nanozyme, the method comprising the following steps: (1) Trivalent iron salt, cobalt salt, hypophosphite and zinc salt were reacted in an alcohol solvent at 45~55℃ in the presence of 2-methylimidazole to obtain ZIF-8 precursor doped with phosphorus, cobalt and iron; (2) The FeCoP-NC diatomic nanozyme is obtained by calcining the ZIF-8 precursor doped with phosphorus, cobalt and iron in an inert gas atmosphere, wherein the calcination temperature is 900~1100℃, for example, 900℃, 950℃, 1000℃, 1050℃, 1100℃ or any temperature between any two of the above.

[0015] Furthermore, in step (2), the inert gas is nitrogen or argon.

[0016] Further, in step (2), the calcination includes first heating to 900-1100℃ at a heating rate of 1-10℃ / min, and then calcining at 900-1100℃ for at least 2 hours. The heating rate is, for example, 1℃ / min, 2℃ / min, 3℃ / min, 4℃ / min, 5℃ / min, 6℃ / min, 7℃ / min, 8℃ / min, 9℃ / min, or 10℃ / min.

[0017] Furthermore, in step (2), the heating rate is 3~7℃ / min.

[0018] Furthermore, in step (1), the ZIF-8 precursor doped with phosphorus, cobalt, and iron is obtained by reacting trivalent iron salt, cobalt salt, hypophosphite, and zinc salt in an alcohol solvent at 45-55°C in the presence of 2-methylimidazole. The reaction temperature is, for example, 45°C, 46°C, 47°C, 48°C, 49°C, 50°C, 51°C, 52°C, 53°C, or 55°C.

[0019] In some embodiments, the ferric salt is ferric nitrate, the cobalt salt is cobalt nitrate, the zinc salt is zinc nitrate, and the hypophosphite is sodium hydrogen phosphate.

[0020] In some embodiments, the alcohol solvent is methanol.

[0021] In some embodiments, the molar ratio of the trivalent iron salt, cobalt salt, zinc salt, 2-methylimidazole and hypophosphite is 1:(0.8~1.2):(50~100):(300~350):(1~5).

[0022] More preferably, the molar ratio of the trivalent iron salt, cobalt salt, zinc salt, 2-methylimidazole and hypophosphite is 1:(0.8~1.2):(60~80):(320~330):(1~5).

[0023] In some embodiments, the ferric salt is fed with Fe(NO3)3·9H2O2, the cobalt salt is fed with CO(NO3)2·6H2O, and the zinc salt is fed with Zn(NO3)2·6H2O.

[0024] Further, the mass ratio of Fe(NO3)3·9H2O2, CO(NO3)2·6H2O, Zn(NO3)2·6H2O, 2-methylimidazole and NaH2PO2 is 1~2:1~1.5:80~90:95~105:1.

[0025] In some specific embodiments, in step (1), the ferric salt, the cobalt salt and the zinc salt are added to a portion of the alcohol solvent and mixed by ultrasound to form solution A; the 2-methylimidazole and the hypophosphite are added to the remaining alcohol solvent and mixed by ultrasound to form solution B; solution A and solution B are mixed, and after ultrasonic treatment, the mixture is first allowed to stand at 45~55℃ for 10~15h, and then stirred for 10~15h. The resulting reaction solution is separated to obtain a solid, and the solid is washed with methanol and dried to obtain the ZIF-8 precursor doped with phosphorus, cobalt and iron.

[0026] The third aspect of this invention provides the application of the above-mentioned FeCoP-NC diatomic nanozyme in a colorimetric sensor.

[0027] Specifically, the FeCoP-NC diatomic nanozyme serves as a catalyst in the colorimetric sensor for the colorimetric reaction of the chromogenic substrate.

[0028] More specifically, the chromogenic substrate includes one or more of 3,3',5,5'-tetramethylbenzidine (TMB), N,N-diethyl-p-phenylenediamine sulfate (DPD), o-phenylenediamine, and 2,2'-azidobis-3-ethylbenzothiazoline-6-sulfonic acid.

[0029] In some embodiments, the temperature for the FeCoP-NC diatomic nanozyme catalytic reaction is 22-28°C. More preferably, it is 24-26°C.

[0030] In some embodiments, the concentration of the FeCoP-NC diatomic nanozyme in the reaction system of the colorimetric sensor is 0.1~0.5 mg / mL.

[0031] In some specific embodiments, the chromogenic substrate in the reaction system of the colorimetric sensor is TMB, the initial pH value of the reaction system is 3-4, and after the reaction is completed, the absorbance value of the reaction system is measured, wherein the absorbance value ranges from 625 to 675 nm.

[0032] In some other embodiments, the chromogenic substrate in the reaction system of the colorimetric sensor is DPD, the initial pH value of the reaction system is 6-8, and after the reaction is completed, the absorbance value of the reaction system is tested, the absorbance value range is 530-570 nm.

[0033] Compared with the prior art, the present invention has the following advantages: This invention improves and optimizes Fe-Co diatomic nanozymes, resulting in phosphorus-doped FeCoP-NC diatomic nanozymes with superior oxidase-like activity. Attached Figure Description

[0034] Figure 1This is a schematic diagram of the FeCoP-NC synthesis process; Figure 2 (a) SEM image of FeCoP-NC; (b) HRTEM image of FeCoP-NC; (c) AC-HAADF-STEM image of FeCoP-NC; (d) magnified view of AC-HAADF-STEM image of FeCoP-NC. Figure 3 EDS spectrum of FeCoP-NC; Figure 4 The graph shows the Fe, Co, and P contents of FeCoP-NC. Figure 5 (a) XRD patterns of FeCoP-NC, Fe-NC, Co-NC and FeCo-NC; (b) Raman spectra of FeCoP-NC, Fe-NC, Co-NC and FeCo-NC; Figure 6 (a) High-resolution X-ray photoelectron spectra of N 1p for Fe-NC, Co-NC, FeCo-NC and FeCoP-NC; (b) High-resolution X-ray photoelectron spectra of P 2p for FeCoP-NC with different phosphorus doping amounts; Figure 7 (a) Nitrogen adsorption-desorption isotherms for Fe-NC, Co-NC, FeCo-NC and FeCoP-NC; (b) Pore size distribution curves for Fe-NC, Co-NC, FeCo-NC and FeCoP-NC. Figure 8 A comparison of the catalytic activities of Fe-NC, Co-NC, FeCo-NC, and FeCoP-NC for TMB; Figure 9 This is a comparison diagram of the activities of FeCoP-NC in Example 1 and Comparative Example 4; Figure 10 The effects of (a) concentration, (c) temperature, and (e) initial pH of FeCoP-NC solution on TMB catalytic activity; (b) concentration, (d) temperature, and (f) initial pH of FeCoP-NC solution on DPD catalytic activity. Figure 11 The steady-state kinetics of FeCoP-NC are shown in the inset, with the Michaelis-Menten equation plotted against the TMB concentration as a double reciprocal. Figure 12(a) UV-Vis spectra of color changes of the TMB + FeCoP-NC system in air, oxygen and nitrogen; (b) Effect of different concentrations of inhibitors on the inhibitory effect of FeCoP-NC activity; (c) EPR spectra of Fe-NC, Co-NC, FeCo-NC and FeCoP-NC; and (d) Comparison of spin concentrations of Fe-NC, Co-NC, FeCo-NC and FeCoP-NC. Figure 13 Impedance diagrams for (a) FeCoP-NC and FeCo-NC; pulse voltammetry for TMB, FeCoP-NC, and FeCoP-NC + TMB; and cyclic voltammetry for TMB, FeCoP-NC, and FeCoP-NC + TMB. Figure 14 This is a schematic diagram of the dual-pathway FeCoP-NC oxidation of TMB; Figure 15 (a) K-edge XANES spectrum of Fe; (b) K-edge XANES spectrum of Co; (c) FT-EXAFS spectrum of Fe; (d) FT-EXAFS spectrum of Co; Figure 16 To investigate the mechanism of organophosphorus pesticides; Figure 17 Optimize heatmaps for ACP and AA2P conditions; Figure 18 Different metal ions have different effects on the color development of (a) TMB and (b) DPD; different antibiotics and pesticides have different effects on the color development of (c) TMB and (d) DPD. Figure 19 Linear plots of malathion for TMB and DPD detection. Detailed Implementation

[0035] The present invention will be further described below with reference to embodiments. However, the present invention is not limited to the following embodiments. The implementation conditions used in the embodiments can be further adjusted according to different requirements of specific use, and the implementation conditions not specified are conventional conditions in this industry. The technical features involved in the various embodiments of the present invention can be combined with each other as long as they do not conflict with each other. The implementation conditions used in the embodiments can be further adjusted according to specific requirements, and the implementation conditions not specified are usually conditions in conventional experiments.

[0036] A range of standards, solvents, or enzymes were obtained from Adams (Shanghai, China), including ferric nitrate nonahydrate (Fe(NO3)3·9H2O, 98.0%), cobalt nitrate hexahydrate (Co(NO3)2·6H2O, 99.0%), 2-methylimidazolium (2-MIM, 98.0%), sodium hypophosphite (NaH2PO2, 95%), methanol (CH3OH, ≥99.0%), dimethyl sulfoxide (DMSO, ≥99.0%), 3,3',5,5'-tetramethylhydrazine (TMB, ≥98.0%), N,N-diethyl-p-phenylenediamine sulfate (DPD, 99%), and 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid). (ABTS, 97.0%), 3,3'-diaminobenzidine tetrahydrochloride (DAB, ≥98.0%), o-phenylenediamine (OPD, 99%), furfuryl alcohol (FFA, 99.0%), ethylenediaminetetraacetic acid (EDTA, 99.5%), isopropanol (IPA, ≥99.7%), p-benzoquinone (PBQ, 99.2%), acid phosphatase (ACP, 0.4–0.5 U / g), L-ascorbic acid-2-phosphate (AA2P, 96%). Malathion (MAL) was purchased from Tansoole (Shanghai, China). Ultrapure water (>18.2 MΩ) was produced by a Milli-Q gradient system (Bedford, MA, USA). All reagents were used without further purification.

[0037] KQ-500DE CNC ultrasonic cleaner (Kunshan Ultrasonic Instruments Co., Ltd., Kunshan), OTF-1200x tube furnace (Hefei Kejing, Hefei), DHG-9070a electric blow drying oven (Xiwen Biotechnology, Shanghai), B15-3 multi-functional constant speed magnetic stirrer (Shanghai Sile Instruments Co., Ltd., Shanghai, China) and PB-10 pH meter (Sartorius Scientific Instruments, Nanjing, China).

[0038] The morphology and structure of FeCoP-NC diatomic nanozymes were characterized using field emission environmental scanning electron microscopy (QUANTAFEG 250, FEI, USA) and transmission electron microscopy (Talo SF200S, Thermo Scientific, USA). Specific surface area was calculated using the BET (Brunauer-Emmett-Teller, BET) method. Electron paramagnetic resonance (EPR) spectroscopy was performed on a Bruker EMX X-band spectrometer equipped with an Oxford variable-temperature cryostat. High-resolution X-ray photoelectron spectroscopy (XPS) was recorded using an X-ray photoelectron spectrometer (ESCALAB 250Xi, Shimadzu, Japan) equipped with a 150 W (15 kV, 10 mA) standard monochromatic light source (AIKR). All XPS peaks were calibrated to C1s (284.8 eV). All UV-Vis absorbances were measured on a UV-2600 spectrophotometer (Shimadzu, Japan).

[0039] Unless otherwise specified, in the test results and analysis below and in the accompanying drawings of this invention, FeCoP-NC refers to FeCoP-NC prepared by the method of Example 1.

[0040] Example 1: This example provides a FeCoP-NC diatomic nanozyme, the preparation method of which is as follows: Figure 1 As shown, the specific steps are as follows: (1) Weigh 0.060 g Fe(NO3)3·9H2O, 0.050 g Co(NO3)2·6H2O and 3.390 g Zn(NO3)2·6H2O and add them to a beaker containing 100 mL CH3OH (analytical grade). Sonicate for 30 min to form a clear solution A.

[0041] (2) Weigh 3.940 g 2-MIM and 40 mg NaH2PO2 and add them to a beaker containing 100 mL CH3OH to obtain a clear solution B.

[0042] (3) Mix solution A and solution B and sonicate for 30 min. Let stand in an oil bath at 50°C for 12 h, then stir at 600 r / min for 12 h, centrifuge, and wash the obtained solid product with methanol 5 times.

[0043] (4) The product after being washed with methanol was dried in a vacuum drying oven at 60°C for 12 hours to obtain the precursor FeCoP-ZIF-8, which is a light purple powder.

[0044] (5) After grinding the precursor FeCoP-ZIF-8 until there are no obvious agglomerates, transfer it to a ceramic boat and heat it at 5℃·min under a N2 atmosphere.-1 The temperature was increased to 1000℃, and the mixture was calcined at 1000℃ for 2 hours to obtain FeCoP-NC diatomic nanozymes in the form of black powder.

[0045] Comparative Example 1: This comparative example provides a Fe-NC nanozyme, the preparation method of which is basically the same as that of Example 1, except that Co(NO3)2·6H2O is not added in step (1) and NaH2PO2 is not added in step (2).

[0046] Comparative Example 2: This comparative example provides a Co-NC nanozyme, the preparation method of which is basically the same as that of Example 1, except that Fe(NO3)3·9H2O is not added in step (1) and NaH2PO2 is not added in step (2).

[0047] Comparative Example 3: This comparative example provides a FeCo-NC nanozyme, the preparation method of which is basically the same as that of Example 1, except that NaH2PO2 is not added in step (2).

[0048] Comparative Example 4: This comparative example provides another method for preparing FeCoP-NC diatomic nanozymes, the steps of which are as follows: (1) Dissolve 0.1 M ZnCl2, 0.005 M FeCl3·6H2O, 0.005 M CoCl2·6H2O and 0.005 M NaH2PO2 in 40 mL of formamide (FA), and then sonicate for 30 minutes to completely dissolve them to obtain a homogeneous mixed solution.

[0049] (2) The mixed solution was transferred to an autoclave and heated at 180°C for 12 hours. After cooling, it was filtered, and the filtered solid was washed alternately with deionized water and ethanol, repeating the washing three times. The washed solid was dried overnight in a vacuum environment at 60°C to obtain the precursor.

[0050] (3) Grind the precursor into a uniform powder and then heat it at 5℃·min under argon protection. -1 The temperature was increased to 900°C at a rising rate and maintained at 900°C for 2 hours.

[0051] (4) The powder sample obtained by natural cooling was heat-treated in 0.5 M sulfuric acid solution at 80°C for 8 hours.

[0052] (5) Use water and ethanol alternately as the solvent until the pH of the supernatant reaches 7. The filtered solid is then vacuum dried at 60°C to obtain FeCoP-NC diatomic nanozyme.

[0053] Nanozyme characterization (1) Characterization of FeCoP-NC diatomic nanozyme in Example 1 Example 1: A precursor FeCoP-ZIF-8 doped with phosphorus, cobalt, and iron was synthesized by a solvothermal method. It was then calcined at 1000°C in a nitrogen atmosphere to evaporate the zinc element from FeCoP-ZIF-8. The resulting powder was the FeCoP-NC nanocomposite. Figure 1 SEM and TEM images show that FeCoP-NC has a regular dodecahedral structure with a diameter of approximately 0.24 μm. Figure 2 (a and b). Numerous high-brightness atomically dispersed points were observed in the AC-HAADF-STEM images on the carbon substrate (marked with yellow circles). Figure 2 (c and d), no metal particles or clusters were observed. Qualitative and quantitative analysis of the elements in the FeCoP-NC material was performed by TEM-EDS and ICP-MS. Fe, Co, P, C, and N elements were uniformly distributed in the nanocomposite ( Figure 3 The mass fractions of Fe, Co, and P were 0.29%wt, 0.53%wt, and 0.1%wt, respectively. Figure 4 ).

[0054] (2) Comparison of FeCoP-NC diatomic nanozyme of Example 1 with Fe-NC nanozyme of Comparative Example 1, Co-NC nanozyme of Comparative Example 2 and FeCo-NC nanozyme of Comparative Example 3 XRD pattern analysis ( Figure 5 As shown in (a), all nanomaterials exhibit characteristic peaks of graphitic carbon, located at 24.5° (002) and 42.5° (010), respectively, and no metal oxides or elemental phases were detected, confirming that they are primarily carbon-based structures. Raman spectroscopy ( Figure 5 b) indicates that FeCoP-NC's I D / I G The ratio (1.129) was significantly higher than that of other samples (Fe-NC, Co-NC and FeCo-NC), indicating that phosphorus doping introduced more carbon defects, thereby promoting the exposure of metal sites and electron transfer.

[0055] XPS spectral analysis showed that, compared with FeCo-NC, the content of graphitic nitrogen (401.4 eV) in P-doped FeCoP-NC was significantly increased, effectively stabilizing Fe / Co atoms and enhancing active sites. Figure 6 (a). The latter exhibits a synergistic effect among pyridine nitrogen (399.1 eV), graphitic nitrogen (401.3 eV), and pyrrole nitrogen (401.4 eV), where the reduced pyrrole nitrogen content in FeCoP-NC may improve catalytic stability by decreasing the adsorption of unstable intermediates. P 2p spectrum ( Figure 6Figure b) shows characteristic peaks of 132.5 eV (Fe / Co-P) and 140.2 eV (PC), confirming the successful phosphorus doping. Meanwhile, following the method of Example 1, the amount of NaH2PO2 added in step (2) was set to 20 mg (in... Figure 6 The middle b is marked as FeCoP 20 -NC), 40 mg (in Figure 6 The middle b is marked as FeCoP 40 -NC), 50 mg (in Figure 6 The middle b is marked as FeCoP 50 -NC), 70 mg (in Figure 6 The middle b is marked as FeCoP 70 -NC), 100 mg (in Figure 6 The middle b is marked as FeCoP 100 Comparing the effects of different P doping methods, the results showed that the PC bond content reached a maximum of 86.75% in FeCoP40-NC. Excessive doping led to structural deformation. These results indicate that appropriate phosphorus doping can optimize the electronic structure and catalytic performance of nanomaterials by adjusting the nitrogen-phosphorus bonding state.

[0056] BET measurement results show that FeCoP-NC has a diameter of 822.67 m. 2 / g specific surface area ( Figure 7 (a), significantly higher than FeCo-NC (317.93 m). 2 / g) and the surface area of ​​other comparative materials. All nanomaterials exhibited type IV isotherms and H3 pressure hysteresis curves, confirming the presence of slit-like mesopores derived from the ZIF-8 precursor. Notably, the average pore size of FeCoP-NC (3.33 nm) falls between that of Fe-NC (2.84 nm) and Co-NC (4.06 nm). Figure 7 (b) Therefore, phosphorus doping synergistically optimizes the pore size distribution and specific surface area, thereby providing an ideal mass transfer channel and active site exposure environment for the catalytic reaction.

[0057] Detection and comparison of oxidase-like activities of nanozymes Comparison of oxidase-like activities of FeCoP-NC in Example 1 with Fe-NC in Comparative Example 1, Co-NC in Comparative Example 2, and FeCo-NC in Comparative Example 3. The UV-Vis absorption of the reaction system was detected at 652 nm. For example... Figure 8As shown, no adsorption peak at 652 nm was observed in the presence of only TMB. Similarly, the solution was colorless when only FeCoP-NC was present. In the presence of both FeCoP-NC and TMB, the solution exhibited a bright blue color, indicating that FeCoP-NC exhibited oxidase-like activity under these conditions. Furthermore, the redox activity of FeCoP-NC was significantly superior to that of Fe-NC, Co-NC, and FeCo-NC. This suggests that P doping increases the number of active sites, promoting the oxidation of TMB.

[0058] (2) Comparison of oxidase-like activities between FeCoP-NC in Example 1 and FeCoP-NC in Comparative Example 4 like Figure 9 The results showed that the oxidase-like activity of FeCoP-NC in Example 1 was significantly higher than that of FeCoP-NC in Comparative Example 4.

[0059] Colorimetric sensor: Optimization of reaction systems for colorimetric sensors (TMB+FeCoP-NC, DPD+FeCoP-NC) To achieve optimal catalytic efficiency, key parameters in the systems (TMB+FeCoP-NC, DPD+FeCoP-NC) were optimized, including incubation temperature, solution pH, and nanozyme concentration. For example... Figure 10 As shown in Figures a and b, an optimal FeCoP-NC concentration of 0.2 mg / mL was achieved, while concentrations exceeding this level led to a decrease in the absorbance of the DPD system. The TMB system maintained high activity over a wide temperature range of 20–60 °C. Figure 10 (c), while the stability of the DPD system decreases above 30℃ ( Figure 10 (d). Therefore, 25°C was chosen as the suitable reaction temperature. The TMB system exhibits the highest activity at pH 3.2 ( Figure 10 (e), while the DPD system exhibits considerable activity at pH 5.0 and 7.0 ( Figure 10 (f). Ultimately, ultrapure water was selected as the reaction medium for the DPD system. Based on time-dependent experiments, 20 minutes was determined to be the optimal incubation time.

[0060] Steady-state dynamics of FeCoP-NC The steady-state kinetics of the colorimetric sensor based on the prepared nanocomposite material were investigated using the typical Michaelis-Menten equation. TMB was selected as the substrate, and each experiment was repeated three times by varying the concentration of one substrate. Figure 11 As is well known, K mThe lower the value, the higher the affinity between the nanozyme and the substrate. As shown in Table 1, the FeCoP-NC diatomic nanozyme of Example 1 provides K at different TMB levels. m The value was 0.29. Therefore, the affinity of FeCoP-NC in Example 1 was significantly improved compared to FeCo-NC, Fe-NC, and Co-NC. Furthermore, FeCoP-NC in Example 1 exhibited higher affinity than ordinary HRP and previously reported CuS and PdNPs@GOF (Table 1). These data indicate that FeCoP-NC in Example 1 greatly enhances oxidase-like activity.

[0061] Table 1. Comparison of Michaelis-Menten constants for the oxidation of TMB catalyzed by different nanozymes

[0062] Mechanism of FeCoP-NC type oxidase activity in Example 1 Free radical mediated To systematically study the active sites and underlying catalytic mechanisms of the FeCoP-NC diatomic nanozyme of Example 1, three control systems (O2 / air / N2) were constructed. Figure 12 (a) A distinct bright blue color was observed under an O2 atmosphere. Combined with the free radical scavenging experiment ( Figure 12 (b) PBQ vs. O2 - The specific removal of O2 led to a 76.8% decrease in system absorbance, initially indicating that O2... - It is the main reactive oxygen species in the catalytic process. EPR analysis ( Figure 12 (c) Quantitatively, phosphorus doping significantly enhances the radical generation ability of FeCoP-NC. This is achieved by calculating the electron spin number (Ns) ( Figure 12 (d), the spin concentration of FeCoP-NC in Example 1 (1.028 × 10⁻⁶). 12 spin mm -3 ) compared to FeCo-NC (9.781×10 11 spin mm -3 The content of Fe-NC increased by 5.1%, and it was higher than that of the single metal composition (Fe-NC: 8.525 × 10⁻⁶). 11 spin mm -3 Co-NC: 2.547×10 11 spin mm -3 Phosphorus exhibited significant advantages. The positive correlation between EPR signal intensity and catalytic activity confirmed that the introduction of phosphorus enhanced the catalytic activity of O2 by modulating the electronic structure. - The high generation efficiency gives the material superior oxidase-like activity.

[0063] Two-electron-mediated A comparison of impedance before and after phosphorus doping showed that the impedance decreased significantly after phosphorus doping, thereby promoting electron transfer. Figure 13 (a) In a 0.1 M Na₂SO₄ electrolyte system, the oxidation of TMB follows a stepwise single-electron transfer mechanism: first, a semiquinone imine radical cation intermediate (TMB) is generated at 0.30 V (relative to Ag / AgCl). + It is then further oxidized at 0.46V (relative to Ag / AgCl) to the stable diamine diimine final product (TMB). 2+ In differential pulse voltammetry (DPV) testing, this process is characterized by two characteristic anodic oxidation peaks at 0.30 V and 0.46 V. Figure 13 (b) After introducing the FeCoP-NC diatomic nanozyme from Example 1, TMB can be efficiently catalytically oxidized to form a blue diamine-diimine charge-transfer complex (oxTMB), resulting in a significant decrease in the TMB oxidation peak current in the DPV curve. Cyclic voltammetry (CV) testing further confirmed that the pure FeCoP-NC system did not have a significant redox peak ( Figure 13 In contrast, the pure TMB system exhibits a typical redox pair in the 0.3–0.6 V potential range. Notably, the characteristic peaks of TMB completely disappear in the FeCoP-NC / TMB coexistence system, suggesting that this nanozyme may directly catalyze the conversion of TMB to oxTMB via a two-electron transfer mechanism. These results comprehensively demonstrate that the FeCoP-NC diatomic nanozyme efficiently catalyzes the oxidation of TMB through a unique electron transfer pathway.

[0064] Based on the above systematic experimental results, it can be inferred that the FeCoP-NC nanocomposite material of Example 1 exhibits a unique dual-pathway synergistic catalytic mechanism in the colorimetric reaction of TMB. Figure 14 In other words, this catalytic system functions through two parallel but complementary reaction pathways: (1) a direct two-electron transfer pathway mediated by the bimetallic active center (Fe / Co), and (2) a pathway primarily driven by superoxide anion radicals (·O2). - The oxidation pathway driven by the generation of free radicals is significantly improved through a dual-pathway synergistic catalytic mechanism, thereby laying the foundation for the construction of a highly sensitive colorimetric system.

[0065] The coordination environment and chemical state of Fe and Co elements in the FeCoP-NC material of Example 1 were systematically studied using X-ray absorption spectroscopy (XAS). Figure 15As shown in Figure a, the Fe K-edge X-ray absorption near-edge structure (XANES) spectral analysis indicates that the absorption edge position of Fe in the FeCoP-NC of Example 1 is between that of the standard samples FeO and Fe2O3, and closer to the edge position of Fe2O3. This characteristic clearly indicates that the Fe element in the material mainly exists in the +3 oxidation state. Similarly, the cobalt K energy edge XANES spectrum ( Figure 15 As shown in b), the absorption edge of cobalt lies between Co₂O₃ and Co₃O₄, and is closer to the absorption energy of Co₂O₃, indicating that cobalt is in the +3 oxidation state. Further analysis using extended X-ray absorption fine structure (EXAFS) spectroscopy... Figure 15 Figures c and d further show that the FeCoP-NC of Example 1 exhibits distinct main peaks at 1.59 Å and 1.22 Å, respectively, corresponding to the coordination bonds of Fe-N / O and Co-N / O, which is consistent with the results of DFT calculations. Notably, no coordination features of Fe-Fe, Co-Co, or Fe-Co metallic bonds were observed at 2.2 Å, providing crucial evidence that Fe and Co atoms are highly dispersed as single atoms in the nitrogen-doped carbon matrix. These structural features provide important atomic-scale structural information for understanding the excellent catalytic performance of this material. The above detection results indicate that the active site of the FeCoP-NC diatomic nanozyme of Example 1 is FeN3P-CoN3P. In contrast, the FeCoP-NC diatomic nanozyme of Comparative Example 4, after the same detection, was determined to have an active site of FeN4P-CoN4P, indicating a significant difference in activity due to the different active sites.

[0066] Application example: The method for detecting organophosphorus pesticide (malathion) residues using a colorimetric sensor based on the FeCoP-NC diatomic nanozyme from Example 1 is as follows: Mechanism for detecting organophosphorus pesticides Acid phosphatase (ACP) is a widely distributed hydrolase in organisms that efficiently catalyzes the hydrolysis of phosphate ester bonds under acidic conditions, thereby achieving substrate dephosphorylation. In this embodiment, ACP specifically hydrolyzes L-ascorbic acid-2-phosphate trisodium salt (AA2P) to highly reducing ascorbic acid (AA). AA further reduces oxidized chromogenic substrates (oxTMB and oxDPD) to colorless products (TMB and DPD), resulting in a significant fading of the solution color. However, the residue of organophosphate compounds (OPs) inhibits the activity of ACP, thereby reducing AA production and weakening its ability to compete with the OXD-like activity of FeCoP-NC nanozymes. Figure 16Therefore, this phenomenon provides a theoretical basis for the colorimetric detection of malathion based on FeCoP-NC nanozymes. Based on the above analysis, the concentrations of ACP and AA2P were optimized to establish feasible detection conditions for OPs. The optimized concentrations were: ACP 0.2 U / mL, AA2P 6 mg / mL. Figure 17 ).

[0067] To verify the selectivity of this detection system, its resistance to various common ions (Ni) was studied in detail. 2+ Al 3+ Na + Cu 2+ K + PO4 3- and NO2 - The anti-interference effect of the system is shown. ΔA (ΔA = A0 - A, where A0 and A represent the absorbance values ​​at 652 nm in the absence and presence of the interfering agent, respectively) only shows a significant change in the presence of MAL, indicating that the system has high specificity for OPs. Figure 18 (a and b). When antibiotic contaminants (chlortetracycline, oxytetracycline, minocycline) and pyrethroid insecticides (phosmet, cypermethrin, allethrin), interference tests also confirmed that the sensing platform's response to OPs was significantly stronger than that to other types of contaminants. Figure 18 (c and d). These phenomena indicate that the sensing platform can be effectively used to detect OPs and has excellent selectivity.

[0068] The colorimetric sensor used in this application example comprises FeCoP-NC, ACP, AA2P, TMB, DPD, purified water, and HAc-NaAc buffer. The reaction system of this colorimetric sensor includes a TMB system with TMB as the chromogenic substrate and a DPD system with DPD as the chromogenic substrate.

[0069] For detection, the reaction system of TMB was prepared as follows: HAc-NaAc buffer solution with pH 3.2 containing 0.2 mg / mL FeCoP-NC, 0.2 U / mL ACP, 6 mg / mL AA2P, 0.75 mM TMB, and 0.75 mM DPD; the water sample to be tested was added to the reaction system; the mixture was mixed and reacted at 25 °C for color development, and the absorbance at 652 nm was detected.

[0070] The reaction system for preparing the DPD system is purified water with pH 7 containing 0.2 mg / mL FeCoP-NC, 0.2 U / mL ACP, 6 mg / mL AA2P, 0.75 mM TMB, and 0.75 mM DPD. The water sample to be tested is added to the reaction system. The mixture is mixed and reacted at 25 °C to develop color, and the absorbance at 550 nm is detected.

[0071] For water samples that are outside the linear range, they can be diluted with purified water before testing.

[0072] The analytical performance of the FeCoP-NC-based colorimetric sensor in this application example was evaluated using TMB and DPD as chromogenic substrates to detect malathion (…). Figure 19 Within the linear range (LR) of 0.01–14 μM, A 652 A satisfactory linear relationship exists between the value and the concentration of malathion (R0). 2 =0.9971). The determined linear equation (LQ) is y = 0.10763x + 1.21156. Similarly, in A 550 A good linear relationship was also observed between the value and the malathion concentration, with a linear range of 0.01–14 μM, LQ of y = 0.01804x + 0.24834, and R0. 2 The limit of detection (LOD) is 0.9942, and the limit of detection (LOD) is 3.3 nm. The high consistency between the two dual-channel detection systems (TMB-652 nm and DPD-550 nm) not only confirms the reliability of the proposed method, but also provides a flexible option for practical applications under various experimental conditions.

[0073] To verify the feasibility of the current method in practical applications, six fruits and vegetables most susceptible to organophosphate residues (oranges, apples, grapes, broccoli, cabbage, and lettuce) and four actual water samples (three from a tributary of the Shihu River in Suzhou, China, and one from a tap water source) were selected. Malathion in the actual samples was detected by high-performance liquid chromatography (HPLC-DAD), and the detection data were used as a standard reference. The excellent analytical performance of malathion yielded spiked recoveries of 92.5%–100.0%, intra-day precision (expressed as relative standard deviation RSD) <3.2%, and inter-day precision <3.9%. HPLC-DAD analysis showed that the recovery rate of malathion incorporation ranged from 95.0% to 102.5% (see Table 2). Compared with HPLC data, the relative accuracy of the newly developed method was calculated to be 97.5%–102.6%, confirming its good reliability.

[0074] Table 2. Determination of malathion in actual water and food samples using the methods and HPLC-DAD in this application example.

[0075] In summary, Example 1 of this invention provides a nanozyme with excellent oxidase-like activity, which can be used in colorimetric sensors to catalyze the colorimetric reaction of chromogenic substrates. These substrates include, but are not limited to, 3,3',5,5'-tetramethylbenzidine (TMB), o-phenylenediamine (OPD), and 2,2'-azidobis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS). This nanozyme has great application potential in environmental monitoring, food industry, and clinical testing.

[0076] The present invention has been described in detail above, with the aim of enabling those skilled in the art to understand and implement the invention. However, this description should not be construed as limiting the scope of protection of the invention. All equivalent changes or modifications made in accordance with the spirit and essence of the invention should be included within the scope of protection of the invention.

Claims

1. A FeCoP-NC diatomic nanoszyme, characterized in that, The FeCoP-NC diatomic nanoszyme has FeN3P-CoN3P active sites, and has oxydase-like activity.

2. The FeCoP-NC diatomic nanoszyme according to claim 1, wherein, The mass fraction of P in the FeCoP-NC diatomic nanoszyme is 0.08-0.12%, the mass fraction of Fe is 0.25%-0.32%, and the mass fraction of Co is 0.5%-0.58%. And / or, the FeCoP-NC diatomic nanoszyme has a whole dodecahedron structure, a surface area of 810~830 m 2 / g, and has a porous structure with an average pore size of 3~3.5 nm.

3. The method for preparing FeCoP-NC diatomic nanoszyme according to claim 1 or 2, characterized in that, The preparation method comprises the following steps: The trivalent iron salt, the cobalt salt, the hypophosphorous acid salt and the zinc salt are reacted at 45-55 DEG C in an alcohol solvent in the presence of 2-methyl imidazole to obtain a ZIF-8 precursor doped with phosphorus, cobalt and iron; The ZIF-8 precursor doped with phosphorus, cobalt and iron is calcined in an inert gas atmosphere to obtain the FeCoP-NC diatomic nanoszyme, and the calcination temperature is 900-1100 DEG C.

4. The method for preparing FeCoP-NC diatomic nanoszyme according to claim 3, characterized in that, In step (2), the calcination comprises first increasing the temperature to 900-1100 DEG C at a temperature increasing rate of 1-10 DEG C / min, and then calcining at 900-1100 DEG C for at least 2 h.

5. The method for preparing FeCoP-NC diatomic nanoszyme according to claim 3, characterized in that, In step (1), the trivalent iron salt is ferric nitrate, the cobalt salt is cobalt nitrate, the zinc salt is zinc nitrate, and the hypophosphorous acid salt is sodium hydrogen phosphate; And / or, the alcohol solvent is methanol; And / or, the molar ratio of the trivalent iron salt, the cobalt salt, the zinc salt, 2-methyl imidazole and the hypophosphorous acid salt is 1:(0.8-1.2):(50-100):(300-350):(1-5).

6. The method for preparing FeCoP-NC diatomic nanoszyme according to claim 5, characterized in that, The trivalent iron salt is fed in the form of Fe(NO3)3.9H2O2, the cobalt salt is fed in the form of CO(NO3)2.6H2O, and the zinc salt is fed in the form of Zn(NO3)2.6H2O.

7. The method for preparing FeCoP-NC diatomic nanoszyme according to claim 6, characterized in that, The mass ratio of the Fe(NO3)3.9H2O2, the CO(NO3)2.6H2O, the Zn(NO3)2.6H2O, the 2-methyl imidazole and the NaH2PO2 is 1-2:1-1.5:80-90:95-105:

1.

8. The method for preparing FeCoP-NC diatomic nanoszyme according to claim 3, characterized in that, In step (1), the trivalent iron salt, the cobalt salt and the zinc salt are added into part of the alcohol solvent, ultrasonically mixed to form a solution A; the 2-methyl imidazole and the hypophosphorous acid salt are added into the remaining alcohol solvent, ultrasonically mixed to form a solution B; the solution A and the solution B are mixed, ultrasonically treated, and then first statically reacted at 45-55 DEG C for 10-15 h, and then stirred and reacted for 10-15 h; the obtained reaction liquid is separated to obtain a solid; the obtained solid is washed with methanol and dried to obtain the ZIF-8 precursor doped with phosphorus, cobalt and iron.

9. Application of the FeCoP-NC diatomic nanoszyme of claim 1 or 2 in a colorimetric sensor.

10. Use according to claim 9, characterized in that, The FeCoP-NC diatomic nanoszyme acts as a catalyst for catalyzing the color development reaction of a color development substrate in the colorimetric sensor, and the color development substrate comprises one or more of 3,3',5,5'-tetramethylbenzidine, N,N-diethyl-p-phenylenediamine sulfate, o-phenylenediamine and 2,2'-azinobis-3-ethylbenzothiazoline-6-sulfonic acid.

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