Method for preparing nanoparticles with oxidase-like activity, products and uses thereof

Abstract

The present application relates to the preparation method of nanoparticles with oxidase-like activity and its products and applications, and belongs to the technical field of phenylenediamine isomer recognition. The present application mainly adopts ZIF-8, Ce 3+ , M 3+ (Eu 3+ , Fe 3+ or Co 3+ ) and Mn-CDs to react, so as to obtain nanoparticles with oxidase-like activity. The nanoparticles prepared by the present application have good oxidase-like activity, and can be applied to catalyze the oxidation reaction of OPD, MPD and PPD, so as to obtain yellow, colorless and purple oxidation products respectively. Therefore, according to the color of the oxidation product formed by the catalysis of the nanoparticles with oxidase-like activity on the oxidation of phenylenediamine, a colorimetric method for identifying and detecting OPD, MPD and PPD is established, which has the characteristics of low cost, high sensitivity, simple operation and high selectivity, and has good application prospect in the detection of OPD and PPD in environmental water samples.

Description

Technical Field

[0001] This invention belongs to the field of phenylenediamine isomer recognition technology, and relates to the preparation method of nanoparticles with oxidase-like activity, as well as their products and applications. Background Technology

[0002] Organic isomers share similar physical and chemical properties due to subtle structural differences, making the simultaneous identification and detection of isomers of organic compounds a significant challenge. o-phenylenediamine (OPD), m-phenylenediamine (MPD), and p-phenylenediamine (PPD) are well-known isomers of phenylenediamine, used as important chemical intermediates or precursors in pesticides, photosensitive materials, pharmaceuticals, and industrial dyes. Numerous reports have confirmed the environmental impact and health hazards of phenylenediamine isomers: OPD is considered a highly toxic and carcinogenic environmental pollutant, posing a significant threat to the environment and human health. OPD is readily soluble in water, easily polluting groundwater and surface water; therefore, monitoring OPD is crucial in environmental analysis. As a typical aromatic amine water pollutant, MPD is difficult to degrade in water and can cause mutagenic, carcinogenic, and teratogenic effects. Exposure to PPD has been proven to cause various immediate allergic reactions. Therefore, monitoring phenylenediamine isomers in the environment is of great importance.

[0003] To date, various methods have been developed for the determination of OPD, MPD, and PPD, such as fluorescence methods, chemiluminescence, surface-enhanced Raman spectroscopy (SERS), and capillary electrophoresis (CE). However, the application of some of these methods is often limited by factors such as complex sample pretreatment, time consumption, and expensive analytical instruments; furthermore, most of these methods can only analyze one phenylenediamine isomer. Therefore, designing sensitive and effective detection strategies to identify and detect phenylenediamine isomers remains a hot research topic. In recent years, colorimetric methods have been widely developed due to their high sensitivity, ease of visual observation, and ease of operation. Converting phenylenediamine isomers into colored compounds with color differences is key to achieving the simultaneous differentiation and detection of three phenylenediamine isomers using colorimetric methods.

[0004] As an important branch of nanozymes, porous materials assembled from metal ions (or clusters) and organic ligands, namely metal-organic frameworks (MOFs), have attracted widespread attention from scientists. MOFs possess advantages such as ultra-large surface area, open metal sites, unsaturated metal centers, and catalytically active linkers, and their potential applications in catalysis and chemical sensing have been extensively studied. Besides utilizing the metals in MOFs as catalytic sites, other metals can be further introduced to provide additional active sites. Heteroatom doping has proven to be an effective and economical way to improve nanozyme activity. For example, doping transition metal elements (such as Mn, Fe, and Co) and rare earth elements (such as Eu, Nd, and Pr) into CeO2 nanomaterials increases oxygen vacancy defects and CeO2 activity through a synergistic effect. 3+ Active sites enable doped nanomaterials to exhibit higher enzyme-mimicking activity. Related studies have shown that all trivalent rare earth ions exhibit similar ion sizes and chemical properties, with Eu... 3+ The ionic radius (0.1066 nm) is between that of Ce 3+ (0.1143 nm) and Ce 4+ Uniform doping of both materials can be achieved within the range of 0.097 nm. Through post-synthesis modification, metal ions, quantum dots, and other materials are combined with MOF materials to prepare functional MOF composite materials with special optical, electrical, or catalytic properties.

[0005] Therefore, materials capable of recognizing all three isomers of p-phenylenediamine can be prepared by combining metal-organic frameworks (MOFs) with metal ion-doped carbon dots. Summary of the Invention

[0006] In view of this, one objective of the present invention is a method for preparing nanoparticles with oxidase-like activity; a second objective of the present invention is to provide nanoparticles with oxidase-like activity; and a third objective of the present invention is to provide the application of nanoparticles with oxidase-like activity in recognizing phenylenediamine isomers.

[0007] To achieve the above objectives, the present invention provides the following technical solution: 1. A method for preparing nanoparticles with oxidase-like activity, the method comprising the following steps: ZIF-8, water-soluble Ce 3+ Salt, water-soluble M 3+ Salt and manganese-doped carbon dots (Mn-CDs) are dissolved in water, reacted with stirring, and then centrifuged, washed, and dried to obtain CeEu-Mn-CDs@ZIF-8 nanoparticles. Water-soluble Mn-CDs are used in this process. 3+ Salt M 3+ For Eu 3+ Fe 3+ or Co3+ Any one of them.

[0008] Preferably, in step (1), the ZIF-8 is prepared according to the following method: water-soluble Zn 2+ ZIF-8 is obtained by adding an alcoholic solution of salt to an alcoholic solution of 2-methylimidazole under stirring, stirring at 30-50°C for more than 10 hours, centrifuging, washing repeatedly with methanol, and then vacuum drying.

[0009] More preferably, the water-soluble Zn 2+ The molar ratio of salt to 2-methylimidazole is 1:6 to 1:10, of which the water-soluble Zn 2+ The salt is Zn(NO3)2·6H2O; The solvent in the alcohol solution includes methanol or ethanol; The centrifugation speed is 8000~12000 rpm and the time is 5~20 min; The vacuum drying temperature is 40~80℃.

[0010] Preferably, the water-soluble Ce 3+ Salts include Ce(NO3)3, CeCl3, or Ce(Ac)3; The water-soluble M 3+ Salts contain any one of M(NO3)3, M2(SO4)3, M(Ac)3, or MCl3.

[0011] Preferably, the manganese-doped carbon dots (Mn-CDs) are prepared by the following method: citric acid and MnCl2·4H2O are dissolved in water, ethylenediamine is added under stirring, and after stirring and mixing evenly, the mixture is placed in a high-pressure reactor and heated at 160~200℃ for more than 8 hours. After cooling to room temperature, the mixture is centrifuged and dialyzed to obtain an aqueous solution of manganese-doped carbon dots (Mn-CDs).

[0012] More preferably, the molar ratio of citric acid, MnCl2·4H2O and ethylenediamine is 1:1:2 to 1:1:6; The high-pressure reactor is a polytetrafluoroethylene high-pressure reactor; The dialysis membrane used in the dialysis process has a specification of 800~1500 Da.

[0013] Preferably, the Zn and water-soluble Ce in the ZIF-8 3+ Ce in salt, water-soluble M 3+ The molar ratio of M in the salt to manganese in the aqueous solution of manganese-doped carbon dots (Mn-CDs) is 1:2:1:6~1:3:3:8.

[0014] Preferably, the stirring reaction time is not less than 8 hours; The centrifugation speed is 8000~12000 rpm and the time is 5~20 min; The drying process specifically involves vacuum drying overnight at a temperature of 40~80℃.

[0015] 2. Nanoparticles with oxidase-like activity prepared according to the above preparation method.

[0016] 3. The above-mentioned nanoparticles with oxidase-like activity are used in recognizing phenylenediamine isomers.

[0017] The beneficial effects of this invention are as follows: This invention discloses a method for preparing nanoparticles with oxidase-like activity, mainly using ZIF-8 (zinc-based MOFs material) and Ce... 3+ M 3+ (Eu) 3+ Fe 3+ or Co 3+ The present invention reacts with Mn-CDs (manganese-doped carbon dots) to obtain nanoparticles with oxidase-like activity. The nanoparticles prepared in this invention exhibit good oxidase-like activity and can be used to catalyze the oxidation reactions of OPD, MPD, and PPD, yielding oxidation products of different colors (oxOPD is yellow, oxMPD is colorless, and oxPPD is purple). Therefore, based on the color of the oxidation products formed by the oxidation of phenylenediamine catalyzed by nanoparticles with oxidase-like activity according to this invention, a colorimetric method for identifying and detecting OPD, MPD, and PPD can be established. This method is characterized by low cost, high sensitivity, simple operation, and high selectivity, and has good application prospects in the detection of OPD and PPD in environmental water samples.

[0018] Other advantages, objectives, and features of the invention will be set forth in part in the description which follows, and in part will be apparent to those skilled in the art from the following examination, or may be learned from practice of the invention. The objectives and other advantages of the invention can be realized and obtained through the following description. Attached Figure Description

[0019] To make the objectives, technical solutions, and advantages of the present invention clearer, the preferred embodiments of the present invention will be described in detail below with reference to the accompanying drawings, wherein: Figure 1 This is a scanning electron microscope (SEM) image of the CeEu-Mn-CDs@ZIF-8 nanoparticles prepared in Example 1. Figure 2 The elemental characterization results are for the CeEu-Mn-CDs@ZIF-8 nanoparticles prepared in Example 1; Figure 3 In the middle, 'a' represents Ce prepared in Example 1. 60 Eu-Mn-CDs@ZIF-8, Ce prepared in Example 2 60 Fe-Mn-CDs@ZIF-8, Ce prepared in Example 3 60 The absorbance changes of Co-Mn-CDs@ZIF-8 and Ce-Mn-CDs@ZIF-8 as nanoparticles in Comparative Example 1 were obtained from the oxidase-like activity study. b represents the Ce-Mn-CDs@ZIF-8 nanoparticles prepared in Example 1. 60 Eu-Mn-CDs@ZIF-8, Ce 40 Eu-Mn-CDs@ZIF-8 and Ce 20 Absorbance variation diagram obtained from the study of oxidase-like activity of Eu-Mn-CDs@ZIF-8 as nanoparticles; Figure 4 For Ce 60 Image (a) and UV-Vis absorption spectrum (b) of the reaction solution formed after Eu-Mn-CDs@ZIF-8 nanoparticles reacted with three common enzymatic chromogenic substrates (including 2,2'-adiazonium(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS), o-phenylenediamine (OPD), and 3,3′,5,5′-tetramethylbenzidine (TMB)). Figure 5 In the middle, 'a' represents Ce. 60 The graph shows the relationship between TMB concentration and initial velocity V for Eu-Mn-CDs@ZIF-8 nanoparticles; b is the corresponding linear double reciprocal curve obtained from a based on the Michaelis equation. Figure 6 In the equation 'a' represents Ce in N2 and air. 60 Absorption spectrum of TMB oxidation catalyzed by Eu-Mn-CDs@ZIF-8 nanoparticles, b represents the effect of tryptophan, thiourea and p-benzoquinone, three free radical scavengers, on Ce. 60 The effect of Eu-Mn-CDs@ZIF-8 nanoparticles on the catalytic oxidation of TMB; Figure 7 (1) shows the color changes of different systems, and (2) shows the UV-Vis absorption spectra of different systems, where system a is Ce 60 Eu-Mn-CDs@ZIF-8 nanoparticles, system b is Ce 60 Eu-Mn-CDs@ZIF-8 nanoparticles + OPD, system c is Ce 60 Eu-Mn-CDs@ZIF-8 nanoparticles + MPD, system d is Ce 60 Eu-Mn-CDs@ZIF-8 nanoparticles + PPD, system e is OPD, system f is MPD, system g is PPD; Figure 8 In the middle, 'a' represents Ce. 60 The absorbance of Eu-Mn-CDs@ZIF-8 nanoparticles and their oxidized OPD as a function of solution pH, b being Ce 60 The absorbance of Eu-Mn-CDs@ZIF-8 nanoparticles and their oxidized OPD as a function of solution temperature, where c represents Ce. 60 The absorbance of Eu-Mn-CDs@ZIF-8 nanoparticles and their oxidized OPD as a function of reaction time, with d representing Ce. 60 The absorbance of Eu-Mn-CDs@ZIF-8 nanoparticles and their oxidized OPD varies with Ce 60 Changes in the concentration of Eu-Mn-CDs@ZIF-8 nanoparticles; Figure 9 In the middle, 'a' represents Ce. 60 The absorbance of Eu-Mn-CDs@ZIF-8 nanoparticles and their oxidized PPD as a function of solution pH, b being Ce 60 The absorbance of Eu-Mn-CDs@ZIF-8 nanoparticles and their oxidized PPD as a function of solution temperature, where c represents Ce. 60 The change in absorbance of Eu-Mn-CDs@ZIF-8 nanoparticles and their oxidized PPD with reaction time, where d represents Ce. 60 The absorbance of Eu-Mn-CDs@ZIF-8 nanoparticles and their oxidized PPD varies with Ce 60 Changes in the concentration of Eu-Mn-CDs@ZIF-8 nanoparticles; Figure 10 In the figure, 'a' represents Ce with a concentration of 0.09 mg / mL. 60 The UV-Vis absorption spectra of the reaction solution when Eu-Mn-CDs@ZIF-8 nanoparticles oxidize OPD at different concentrations (0.2~75μM) are as follows (inset shows the color change of the reaction solution containing different concentrations of OPD), b represents the concentration of Ce at 0.09 mg / mL. 60 The absorbance of Eu-Mn-CDs@ZIF-8 nanoparticles at 443 nm changes when oxidizing different concentrations (0.2~75 μM) of OPD (inset shows the standard curve fitted between absorbance at 443 nm and OPD concentration), and c represents Ce at a concentration of 0.17 mg / mL. 60 The UV-Vis absorption spectra of the reaction solution when Eu-Mn-CDs@ZIF-8 nanoparticles oxidize PPD at different concentrations (0.7~300μM) are as follows (the inset shows the color changes of the reaction solution containing different concentrations of PPD), and d represents the concentration of Ce at 0.17 mg / mL. 60The absorbance of Eu-Mn-CDs@ZIF-8 nanoparticles at 513 nm changes when oxidizing PPD at different concentrations (0.2~75 μM) (the inset shows the standard curve of absorbance at 513 nm fitted to PPD concentration). Figure 11 For Ce 60 The selectivity of Eu-Mn-CDs@ZIF-8 nanoparticles for OPD and PPD is shown in the inset photographs, which depict the colors of different solutions. Detailed Implementation

[0020] The following specific examples illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention. It should be noted that the illustrations provided in the following embodiments are only schematic representations of the basic concept of the present invention. Unless otherwise specified, the following embodiments and features can be combined with each other.

[0021] The relevant information of the experimental instruments and reagents involved in the following embodiments is shown in Tables 1 and 2, respectively.

[0022] Table 1 Experimental Apparatus

[0023] Table 2 Experimental Drugs

[0024] Example 1 The specific preparation method of a nanoparticle with oxidase-like activity (CeEu-Mn-CDs@ZIF-8) is as follows: (1) Preparation of ZIF-8: 1782 mg (5.99 mmol) Zn(NO3)2·6H2O and 3942 mg (48 mmol) 2-methylimidazole were dissolved in 50 mL of methanol, respectively. The solutions were sonicated for 10 min to obtain zinc ion methanol solution and 2-methylimidazole methanol solution, respectively. The zinc ion methanol solution was added to the 2-methylimidazole methanol solution under magnetic stirring and stirred at 35 °C for 12 h. After centrifugation at 12000 rpm for 5 min, the solution was washed three times with methanol and then vacuum dried overnight at 60 °C to obtain ZIF-8 (zinc-based MOFs material). (For the specific preparation method, please refer to "Wang, M.; Zhu, H.; Liu, B.; Hu, P.; Pan, J.; Niu, X., Bifunctional Mn-Doped N-Rich CarbonDots with Tunable Photoluminescence and Oxidase-Mimetic Activity Enabling Bimodal Ratiometric Colorimetric / Fluorometric") Detection of Nitrite. ACS Applied Materials & Interfaces 2022, 14 (39), 44762-44771” contains the recorded content.

[0025] (2) Preparation of Mn-CDs (manganese-doped carbon dots): 5254 mg (2.5 mmol) of citric acid and 4948 mg (2.5 mmol) of MnCl2·4H2O were fully dissolved in 10 mL of distilled water. 10 mmol of ethylenediamine was added while stirring. After stirring for 10 min, the mixture was transferred to a polytetrafluoroethylene high-pressure reactor and heated at 180 °C for 10 h. After the reaction was completed, the mixture was naturally cooled to room temperature. After centrifugation at 10000 rpm for 10 min, the mixture was dialyzed using a 1000 Da dialysis membrane for further purification to remove large particles. After purification, Mn-CDs (manganese-doped carbon dots) were obtained and stored in a refrigerator at 4 °C for later use. (For details of this preparation method, please refer to "Wang, M.; Zhu, H.; Liu, B.; Hu, P.; Pan, J.; Niu, X., Bifunctional Mn-Doped N-Rich Carbon Dots with Tunable Photoluminescence and Oxidase-Mimetic Activity Enabling") Bimodal Ratiometric Colorimetric / FluorometricDetection of Nitrite. ACS Applied Materials&Interfaces 2022, 14 (39), 44762-44771” contains the recorded content.

[0026] (3) Preparation of nanoparticles with oxidase-like activity (Ce 60 Eu-Mn-CDs@ZIF-8: First, 60 mg of ZIF-8, 60 mg (0.138 mmol) Ce(NO3)3·6H2O, 60 mg (0.135 mmol) Eu(NO3)3·6H2O, and 1.5 mL of Mn-CDs were added to 30 mL of deionized water. The mixture was reacted under magnetic stirring for 12 h. After the reaction, the mixture was centrifuged at 12000 rpm for 5 min, washed several times with ultrapure water, and dried overnight in a vacuum oven at 60 ℃ to obtain nanoparticles with oxidase-like activity (Ce). 60 Eu-Mn-CDs@ZIF-8).

[0027] By changing the "60 mg (0.138 mmol) Ce(NO3)3·6H2O" added in step (3) above to "20 mg (0.138 mmol) Ce(NO3)3·6H2O" and "40 mg (0.138 mmol) Ce(NO3)3·6H2O" respectively, while keeping other conditions and methods unchanged, nanoparticles with different Ce doping amounts were obtained.20 Eu-Mn-CDs@ZIF-8 and Ce 40 Eu-Mn-CDs@ZIF-8. This shows that adjusting Ce during the preparation process... 3+ The amount of Ce added can be appropriately adjusted to control the Ce content in nanoparticles with oxidase-like activity.

[0028] The CeEu-Mn-CDs@ZIF-8 nanoparticles prepared in Example 1 above were characterized by scanning electron microscopy (SEM), and the results are as follows: Figure 1 As shown. From Figure 1 The SEM images show that the CeEu-Mn-CDs@ZIF-8 nanoparticles are nanospheres with a sheet-like surface structure. From the collapsed areas of some nanoparticles in the SEM images, it can be observed that the nanospheres may have a hollow structure. The elemental composition of the CeEu-Mn-CDs@ZIF-8 nanoparticles prepared in Example 1 was characterized using EDS energy dispersive spectroscopy, and the results are as follows: Figure 2 As shown. From Figure 2 The EDS elemental distribution images confirm that C, Eu, O, Ce, Zn, Mn and N are uniformly distributed in the nanoparticles prepared in Example 1 above, with mass percentages of 46.7%, 16.2%, 13.4%, 12.7%, 5.4%, 3.3% and 2.4%, respectively. Figure 1 and Figure 2 The characterization results all indicate that the method described in Example 1 above successfully prepared CeEu-Mn-CDs@ZIF-8 nanoparticles.

[0029] Example 2 A nanoparticle with oxidase-like activity (Ce 60 Fe-Mn-CDs@ZIF-8), in step (3) of the preparation method of Example 1, "60 mg (0.135 mmol) Eu(NO3)3·6H2O" is modified to "46.8 mg (0.135 mmol) Fe(NO3)3·6H2O", and the remaining conditions and methods are the same as in Example 1.

[0030] Example 3 A nanoparticle with oxidase-like activity (Ce 60 Co-Mn-CDs@ZIF-8), in step (3) of the preparation method of Example 1, "60 mg (0.135 mmol) Eu(NO3)3·6H2O" is modified to "47.6 mg (0.135 mmol) Co(NO3)3·6H2O", and the remaining conditions and methods are the same as in Example 1.

[0031] After multiple experiments, Ce(NO3)3 in the preparation methods of Examples 1-3 was replaced with CeCl3 or Ce(Ac)3, and M(NO3)3 was replaced with any one of M2(SO4)3, M(Ac)3, or MCl3 (where M... 3+ For Eu 3+ Fe 3+ or Co 3+ (any one of the following), and Zn in ZIF-8 and water-soluble Ce 3+ Ce in the salt (Ce(NO3)3), water-soluble M 3+ Nanoparticles with oxidase-like activity can also be obtained by varying the molar ratio of M (Eu, Fe or Co) in salt (Eu(NO3)3) and manganese in aqueous solution of manganese-doped carbon dots (Mn-CDs) between 1:2:1:6 and 1:3:3:8.

[0032] Comparative Example 1 A nanoparticle (Ce-Mn-CDs@ZIF-8) was prepared in step (3) of Example 1 without adding Eu(NO3)3·6H2O, and the remaining conditions and methods were the same as in Example 1.

[0033] Performance testing 1. The effect of different metal doping amounts on the activity of nanoparticle-based oxidases was studied, and the specific methods are shown below: Methods for studying the activity of nanoparticle-based oxidases: 50 μL of 1 mg / mL aqueous solution of nanoparticles, 50 μL of 1 mM OPD, and 650 μL of Britton-Robinson buffer (pH=4.4) were mixed. The final volume of the solution was adjusted to 1 mL with distilled water to form a mixed solution. After incubating the mixture at 25 °C for 5 min, the absorption spectrum of the reaction solution in the wavelength range of 300–600 nm was scanned using a UV-Vis spectrophotometer, and the absorbance of the mixed solution at 443 nm was analyzed.

[0034] Ce prepared in Example 1 60 Eu-Mn-CDs@ZIF-8, Ce prepared in Example 2 60 Fe-Mn-CDs@ZIF-8, Ce prepared in Example 3 60 Co-Mn-CDs@ZIF-8 and Ce-Mn-CDs@ZIF-8 in Comparative Example 1 were used as nanoparticles for oxidase-like activity studies, and the absorbance changes were obtained as follows: Figure 3 As shown in Figure a. From Figure 3As can be seen from Figure a, compared with the oxidase-like activity of Ce-Mn-CDs@ZIF-8 nanoparticles, the doping of Eu, Fe and Co elements can significantly improve the oxidase-like activity of the formed nanoparticles. The main reason is that after doping with Eu, Fe and Co elements, they can have a synergistic effect with Ce, thereby improving the oxidase-like activity. The degree of improvement mainly depends on the synergistic effect between the doped metal and Ce (e.g., CeEu-Mn-CDs@ZIF-8 nanoparticles have the best catalytic activity, indicating that in this MOF material, the synergistic effect between Eu and Ce makes it exhibit higher oxidase-like activity).

[0035] 2. The effect of different Ce ion doping amounts on the activity of nanoparticle-based oxidases was studied, and the specific methods are shown below: The Ce prepared in Example 1 60 Eu-Mn-CDs@ZIF-8, Ce 40 Eu-Mn-CDs@ZIF-8 and Ce 20 Eu-Mn-CDs@ZIF-8 were used as nanoparticles to study oxidase-like activities, and the absorbance changes were obtained as follows: Figure 3 As shown in b. From Figure 3 As can be seen from Figure b, the oxidase-like activity of the nanoparticles gradually increases with the increase of cerium content, indicating that the Ce content is the key factor affecting the oxidase-like activity in this nanoparticle material system.

[0036] Additionally, Ce 60 Eu-Mn-CDs@ZIF-8 nanoparticles were reacted with three common enzymatic chromogenic substrates (including 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS), o-phenylenediamine (OPD), and 3,3',5,5'-tetramethylbenzidine (TMB)). The specific method involved reacting 50 μL of 1 mg / mL Ce... 60 Eu-Mn-CDs@ZIF-8 nanoparticles, solutions containing chromogenic substrates for the enzymatic reaction (50 μL of 1 mM ABTS solution, 50 μL of 1 mM OPD solution, and 90 μL of 1 mM TMB solution, respectively), and 650 μL of Britton-Robinson buffer at pH 4.4 were mixed. The final volume of the solution was adjusted to 1 mL with distilled water to obtain the mixture. The mixture was incubated at 25°C for 5 min. Images of each reaction solution are shown below. Figure 4 As shown in Figure a; the absorption spectra of each reaction solution were scanned using a UV-Vis spectrophotometer, and the UV-Vis absorption spectra of each reaction solution are shown in Figure a. Figure 4 As shown in b. From Figure 4 It can be seen that C60 eEu-Mn-CDs@ZIF-8 nanoparticles effectively promote the oxidation of three chromogenic substrates (including 2,2'-adiazonium(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS), o-phenylenediamine (OPD), and 3,3',5,5'-tetramethylbenzidine (TMB)), yielding oxidation products of corresponding colors, further demonstrating that C 60 eEu-Mn-CDs@ZIF-8 nanoparticles exhibit high oxidase-like activity.

[0037] Similarly, tests on other nanomaterials prepared using the method of this invention revealed that they also exhibited high oxidase-like activity.

[0038] 3. Enzyme kinetics study of nanoparticle-based oxidases With Ce 60 Taking Eu-Mn-CDs@ZIF-8 as an example, the analysis was conducted, and the enzyme kinetics experiment was performed using 50 μL of Ce at a concentration of 1 mg / mL. 60 Enzyme kinetics experiments were conducted in the presence of Eu-Mn-CDs@ZIF-8 nanoparticles by adding 50 μL of 3,3',5,5'-tetramethylbenzidine (TMB) substrate at different concentrations (final concentrations of 20–90 μM) to Britton-Robinson buffer at pH 4.4. The absorbance (A) of the mixed solution at 652 nm was obtained using a UV-Vis spectrophotometer. 652 ).

[0039] Figure 5 In the middle, 'a' represents Ce. 60 The graph for Eu-Mn-CDs@ZIF-8 nanoparticles is a graph showing the relationship between TMB concentration and initial velocity V. Graph b is the corresponding linear double reciprocal curve obtained from graph a based on the Michaelis equation. Figure 5 a indicates Ce 60 The oxidation of TMB catalyzed by Eu-Mn-CDs@ZIF-8 nanoparticles is a typical Michaelis-Menten reaction. According to the Michaelis-Menten equation (1 / V = (Km / Vmax) / [S] + 1 / Vmax), where... V Initial velocity (unit: Ms) -1 ), V max Maximum reaction rate (in Ms) -1 [S] represents the substrate concentration (in mM). K m This is the Michaelis constant (in mM). Calculated using the Lineweaver-Burk double reciprocal curve. K m Value andV max The values ​​were 0.059 mM and 7.2 × 10⁻⁶, respectively. -8 Ms -1 . K m The affinity value is an important indicator for evaluating the affinity between an enzyme and its substrate. K m The lower the value, the stronger the affinity between the enzyme and the substrate.

[0040] Comparison of several oxidase-like nanomaterials using TMB as a substrate K m Value and V max The values ​​are shown in Table 3. As can be seen from the results in Table 3, the nanoparticle-mimicking enzyme of this application exhibits lower [efficacy / value] in its interaction with the substrate. K m value( K m A lower Ce value indicates a stronger affinity between the enzyme and its substrate. 60 Eu-Mn-CDs@ZIF-8 nanoparticles exhibit high affinity for the reaction substrate (TMB) as an oxidase-like enzyme.

[0041] Table 3. Comparison of different types of oxidase nanomaterials using TMB as a substrate K m Value and V max value

[0042] Reference 1 is from "Liu, Y.; Zhou, M.; Cao, W.; Wang, X.; Wang, Q.; Li, S.; Wei, H., Light-Responsive Metal–Organic Framework as an Oxidase Mimic for Cellular Glutathione Detection". Analytical Chemistry 2019, 91(13), 8170-8175”, Reference 2 is from “Lai, X.; Shen, Y.; Gao, S.; Chen, Y.; Cui, Y.; Ning, D.; Ji, X.; Liu, Z.; Wang, L., The Mn-modified porphyrin metal-organic framework with enhanced oxidase-like activity for sensitively colorimetric detection of glutathione. Biosensors and Bioelectronics 2022, 213 .”, Reference 3 is from “Lu, W.; Chen, J.; Kong, L.; Zhu, F.; Feng, Z.; Zhan, J., Oxygen vacancies modulation Mn3O4 nanozyme with enhanced oxidase-mimicking performance for L-cysteine detection. Sensors and Actuators B: Chemical 2021, 333 ”, Reference 4 is from “Li, J.; Zhou, Y.; Xiao, Y.; Cai, S.; Huang, C.; Guo, S.; Sun, Y.; Song, R.-B.; Li, Z., Carbon dots as light-responsive oxidase-like nanozyme for colorimetric detection of total antioxidant capacity in fruits. Food Chemistry 2023, 405 ”, Reference 5 is from “Xu, X.; Luo, J.; Wei, S.; Zou, X.; Niu, X.; Pan, J., Three-dimensional flower-like multifunctional adsorbents with excellent sorptive removal and colorimetric detection of arsenate. Chemical Engineering Journal 2020, 398 ”.

[0043] Similarly, tests on other nanomaterials prepared using the method of this invention revealed that they also exhibit high affinity for TMB as oxidase-like enzymes.

[0044] 4. Study on the mechanism of oxidase-like activity of nanoparticles with oxidase-like activity Enzyme-like catalytic reactions have two possible mechanisms: free radical generation or electron transfer processes. It is speculated that the oxidase-like activity of the nanoparticles of this invention may stem from their ability to promote the conversion of dissolved oxygen into reactive oxygen species (ROS) during the reaction. To investigate the oxidase-like catalytic mechanism of the nanoparticles of this invention, Ce prepared in Example 1 was used... 60 Taking Eu-Mn-CDs@ZIF-8 as an example, TMB was selected as the chromogenic substrate for the study.

[0045] Study on the effect of dissolved oxygen content on Ce 60 The effect of Eu-Mn-CDs@ZIF-8 nanoparticles on the color change of TMB catalysis was investigated. High and low concentrations of dissolved oxygen were obtained by bubbling air or N2 into the reaction solution for 30 min, respectively, to measure the effect of Ce. 60 Experiments on the catalytic oxidation of TMB by Eu-Mn-CDs@ZIF-8 nanoparticles under different dissolved oxygen contents (specific method: First, add 650 μL of Britton-Robinson buffer (pH=4.4), 90 μL of 1 mM TMB solution, and 150 μL of 1 mg / mL CeEu-Mn-CDs@ZIF-8 nanoparticle solution to two 1.5 mL centrifuge tubes in sequence. Then, adjust the total volume of the solution to 1 mL with distilled water and mix thoroughly using a vortex mixer to obtain a mixed solution. Then, introduce air and N2 into the mixed solution for 30 min to obtain high and low concentrations of dissolved oxygen, respectively. After the reaction is completed, record the absorption spectrum of the reaction mixture).

[0046] Subsequently, free radical scavenging experiments were used to verify the intermediate products generated during the oxidation reaction, with tryptophan, thiourea, and p-benzoquinone being used as agents for capturing singlet oxygen. 1 O2), hydroxyl radicals (·OH), and superoxide anion radicals (·O2). -The scavenging agent was prepared by adding 650 μL of Britton-Robinson buffer (pH=4.4), 90 μL of 1 mM TMB solution, and 150 μL of 1 mg / mL CeEu-Mn-CDs@ZIF-8 nanoparticle solution to three 1.5 mL centrifuge tubes. Then, 50 μL of tryptophan, thiourea, and p-benzoquinone at concentrations of 2 mM, 10 mM, and 20 mM, respectively, were added to the three centrifuge tubes. The total volume of the solution was adjusted to 1 mL with distilled water, and the mixture was vortexed to obtain a homogeneous solution. The solution was incubated at 25 °C for 5 min, and the absorbance of the reaction mixture at 652 nm was measured.

[0047] Figure 6 In the equation 'a' represents Ce in N2 and air. 60 Absorption spectra of TMB oxidation catalyzed by Eu-Mn-CDs@ZIF-8 nanoparticles, b represents the effect of three free radical scavengers—tryptophan, thiourea, and p-benzoquinone—on Ce2+. 60 The effect of Eu-Mn-CDs@ZIF-8 nanoparticles on the catalytic oxidation of TMB. Figure 6 As can be seen from Figure a, compared to the lower concentration of dissolved oxygen under N2, the system exhibits a stronger catalytic effect under the higher concentration of dissolved oxygen under air, as evidenced by a significant increase in absorbance at 652 nm. These experimental results demonstrate that Ce... 60 The efficiency of Eu-Mn-CDs@ZIF-8 nanoparticles in catalyzing the oxidation of TMB depends on the dissolved oxygen content. During the oxidation process, singlet oxygen (… 1 O2), hydroxyl radicals (•OH), and superoxide anion radicals (•O2). - ) is a possible active substance. Subsequently, free radical scavenging experiments verified the intermediate products generated during the oxidation reaction; tryptophan, thiourea, and p-benzoquinone were used to capture singlet oxygen ( 1 O2), hydroxyl radicals (·OH), and superoxide anion radicals (·O2). - (A cleaning agent.) Figure 6 As can be seen from Figure b, the relative activity of the system did not change significantly in the presence of tryptophan and thiourea. However, the relative activity of the system decreased significantly after the addition of p-benzoquinone, indicating that the superoxide anion radical (·O2) is a significant factor. - This plays a major role in the catalytic reaction. Similarly, similar conclusions can be drawn from corresponding studies on other nanoparticles of this invention.

[0048] Based on the above research, it can be found that the mechanism by which the nanoparticles of the present invention possess oxidase-like activity is as follows: the nanoparticles of the present invention possess oxidase-like activity can promote the conversion of dissolved oxygen into active superoxide anion free radicals (•O2) during the reaction process. - This, in turn, promotes the oxidation process of the reaction substrate.

[0049] 5. The effect of nanoparticles with oxidase-like activity on the identification of phenylenediamine isomers. OPD, MPD, and PPD can be oxidized to produce substances of different colors; this property can be used to identify these three phenylenediamine isomers. Ce was prepared as described in Example 1. 60 Taking Eu-Mn-CDs@ZIF-8 nanoparticles as an example, their effectiveness in identifying phenylenediamine isomers was studied. The specific identification method was as follows: 650 μL of Britton-Robinson buffer solution (pH=7.0), 50 μL of phenylenediamine solutions of different concentrations (OPD, MPD, and PPD, respectively), and 170 μL of Ce2-2000 styrene solution (1 mg / mL) were prepared. 60 Eu-Mn-CDs@ZIF-8 nanoparticles were mixed in a 1.5 mL sample tube and reacted at 25°C for 16 minutes. The phenylenediamine isomers were then distinguished by color. (In this method, the concentrations of OPD, MPD, and PPD were 250 mM, and the pH of the Britton-Robinson buffer was 7.0.) Ce2 was prepared in Example 1. 60 The concentration of Eu-Mn-CDs@ZIF-8 nanoparticles was 0.17 mg / mL.

[0050] Figure 7 (1) shows the color changes of different systems, and (2) shows the UV-Vis absorption spectra of different systems, where system a is Ce 60 Eu-Mn-CDs@ZIF-8 nanoparticles, system b is Ce 60 Eu-Mn-CDs@ZIF-8 nanoparticles + OPD, system c is Ce 60 Eu-Mn-CDs@ZIF-8 nanoparticles + MPD, system d is Ce 60 Eu-Mn-CDs@ZIF-8 nanoparticles + PPD, system e is OPD, system f is MPD, system g is PPD. From Figure 7 It can be seen that in Ce 60In the presence of Eu-Mn-CDs@ZIF-8 nanoparticles, OPD and PPD are oxidized to produce yellow (oxOPD) and purple (oxPPD) products, with corresponding absorption peaks at 443 nm and 513 nm, respectively. However, no obvious color or absorption peak was observed for the oxidation product of MPD. This indicates that these three phenylenediamine isomers can be oxidized in Ce... 60 Different colored oxidation products are generated in the presence of Eu-Mn-CDs@ZIF-8 nanoparticles, which can be visualized and identified.

[0051] Similarly, when OPD, MPD, and PPD were oxidized using other nanoparticles with oxidase-like activity of the present invention, the changes in color and absorption peaks were similar to the results described above, indicating that the nanoparticles with oxidase-like activity of the present invention can achieve the purpose of identification by oxidizing OPD, MPD, and PPD.

[0052] 6. Optimization of OPD and PPD detection conditions using nanoparticles with oxidase-like activity Since the oxidation products of MPD did not show obvious color and absorption peaks, the experimental conditions for MPD will not be optimized for the time being. Instead, the Ce2O3 product prepared in Example 1 will be used. 60 Taking Eu-Mn-CDs@ZIF-8 nanoparticles as an example, we optimized the detection conditions for OPD and PPD. To obtain the optimal experimental conditions for quantitative analysis of OPD and PPD, we explored the effects of pH, temperature, reaction time, and Ce2O3. 60 The effect of Eu-Mn-CDs@ZIF-8 nanoparticle dosage on OPD and PPD detection.

[0053] (1) Ce 60 Effect of varying conditions on absorbance of Eu-Mn-CDs@ZIF-8 nanoparticles oxidizing OPD The Britton-Robinson buffer in the above OPD identification system was varied to obtain the absorbance of the oxidation products at 443 nm at different pH values, such as... Figure 8 As shown in figure a. It can be seen from the figure that Ce alone... 60 Eu-Mn-CDs@ZIF-8 nanoparticles showed no significant change in absorbance within the studied pH range (pH 2.4–9.3), indicating good pH stability. However, during catalytic OPD oxidation, the absorbance exhibited a trend of first increasing and then decreasing within the pH range of 2.4–9.3, with the highest absorbance at pH 4.4, indicating that Ce... 60 Eu-Mn-CDs@ZIF-8 nanoparticles exhibited the best catalytic activity at pH=4.4.

[0054] The reaction temperature in the above system for identifying OPD was varied, and the absorbance of the oxidation products at 443 nm was obtained at different temperatures, such as... Figure 8 As shown in figure b. It can be seen from the figure that Ce alone... 60 The absorbance of Eu-Mn-CDs@ZIF-8 nanoparticles did not change significantly within the studied temperature range (25~55℃), indicating that the nanomaterial has good temperature stability. Furthermore, during catalytic OPD oxidation, the absorbance remained essentially unchanged within the stable temperature range of 25~55℃, indicating that Ce... 60 The catalytic activity of Eu-Mn-CDs@ZIF-8 nanoparticles is less affected by temperature changes. Therefore, 45℃, with a slightly higher absorbance value, can be selected as the optimal experimental temperature.

[0055] By varying the reaction time in the above system for identifying OPD, the absorbance of the oxidation products at 443 nm was obtained at different reaction times, such as... Figure 8 As shown in Figure c. It can be seen from the figure that Ce alone... 60 Eu-Mn-CDs@ZIF-8 nanoparticles showed no significant change in absorbance across different reaction time ranges (0–24 min), indicating good time stability. However, during the catalytic oxidation of OPD, the absorbance of Ce... 60 In the presence of Eu-Mn-CDs@ZIF-8 nanoparticles, OPD is rapidly oxidized, and the absorbance of the system increases rapidly within the first 2 minutes of the reaction. After 6 minutes, the absorbance gradually stabilizes with increasing reaction time. Therefore, to obtain a more stable signal, 22 minutes was selected as the optimal reaction time.

[0056] Ce in the above-mentioned system for identifying OPD 60 By varying the amount of Eu-Mn-CDs@ZIF-8 nanoparticles, different Ce values ​​were obtained. 60 The absorbance of the oxidation product at 443 nm under different amounts of Eu-Mn-CDs@ZIF-8 nanoparticles, such as Figure 8 As shown in d. From the figure, it can be seen that Ce 60 Different amounts of Eu-Mn-CDs@ZIF-8 nanoparticles (concentration range of 0.07 mg / ml to 0.13 mg / ml) resulted in only a slight increase in the absorbance of the oxidation products, remaining essentially unchanged. This indicates that Ce... 60 The amount of Eu-Mn-CDs@ZIF-8 nanoparticles had little effect on the absorbance of the system. This is because the amount of Ce... 60 As the amount of Eu-Mn-CDs@ZIF-8 nanoparticles increased, the blank value gradually increased. Therefore, a Ce concentration of 0.09 mg / mL can be selected. 60Eu-Mn-CDs@ZIF-8 nanoparticles are the optimal dosage.

[0057] In summary, Ce 60 The optimal experimental conditions for detecting OPD using Eu-Mn-CDs@ZIF-8 nanoparticles are pH=4.4, reaction temperature 45℃, reaction time 22 min, and Ce... 60 The concentration of Eu-Mn-CDs@ZIF-8 nanoparticles was 0.09 mg / mL.

[0058] (2) Ce 60 Effect of varying conditions on absorbance of Eu-Mn-CDs@ZIF-8 nanoparticles oxidizing PPD The Britton-Robinson buffer in the above PPD identification system was varied to obtain the absorbance of the oxidation products at 513 nm at different pH values, such as... Figure 9 As shown in figure a. It can be seen from the figure that Ce alone... 60 The absorbance of Eu-Mn-CDs@ZIF-8 nanoparticles did not change significantly within the studied pH range (pH 2.4–9.3), indicating that the nanomaterial has good pH stability. However, during the catalytic oxidation of PPD, the absorbance showed an increasing trend within the pH range of 2.4–9.3, reaching a maximum at pH 7.0. Above pH 7.0, the absorbance decreased with increasing pH. Therefore, pH 7.0 was selected as the optimal reaction pH for subsequent experiments.

[0059] The reaction temperature in the above system for identifying PPD was varied, and the absorbance of the oxidation product at 513 nm was obtained at different temperatures, such as... Figure 9 As shown in figure b. It can be seen from the figure that Ce alone... 60 The absorbance of Eu-Mn-CDs@ZIF-8 nanoparticles did not change significantly within the studied temperature range (25~55℃), indicating that the nanomaterial has good temperature stability. Furthermore, during the catalytic oxidation of PPD, the absorbance remained essentially unchanged within the stable temperature range of 25~55℃, indicating that Ce... 60 The catalytic activity of Eu-Mn-CDs@ZIF-8 nanoparticles is less affected by temperature changes. Therefore, 25℃, with a slightly higher absorbance value, can be selected as the optimal experimental temperature.

[0060] By varying the reaction time in the above system for identifying PPD, the absorbance of the oxidation product at 513 nm was obtained at different reaction times, such as... Figure 9 As shown in Figure c. It can be seen from the figure that Ce alone... 60Eu-Mn-CDs@ZIF-8 nanoparticles showed no significant change in absorbance across different reaction time ranges (0–24 min), indicating good time stability. However, during the catalytic oxidation of PPD, the absorbance of Ce... 60 In the presence of Eu-Mn-CDs@ZIF-8 nanoparticles, PPD is rapidly oxidized, and the absorbance of the system increases rapidly within the first 2 minutes of the reaction. After 10 minutes, the absorbance gradually stabilizes with increasing reaction time. Therefore, to obtain a more stable signal, 16 minutes was selected as the optimal reaction time.

[0061] Ce in the above-mentioned system for identifying PPD 60 By varying the amount of Eu-Mn-CDs@ZIF-8 nanoparticles, different Ce values ​​were obtained. 60 The absorbance of the oxidation product at 513 nm under different amounts of Eu-Mn-CDs@ZIF-8 nanoparticles, such as Figure 9 As shown in d. From the figure, it can be seen that Ce 60 The absorbance of the oxidation product obtained from the reaction varied with the amount of Eu-Mn-CDs@ZIF-8 nanoparticles (concentration range of 0.13 mg / ml to 0.2 mg / ml). 60 The concentration of Eu-Mn-CDs@ZIF-8 nanoparticles increases with the increase of Ce. 60 When the concentration of Eu-Mn-CDs@ZIF-8 nanoparticles is 0.17 mg / mL, the increase in absorbance of the system decreases.

[0062] In summary, Ce 60 The optimal experimental conditions for detecting PPD using Eu-Mn-CDs@ZIF-8 nanoparticles are: pH = 7.0, reaction temperature = 25℃, reaction time = 16 min, and Ce... 60 The concentration of Eu-Mn-CDs@ZIF-8 nanoparticles was 0.17 mg / mL.

[0063] The oxidation of OPD and PPD using other nanoparticles of this invention under different conditions showed their variation trends with Ce. 60 The Eu-Mn-CDs@ZIF-8 nanoparticles are essentially similar.

[0064] 7. Establish a standard curve for detecting OPD and PPD using nanoparticles with oxidase-like activity. Under the aforementioned optimal experimental conditions, the effects of Ce were investigated. 60The sensitivity of colorimetric detection of OPD and PPD using Eu-Mn-CDs@ZIF-8 nanoparticles as an example of nanoparticles with oxidase-like activity was demonstrated. The specific method was as follows: For OPD detection, 650 μL of pH 4.4 Britton-Robinson buffer, 50 μL of OPD at different concentrations, and 90 μL of 1 mg / mL CeEu-Mn-CDs @ ZIF-8 nanoparticles were added sequentially to a 1.5 mL centrifuge tube. The total volume of the solution was adjusted to 1 mL with distilled water. After thorough mixing using a vortex mixer, the reaction solution was reacted at 45 ℃ for 22 min. Finally, the absorption spectrum of the reaction solution in the wavelength range of 300 nm to 600 nm was scanned using a UV-Vis spectrophotometer, and the absorbance at 443 nm was analyzed. The PPD detection procedure involved adding 650 μL of pH 7.0 Britton-Robinson buffer, 50 μL of PPD at different concentrations, and 170 μL of 1 mg / mL CeEu-Mn-CDs @ ZIF-8 nanoparticles sequentially to a 1.5 mL centrifuge tube. The total volume was adjusted to 1 mL with distilled water. After vortexing the mixture thoroughly, it was incubated at 25 °C for 16 min. The absorption spectrum of the reaction solution in the wavelength range of 400 nm to 600 nm was then scanned using a UV-Vis spectrophotometer, and the absorbance at 513 nm was analyzed.

[0065] Figure 10 In the figure, 'a' represents Ce with a concentration of 0.09 mg / mL. 60 The UV-Vis absorption spectra of the reaction solution when Eu-Mn-CDs@ZIF-8 nanoparticles oxidize OPD at different concentrations (0.2~75μM) are as follows (inset shows the color change of the reaction solution containing different concentrations of OPD), b represents the concentration of Ce at 0.09 mg / mL. 60 The absorbance of Eu-Mn-CDs@ZIF-8 nanoparticles at 443 nm changes when oxidizing different concentrations (0.2~75 μM) of OPD (inset shows the standard curve fitted between absorbance at 443 nm and OPD concentration), and c represents Ce at a concentration of 0.17 mg / mL. 60 The UV-Vis absorption spectra of the reaction solution when Eu-Mn-CDs@ZIF-8 nanoparticles oxidize PPD at different concentrations (0.7~300μM) are as follows (the inset shows the color changes of the reaction solution containing different concentrations of PPD), and d represents the concentration of Ce at 0.17 mg / mL. 60The absorbance changes at 513 nm of the reaction solution of Eu-Mn-CDs@ZIF-8 nanoparticles when oxidizing different concentrations (0.2~75 μM) of PPD (the inset shows the standard curve fitted between absorbance at 513 nm and PPD concentration). Figure 10 As can be seen from Ce 60 In the experimental system of Eu-Mn-CDs@ZIF-8 nanoparticles catalyzing OPD and PPD, the absorbance of the solution at 443 nm and 513 nm gradually increased with increasing OPD and PPD concentrations, respectively, showing a good linear relationship within a certain concentration range. The linear regression equation between OPD concentrations of 0.2–55 μM and absorbance at 443 nm is A. 443 = 0.00958 c OPD +0.07854 (R) 2 = 0.9938) Figure 10 (As shown in a and b); and the linear regression equation between PPD concentration range of 0.7~275 μM and absorbance at 513 nm is A. 513 =0.0004297 c PPD +0.09785 (R) 2 = 0.9922)( Figure 10 (As shown in c and d). Furthermore, Ce of OPD and PPD was calculated based on the relative standard deviation of the blank values ​​and the slope of the standard curve. 60 The detection limits for Eu-Mn-CDs@ZIF-8 nanoparticles were 66.7 nM and 233 nM (3σ / k , n = 11, where 3σ is the standard deviation of three repeated measurements of the blank sample. k The slope of the standard curve, n (This refers to the number of parallel determinations). Correspondingly, the color change of the reaction solution can be used to initially determine the concentrations of OPD and PPD through visual identification. Figure 10 (As shown in the illustrations in b and d). Furthermore, compared to other OPD and PPD detection methods (Table 4), Ce-based methods... 60 The colorimetric analysis method for Eu-Mn-CDs@ZIF-8 nanoparticles exhibits advantages such as high sensitivity, wide linear range, and low detection limit.

[0066] Table 4 Comparison of the effects of different detection methods on OPD and PPD

[0067] Wherein, Reference 6 is "Gao, L. F.; Lin, X.; Hai, X.; Chen, X. W.; Wang, J. H.,Polymeric Ionic Liquid-Based Fluorescent Amphiphilic Block Copolymer Micellefor Selective and Sensitive Detection of p-Phenylenediamine. ACS Appl Mater Interfaces 2018, 10 (49), 43049-43056", Reference 7 is "Ma, P.; Liang, F.; Wang, D.;Yang, Q.; Cao, B.; Song, D.; Gao, D.; Wang, X., Selective determination of o-phenylenediamine by surface-enhanced Raman spectroscopy using silvernanoparticles decorated with α-cyclodextrin. Microchimica Acta 2014, 182 (1-2),167-174", Reference 8 is "Li, N.; Gu, Y.; Gao, M.; Wang, Z.; Xiao, D.; Li, Y.; Lin,R.; He, H., Colorimetric determination of o-phenylenediamine in water samplesbased on the formation of silver nanoparticles as a colorimetricprobe. Spectrochim Acta A Mol Biomol Spectrosc 2015, 140 , 328-33", Reference 9 is "Shi,B.; Su, Y.; Zhang, L.; Huang, M.; Li, X.; Zhao, S., Facilely prepared Fe3O4 / nitrogen-doped graphene quantum dot hybrids as a robust nonenzymatic catalystfor visual discrimination of phenylenediamine isomers. Nanoscale 2016, 8 (20),10814-10822", Document 10 is "Lin, L.; Xiao, Y.; Wang, Y.; Zeng, Y.; Lin, Z.; Chen, Mikrochim Acta 2019, 186 (5), 288.

[0068] Using other nanoparticles of the present invention to oxidize OPD and PPD at different concentrations, the concentrations of OPD and PPD showed a good linear relationship with the absorbance at 443 nm and 513 nm, respectively, within a certain range.

[0069] 8. Selectivity study of OPD and PPD by nanoparticles with oxidase-like activity To evaluate the selectivity of colorimetric detection of OPD and PPD for nanoparticles with oxidase-like activity, some common potential interfering substances (such as Cu) were investigated. 2+ Mg 2+ SO4 2- ,ClO - The effects of glutamic acid (Glu), proline (Pro), cysteine ​​(Cys), catechol (CC), resorcinol (RC), hydroquinone (HQ), melamine (MA), and m-phenylenediamine (MPD) on the determination of OPD and PPD were investigated. During the detection process, the concentration of interfering substances was 10 times that of the test substances (OPD concentration was 50 μM, PPD concentration was 250 μM). Similarly, Ce... 60 Taking Eu-Mn-CDs@ZIF-8 nanoparticles as an example, the OPD detection method was as follows: [The text abruptly shifts to a different topic] ... containing Ce... 60 Eu-Mn-CDs@ZIF-8 nanoparticles (concentration 0.09 mg / ml) were added to Britton-Robinson buffer (pH=4.4) with OPD and various interfering agents (such as Cu). 2+ Mg 2+ SO4 2- ,ClO -The following substances were found to be glutamic acid (Glu), proline (Pro), cysteine ​​(Cys), catechol (CC), resorcinol (RC), hydroquinone (HQ), melamine (MA), and m-phenylenediamine (MPD); the detection method for OPD is as follows: [The text abruptly ends here, likely due to an incomplete sentence or missing information.] 60 PPD and various interfering agents (such as Cu) were added to Britton-Robinson buffer (pH=7.0) containing Eu-Mn-CDs@ZIF-8 nanoparticles (concentration of 0.17 mg / ml). 2+ Mg 2+ SO4 2- ,ClO - Glutamic acid (Glu), proline (Pro), cysteine ​​(Cys), catechol (CC), resorcinol (RC), hydroquinone (HQ), melamine (MA), and m-phenylenediamine (MPD)).

[0070] Figure 11 For Ce 60 The selectivity of Eu-Mn-CDs@ZIF-8 nanoparticles for OPD and PPD is shown in the inset photographs, which depict the colors of different solutions. Figure 11 It can be seen that, and with Ce 60 Based on Eu-Mn-CDs@ZIF-8 nanoparticles (concentration 0.17 mg / ml) in Britton-Robinson buffer (pH=7.0) (Control), the addition of both OPD and PPD significantly increased absorbance, while the addition of other interfering ions did not show a significant increase in absorbance; and it contained Ce 60 Eu-Mn-CDs@ZIF-8 nanoparticles (concentration 0.17 mg / ml) in Britton-Robinson buffer (pH=7.0) (Control) are colorless and do not absorb in the visible light region. The color changes upon the addition of other interfering ions, but turns yellow and purple upon the addition of OPD and PPD. Therefore, compared to nanomaterials that are inherently colored, the nanomaterials of this invention exhibit lower background interference when used for visual colorimetry.

[0071] Selectivity tests of OPD, PPD, and interfering ions were performed using other nanoparticles of this invention, and the results were consistent with Ce. 60 The Eu-Mn-CDs@ZIF-8 nanoparticles are essentially similar.

[0072] 9. Using nanoparticles with oxidase-like activity for detection in specific water samples. For the detection of OPD and PPD in actual water samples, the tap water sample came from the laboratory of the School of Chemistry, Chongqing Normal University, and was not further purified before testing. The lake water sample came from Sanchun Lake, Chongqing Normal University, and was filtered with a 0.22μm filter membrane to remove solid impurities before testing. All water samples were stored in brown glass bottles at 4℃ and kept in a refrigerator at 4℃ for later use.

[0073] OPD and PPD in tap water and lake water were quantitatively analyzed using the standard addition method. 5 μM, 25 μM, and 50 μM OPD and 50 μM, 150 μM, and 250 μM PPD were added to the tap water and lake water samples, respectively. Ce was also used as the standard addition method. 60 Taking Eu-Mn-CDs@ZIF-8 nanoparticles as an example, the detection method of OPD in water samples was as follows: at 45℃, 90 L of Ce with a concentration of 1 mg / mL was added to a centrifuge tube. 60 Eu-Mn-CDs@ZIF-8 nanoparticle solution and 650 mL Britton-Robinson buffer (pH=4.4) were mixed, followed by the addition of OPD solutions of different concentrations (5 μM, 25 μM, 50 μM), and then 120 mL of actual water sample was added. After reacting for 22 min, the corresponding UV-Vis absorption spectra were measured. The detection method for PPD in the water sample was as follows: at 25℃, 170 μL of Ce2 (1 mg / mL) solution was added to a centrifuge tube. 60 Eu-Mn-CDs@ZIF-8 nanoparticle solution and 650 μL Britton-Robinson buffer (pH=7.0) were added, followed by the addition of PPD solutions of different concentrations (50 μM, 150 μM, 250 μM), and finally 120 μL of actual water sample. After reacting for 22 min, the corresponding UV-Vis absorption spectra were measured.

[0074] The above experiments were performed in triplicate, and the experimental data were recorded. The spiked recovery rate was calculated according to the formula: Spiked recovery rate = (Spiked sample value - Sample value) ÷ Spiked amount × 100%. The results are shown in Tables 5 and 6. The spiked recoveries of OPD and PPD were 98.9%~102.1% and 97.1~102.3%, respectively, with relative standard deviations of less than 5% (n=3). This indicates that the colorimetric method has high accuracy in detecting OPD and PPD in environmental samples and is feasible for quantitative analysis of OPD and PPD in natural water samples.

[0075] Table 5 Summary of OPD Spike Recovery Rates in Actual Environmental Water Samples

[0076] Table 6 Summary of PPD Spike Recovery Rates in Actual Environmental Water Samples

[0077] In summary, this invention provides a method for preparing nanoparticles with oxidase-like activity. The prepared nanoparticles can catalyze the oxidation of OPD, MPD, and PPD to produce oxidation products of different colors, which can be used for the visual identification and detection of phenylenediamine isomers. The nanoparticles with oxidase-like activity of this invention are used as colorimetric probes to detect OPD and PPD, with Ce... 60 Taking Eu-Mn-CDs@ZIF-8 nanoparticles as an example, the linear ranges obtained were 0.2-55 μM and 0.7-275 μM, respectively, and the detection limits were 66.7 nM and 233 nM, respectively, demonstrating high sensitivity and selectivity. Simultaneously, the nanoparticles of this invention with oxidase-like activity can be successfully used as colorimetric nanoprobes for the detection of OPD and PPD in environmental water samples, taking Ce... 60 Taking Eu-Mn-CDs@ZIF-8 nanoparticles as an example, the recoveries were 98.9–102.1% and 97.1–102.3%, respectively. This method has potential applications in the fields of biosensing and environmental monitoring.

[0078] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. A method for preparing nanoparticles with oxidase-like activity, characterized in that, The preparation method includes the following steps: ZIF-8, water-soluble Ce 3+ Salt, water-soluble M 3+ Salt and manganese-doped carbon dots dissolve in water and react with stirring, in which the water-soluble M... 3+ Salt M 3+ For Eu 3+ or Fe 3+ After centrifugation, washing, and drying, CeEu-Mn-CDs@ZIF-8 or CeFe-Mn-CDs@ZIF-8 nanoparticles can be obtained. The manganese-doped carbon dots are prepared by the following method: citric acid and MnCl2·4H2O are dissolved in water, ethylenediamine is added under stirring, and after stirring and mixing evenly, the mixture is placed in a high-pressure reactor and heated at 160~200℃ for more than 8 hours. After cooling to room temperature, the mixture is centrifuged and dialyzed to obtain manganese-doped carbon dots.

2. The preparation method according to claim 1, characterized in that, In step (1), the ZIF-8 is prepared according to the following method: water-soluble Zn 2+ ZIF-8 is obtained by adding an alcoholic solution of salt to an alcoholic solution of 2-methylimidazole under stirring, stirring at 30-50°C for more than 10 hours, centrifuging, washing repeatedly with methanol, and then vacuum drying.

3. The preparation method according to claim 2, characterized in that, The water-soluble Zn 2+ The molar ratio of salt to 2-methylimidazole is 1:6 to 1:10, of which the water-soluble Zn 2+ The salt is Zn(NO3)2·6H2O; The solvent in the alcohol solution includes methanol or ethanol; The centrifugation speed is 8000~12000 rpm and the time is 5~20 min; The vacuum drying temperature is 40~80℃.

4. The preparation method according to claim 1, characterized in that, The water-soluble Ce 3+ The salt contains any one of Ce(NO3)3, CeCl3, or Ce(Ac)3; The water-soluble M 3+ The salt contains any one of M(NO3)3, M2(SO4)3, M(Ac)3 or MCl3.

5. The preparation method according to claim 1, characterized in that, The molar ratio of citric acid, MnCl2·4H2O and ethylenediamine is 1:1:2 to 1:1:6; The high-pressure reactor is a polytetrafluoroethylene high-pressure reactor; The dialysis membrane used in the dialysis process has a specification of 800~1500 Da.

6. The preparation method according to claim 1, characterized in that, The Zn and water-soluble Ce in ZIF-8 3+ Ce in salt, water-soluble M 3+ The molar ratio of M in the salt to manganese in the manganese-doped carbon dots is 1:2:1:6~1:3:3:

8.

7. The preparation method according to claim 1, characterized in that, The stirring reaction time shall be no less than 8 hours; The centrifugation speed is 8000~12000 rpm and the time is 5~20 min; The drying process specifically involves vacuum drying overnight at a temperature of 40~80℃.

8. Nanoparticles with oxidase-like activity prepared by the preparation method according to any one of claims 1 to 7.

9. The application of the nanoparticles with oxidase-like activity as described in claim 8 in recognizing phenylenediamine isomers.

Images