Synthesis of a carbon dot-based nanozyme and its cascade detection application

Abstract

The purpose of this invention is to synthesize a carbon dot-based nanozyme and its cascade detection application. Using ascorbic acid and 1,3,5-pyromellitic acid as raw materials, ethylenediamine as a nitrogen dopant, and ferric chloride as a metal source, a one-step microwave synthesis method is employed in a microwave reactor (glass material). This method solves the problem of high energy consumption caused by the need for high temperature, high pressure, or long reaction time in existing technologies. Characterization and analysis of its morphology, structure, and properties revealed that the prepared iron-nitrogen co-doped carbon dot-based nanozyme consists of two-dimensional spherical carbon nanoparticles with excellent Fe-N... x The active site exhibits POD-like enzyme activity, with catalytic activity exceeding that of most reported nanozymes. Furthermore, this invention synergistically utilizes its peroxidase-like catalytic activity and photodynamic effect to significantly enhance reactive oxygen species (ROS) generation, achieving highly sensitive detection of organophosphorus pesticides and significantly reducing their detection limits. This synergistic strategy provides a new approach for expanding the application of nanomaterials in chemical analysis and sensing.

Description

Technical Field

[0001] This invention relates to the field of cascade detection applications of carbon nanomaterials, specifically to the synthesis of carbon dot-based nanozymes and their cascade detection applications. Background Technology

[0002] Nanozymes are a class of nanomaterials with biocatalytic functions. Their unique nanostructure not only endows them with excellent catalytic performance but also gives them higher stability and greater potential for large-scale production compared to natural enzymes. Currently, nanozymes have shown broad application prospects in various fields such as catalytic medicine, sensing and detection, green synthesis, new energy, and environmental governance. Despite these advantages and huge application potential, nanozyme research still faces many challenges: First, as the particle size decreases, the specific surface energy of nanozymes increases significantly, leading to the spontaneous aggregation of metal single atoms into clusters or particles; second, the substrate specificity and selectivity of nanozymes are not yet ideal and urgently need further optimization; third, their catalytic mechanism is not yet fully elucidated, resulting in a lack of unified theoretical guidance for the rational design of nanozymes. Therefore, selecting carriers with abundant anchoring sites and strong binding capabilities to stabilize single atoms is crucial for the preparation of nanozymes. In addition, in-depth exploration of the catalytic mechanism of nanozymes and the realization of the rational design and controllable synthesis of novel nanozymes remain key scientific issues that urgently need to be addressed in this field.

[0003] Among numerous nanomaterials, carbon dots (CDs) are a collective term for a class of novel luminescent carbon-based nanomaterials, encompassing carbon nanodots (CNDs), carbonized polymer dots (CPDs), carbon quantum dots (CQDs), and graphene quantum dots (GQDs), all typically with sizes below 10 nm. Due to their low toxicity, high chemical stability, ease of synthesis, and good biocompatibility, carbon dots have attracted widespread attention in various research fields. As an important branch of nanozymes, carbon dot-based nanozymes exhibit high catalytic activity due to their significant small size effect and abundant surface active sites. More importantly, the well-defined electronic structure and geometry of carbon dots facilitate the precise regulation and optimization of their catalytic active sites. Currently, research on carbon dot-based nanozymes mainly focuses on their peroxidase-like catalytic properties, a direction that has become a research hotspot in the field.

[0004] Based on the above background, carbon dot-based nanozymes have been widely used in various fields such as biosensing, disease diagnosis and treatment, and environmental monitoring due to their advantages of simple preparation process, adjustable catalytic activity, and good biocompatibility. However, they still face several bottlenecks in synthesis and application: (1) In existing literature, the synthesis conditions of nanozymes are often quite harsh, involving high temperature, high pressure, or long reaction time, resulting in high energy consumption and low synthesis efficiency; (2) The catalytic activity of some carbon dot-based nanozymes is not ideal, which limits their practical application under physiological conditions. This invention solves the problem of high energy consumption caused by the need for high temperature, high pressure, or long reaction time in the prior art, improves the activity of POD-like enzymes, and reduces the detection limit of organophosphorus pesticides. Summary of the Invention

[0005] This invention provides a carbon dot-based nanozyme with peroxidase-like (POD) enzyme activity, overcoming the shortcomings of the prior art. This invention also provides a method for preparing and applying the carbon dot-based nanozyme: a simple one-step microwave synthesis method yields carbon dot-based nanozymes (Fe-CDs) in just 35 minutes. These nanozymes have a distinct two-dimensional lattice structure of spherical nanoparticles, are well dispersed in aqueous solution, and under irradiation at a specific wavelength, can generate a large amount of ROS in a short time, the type of which is singlet oxygen (ROS). 1 O2), which, in synergy with the properties of POD-like enzymes, can enhance the limit of detection (LOD) of cascade detection of organophosphorus pesticides (OPs), and is of great significance for promoting the practical application of nanozymes in fields such as bioanalysis and environmental monitoring.

[0006] The technical solution adopted to achieve the above-mentioned objectives of this invention is as follows:

[0007] This invention provides a method for preparing carbon dot-based nanozymes, comprising the following steps: using ascorbic acid and 1,3,5-pyromellitic acid as raw materials, ethylenediamine as a nitrogen dopant, and ferric chloride as a metal source, the nanozymes are prepared by a one-step microwave synthesis method in a microwave reactor (glass material).

[0008] The above method can be used to prepare carbon dot-based nanozymes with photodynamic and peroxidase-like (POD) activities, which can then be used for cascade detection applications. The preparation method provided by this invention increases ROS generation during the preparation process through light irradiation and simultaneously possesses POD-like enzyme activity, catalyzing the production of large amounts of ROS from H₂O₂, thus broadening its application range in the field of biosensing.

[0009] Furthermore, the following steps may be included:

[0010] (1) Dissolve ascorbic acid, 1,3,5-pyromellitic acid and ferric chloride in 10 mL of water to obtain a mixed solution;

[0011] (2) Add ethylenediamine to the mixed solution to obtain the reaction solution;

[0012] (3) The reaction liquid is introduced into a microwave reactor for microwave reaction treatment, cooled, and a solid-liquid mixture is obtained;

[0013] (4) The solid-liquid mixture was filtered, purified and dried to obtain carbon dot-based nanozymes.

[0014] Furthermore, in step (1), the molar ratio of ethylenediamine to ferric chloride is 4:1, wherein 0.1761 g ascorbic acid, 0.2101 g 1,3,5-pyromellitic acid and 0.0406 g anhydrous ferric chloride, 67 μL ethylenediamine and 10 mL water are used.

[0015] Furthermore, in step (4), the precipitate is removed by filtration through a 0.45 μm filter head; purification is carried out by dialysis, and the molecular cutoff of the dialysis bag used is 500 Da.

[0016] This invention provides a carbon dot-based nanozyme, which is prepared using the method described above.

[0017] This invention provides carbon dot-based nanozymes in organophosphorus pesticides

[0018] This invention provides a carbon dot-based nanozyme that can detect one or more of chlorpyrifos, dichlorvos, and trichlorfon.

[0019] Compared with the prior art, the beneficial effects obtained by the present invention include:

[0020] 1. This invention provides a carbon dot-based nanozyme, specifically a synthesized carbon dot-based nanozyme (Fe-CDs) with POD-like enzyme activity. Through a unique structural design, the active centers of Fe-Nx with POD-like activity are encapsulated within a π-π conjugated carbon framework, dispersing the active sites and improving the catalytic activity of the nanozyme. Furthermore, the dried solid Fe-CDs exhibit a nanoflower structure, possessing a larger specific surface area, providing rapid mass transfer channels, enhancing electron transfer, and utilizing confinement effects to fully expose the catalytic active sites, thereby improving catalytic activity. This provides an effective structural design approach to address the current problems of insufficient catalytic activity and poor substrate selectivity in nanozymes.

[0021] 2. The Fe-CDs synthesized in this invention not only possess POD-like catalytic activity, but also, under irradiation at a specific wavelength, can generate a large amount of ROS in a short time, the type of which is singlet oxygen (ROS). 1 O2), which, in synergy with the properties of POD-like enzymes, can enhance the limit of detection (LOD) of cascade detection of organophosphorus pesticides (OPs).

[0022] 3. The method for preparing carbon dot-based nanozymes with POD-like enzyme activity provided by this invention has advantages such as simple equipment, rapid synthesis, reduced energy consumption, and optimized production process. Its synthesis steps are few, achieving a one-step reaction with a short reaction time (35 min). For the synthesis of carbon dot-based nanozymes, the microwave-assisted method can achieve rapid nucleation and growth of carbon dots in a very short time, which is beneficial for obtaining nanomaterials with uniform size and sufficient exposure of catalytic active sites, while also providing the possibility for low-energy, continuous production. Compared with hydrothermal methods, its heating temperature is significantly lower and the reaction time is significantly shortened, which greatly promotes the development of chemical reactions and the synthesis of inorganic materials. Attached Figure Description

[0023] Figure 1 This is a flowchart of the synthesis process of Fe-CDs.

[0024] Figure 2 This is a SEM image of Fe-CDs solid under a scanning electron microscope.

[0025] Figure 3 These are TEM and HRTEM images of Fe-CDs under a high-resolution transmission electron microscope.

[0026] Figure 4 This is the absorption spectrum of Fe-CDs under ultraviolet light.

[0027] Figure 5 The image shows the excitation / emission spectra of Fe-CDs in a fluorescence spectrometer.

[0028] Figure 6 Here is the XRD pattern of Fe-CDs.

[0029] Figure 7 This is the Raman plot of Fe-CDs.

[0030] Figure 8 UV images of Fe-CDs under different conditions to verify the feasibility of nanozymes.

[0031] Figure 9 For the generation of Fe-CDs under different conditions 1 EPR graph of O2.

[0032] Figure 10 EPR diagrams of Fe-CDs generating ·OH under different conditions.

[0033] Figure 11 Linearity graph for Fe-CDs detection of organophosphorus pesticides. Detailed Implementation

[0034] The present invention will be specifically described below through embodiments. It should be noted that the following embodiments are only used to further illustrate the present invention and should not be construed as limiting the scope of protection of the present invention. Those skilled in the art can make some non-essential improvements and adjustments to the present invention based on the above description.

[0035] Example 1

[0036] A method for preparing carbon dot-based nanozymes with POD-like enzyme activity, the reaction flow diagram of which is provided below. Figure 1 The preparation steps include the following:

[0037] (1) Dissolve 0.1761 g ascorbic acid, 0.2101 g 1,3,5-pyromellitic acid and 0.0406 g anhydrous ferric chloride in 10 mL of ultrapure water.

[0038] (2) Add 67 μL of ethylenediamine to the mixture in (1).

[0039] (3) The mixture was placed in a microwave synthesizer for a one-step microwave synthesis reaction at a temperature of 90 °C for 35 min. After the reaction was completed, the mixture was cooled to room temperature to obtain a solid-liquid mixture.

[0040] (4) The mixture in (3) was filtered through a 0.45 μm filter to remove the precipitate and obtain a crude solution of carbon dot-based nanozyme.

[0041] (5) The solution in (4) was purified by dialysis. The molecular cutoff of the dialysis bag was 500 Da, and the dialysis was performed for 3 h to remove the precursor substances and obtain a relatively pure carbon dot-based nanozyme solution.

[0042] (6) Finally, the solution in step (5) was freeze-dried to obtain carbon dot-based nanozyme Fe-CDs powder with high purity.

[0043] Example 2

[0044] The present invention also conducted the following characterization experiments:

[0045] like Figure 2 The image shows a SEM image of the Fe-CDs synthesized in this invention. The SEM reveals a spherical nanoflower structure, which has a larger specific surface area, provides a fast mass transfer channel, enhances electron transfer, and utilizes the confinement effect to fully expose the catalytic active sites, thereby improving catalytic activity.

[0046] like Figure 3The images shown are TEM and HRTEM images of the Fe-CDs synthesized in this invention. The TEM images show that the Fe-CDs are spherical, monodisperse particles with an average particle size of 5.93 nm. Furthermore, the Fe-CDs exhibit clear two-dimensional lattice fringes with a lattice spacing of 0.21 nm, corresponding to the (100) crystal plane of the graphite structure.

[0047] like Figure 4 The image shows the UV-vis spectrum of the Fe-CDs synthesized in this invention. The results indicate that HPGQD exhibits absorption bands at 283 nm and 395 nm. The strong absorption (283 nm) is attributed to the π−π* transition of the C=C bond in the core of the graphene nanoparticles. The weak absorption (395 nm) is the result of the combined effects of the n−π* transition of the C=C / C=O bond and the π−π* charge transfer transition at the edge (edge ​​band).

[0048] like Figure 5 The figure shows the PL spectrum of the Fe-CDs synthesized in this invention. The results indicate that the optimal excitation wavelength for Fe-CDs is 390 nm, and the maximum emission wavelength obtained under this excitation is 482 nm, exhibiting typical blue fluorescence.

[0049] like Figure 6 The image shows the XRD pattern of the Fe-CDs synthesized in this invention. The XRD pattern exhibits a characteristic diffraction peak at 2θ = 25.8°, corresponding to the (002) crystal plane of graphitized carbon. This diffraction peak is sharp and intense, indicating that the synthesized Fe-CDs have a high degree of graphitization crystallinity.

[0050] like Figure 7 The image shows the Raman spectroscopy plot of the Fe-CDs synthesized in this invention. The Fe-CDs at 1350 cm⁻¹... -1 and 1580 cm -1 Two characteristic peaks appear at the point, corresponding to the D band (defect / disorder structure) and G band (sp) of carbon materials, respectively. 2 (In-plane vibrations of hybrid carbon). The intensity ratio of the D band to the G band (I) was calculated. D / I G The value is 0.52. This moderate I... D / I G The values ​​indicate that although Fe-CDs contain a certain degree of lattice defects or edge structures, they are still predominantly highly graphitized sp. 2 The carbon network serves as the main framework.

[0051] The carbon dot-based nanozyme preparation method provided by this invention yields a product with a large specific surface area, uniform particle size distribution, excellent optical properties, and high crystallinity, which fully demonstrates the potential for peroxidase-like catalysis.

[0052] Example 3

[0053] The present invention also conducted the following mechanistic experimental studies:

[0054] 5 μL Fe-CDs (0.5 mg mL) -1 Add 0.1 mol / L HAc-NaAc buffer solution. -1 (pH = 3) and 100 μL H2O2 (10 mmol L -1 Add 10 μL of the trapping agent 2,2,6,6-tetramethylpiperidine (TEMP, 100 mmol / L) to the mixture above. -1 ) or 5,5-dimethyl-1-pyrrolline-N-oxide (DMPO, 100 mmol L -1 The samples were thoroughly mixed. A capillary tube was used to take a sample, which was then encased in a quartz protective tube and assembled into a test sample. The sample was placed in the resonant cavity of an electron paramagnetic resonance (EPR) spectrometer. Signals were first acquired in darkness, and then the sample was irradiated with a 300 W xenon lamp for 30 seconds before a second detection.

[0055] like Figure 8 The image shows UV-Vis images of the Fe-CDs synthesized in this invention under different conditions to verify the feasibility of nanozymes. The experimental results show that a significant absorption peak at 652 nm only appears when H2O2, TMB, and Fe-CDs coexist, while no obvious signal is observed in the control group lacking any component, confirming that Fe-CDs itself has inherent POD-like catalytic activity.

[0056] like Figure 9 The singlet oxygen of Fe-CDs synthesized in this invention ( 1 ESR diagram of H2O2. Fe-CDs can be decomposed under the catalysis of peroxidase-like enzymes to generate H2O2. 1 O2; simultaneously, Fe-CDs can also be directly generated under photodynamic action. 1 O2. When peroxidase-like catalysis and photodynamic effect work synergistically, in the system 1 The amount of O2 generated was significantly higher than that of a single pathway, demonstrating a clear synergistic enhancement effect.

[0057] like Figure 10The figure shows the ESR of the hydroxyl radical (·OH) of the Fe-CDs synthesized in this invention. Studies have shown that Fe-CDs possess peroxidase-like (POD) activity, capable of catalyzing the decomposition of H₂O₂ to generate ·OH. Notably, under photodynamic catalysis, Fe-CDs do not directly generate ·OH via this pathway. However, when the POD-like enzyme activity and photodynamic effect work synergistically, the amount of ·OH generated in the system is significantly higher than the sum under either photodynamic or enzymatic catalysis alone, exhibiting a clear synergistic mechanism.

[0058] Example 4

[0059] Add 40 μL of AChE (100 mU·mL) -1 ) and 50 μL of different concentrations of OPs (0.1–10000 ng·mL) -1 Mix and incubate at 37°C for 60 min; then add 40 μL of thioacetylcholine (ATCh, 2 mmol·L⁻¹). -1 Continue incubation for 60 min to allow residual AChE to catalyze the hydrolysis of ATCh. Then, add 50 μL of acetate-sodium acetate buffer (0.1 mol·L⁻¹) to the above reaction solution (total volume 130 μL). -1 , pH 3.0), 5 μL Fe-CDs (0.5 mg·mL -1 ), 50 μL H2O2 (10 mmol·L -1 For standard colorimetric assays, immediately add 50 μL of TMB (1 mmol·L⁻¹). -1 After incubation for 5 min, the absorbance at 652 nm was measured; for illumination-assisted detection, a 520 nm laser (power density 20 mW·cm⁻¹) was first used. -2 Irradiate for 1 min, then immediately add 50 μL of TMB (1 mmol·L⁻¹). -1 After incubation for 5 min, the absorbance at 652 nm was measured. By comparing the changes in absorbance after treatment with different concentrations of OPs, the inhibition rate of OPs on AChE activity can be quantitatively evaluated, and the limit of detection (LOD) under the two detection modes can be calculated based on the dose-response curve, thereby revealing the synergistic effect of light and POD enzyme on enhancing detection sensitivity.

[0060] like Figure 11 The figure shows the linear relationship between the Fe-CDs synthesized in this invention and the detection of organophosphorus pesticides using cascade detection. The results show a linear relationship between the concentration and absorbance of organophosphorus pesticides, and the detection limit was calculated to be 0.16 ng / mL. -1 .

[0061] In the above embodiments, the synthesized carbon dot-based nanozymes Fe-CDs possess good hydrophilicity, while also exhibiting peroxidase-like catalytic activity and photodynamic ROS generation characteristics. By synergistically combining these two properties, more ROS can be generated. Therefore, they can be applied to the detection of organophosphorus pesticides.

[0062] The above description is merely a selection of preferred embodiments of this disclosure and an explanation of the technical principles employed. Those skilled in the art should understand that the scope of the invention involved in the embodiments of this disclosure is not limited to technical solutions formed by specific combinations of the above-described technical features, but should also cover other technical solutions formed by arbitrary combinations of the above-described technical features or their equivalents without departing from the above-described inventive concept. For example, technical solutions formed by substituting the above-described features with (but not limited to) technical features with similar functions disclosed in the embodiments of this disclosure.

Claims

1. Synthesis of carbon dot-based nanozymes: using ascorbic acid and 1,3,5-pyromellitic acid as raw materials, ethylenediamine as nitrogen dopant, and ferric chloride as metal source, the nanozymes were prepared by a one-step microwave synthesis method in a microwave reactor (glass material).

2. Synthesis process of carbon dot-based nanozymes:

3. The method for preparing a carbon dot-based nanozyme according to claim 1 or 2, characterized in that, The molar ratio of ethylenediamine to ferric chloride is 4:1, consisting of 0.1761 g ascorbic acid, 0.2101 g 1,3,5-pyromellitic acid, 0.0406 g anhydrous ferric chloride, 67 μL ethylenediamine, and 10 mL water.

4. The method for preparing a carbon dot-based nanozyme according to claim 1 or 2, characterized in that, In the one-step microwave synthesis process, the reaction temperature is 90℃ and the heating time is 35 min.

5. The method for preparing a carbon dot-based nanozyme according to claim 1 or 2, characterized in that, Includes the following steps (1) Dissolve ascorbic acid, 1,3,5-pyromellitic acid and ferric chloride in 10 mL of water to obtain a mixed solution; (2) Add ethylenediamine to the mixed solution to obtain the reaction solution; (3) The reaction solution is placed into a microwave reactor for microwave reaction treatment, cooled, and a solid-liquid mixture is obtained; (4) The solid-liquid mixture was filtered, purified and dried to obtain carbon dot-based nanozymes.

6. The method for preparing a carbon dot-based nanozyme according to claim 5, characterized in that, In step (4), the precipitate is removed by filtration through a 0.45 μm filter head; purification is carried out by dialysis, and the molecular cutoff of the dialysis bag used is 500 Da.

7. The application of carbon dot-based nanozymes in cascade detection, characterized in that, The carbon dot-based nanozyme is prepared using any one of the methods described in 1-6.

8. The application according to claim 7, characterized in that, The carbon dot-based nanozyme uses photodynamic and POD-like enzymes to synergistically increase the generation of reactive oxygen species (ROS), thereby enhancing the activity of the POD enzyme and lowering the detection limit for OPs.

9. The application according to claim 7 or 8, characterized in that, It is used for any one or more of chlorpyrifos, dichlorvos, and trichlorfon.

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