Copper-carbon quantum dot nano-enzyme as well as preparation method and application thereof

Abstract

The invention relates to the technical field of optical sensing, in particular to a copper-carbon quantum dot nano-enzyme and a preparation method and application thereof. Poria cocos and Cu < 2 + > are taken as raw materials, and the copper-carbon quantum dots with excellent peroxidase activity are prepared by adopting a one-pot hydrothermal method and can be used for sensitively, accurately and selectively detecting glyphosate. Cu < 2 + > is combined with oxygen-rich and nitrogen-rich components in poria cocos to generate an oxidation-reduction reaction, CuCDs with Cu < 2 + > / Cu < + > are obtained, Cu < 2 + > doping changes electron cloud distribution of the CDs, the electron transmission capability of the CuCDs is enhanced, and Cu < 2 + > and the CDs have a synergistic effect to generate three ROS (. OH,. O2 <-> and < 1 > O2), so that excellent peroxidase activity is exerted. The glyphosate is chelated with hydroxyl, amino, carbonyl, carboxyl and Cu on the surface of the CuCDs to occupy active sites, so that the active sites are poisoned, the peroxidase activity of the CuCDs is selectively inhibited, and a powerful choice is provided for simply, quickly, sensitively and selectively detecting the glyphosate.

Description

Technical Field

[0001] The present invention relates to the field of optical sensing technology, and in particular to a copper-carbon quantum dot nanozyme and a preparation method and application thereof. Background Art

[0002] The information disclosed in this background technology section is only intended to enhance the understanding of the overall background of the invention, and should not necessarily be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art.

[0003] Glyphosate (N-(phosphomethylethyl)glycine) has become the most popular herbicide due to its high efficiency, low toxicity and wide applicability. Long-term overuse and abuse have caused glyphosate to remain in the environment, posing an extremely serious threat to the ecosystem and even human health. Therefore, there is an urgent need to develop a simple, rapid, sensitive and reliable detection method to monitor glyphosate in agricultural products.

[0004] The main methods used for glyphosate detection include: liquid / gas chromatography coupled with mass spectrometry, enzyme-linked immunosorbent assay, surface-enhanced Raman scattering analysis, electrochemistry, and fluorescence and colorimetry. In recent years, fluorescence and colorimetric biosensors have become the most popular and dominant strategies due to their high sensitivity, rapid analysis, simple operation, on-site detection, and reliable results. Summary of the invention

[0005] In order to overcome the above problems, the present invention provides a copper-carbon quantum dot nanozyme and a preparation method and application thereof.

[0006] In order to achieve the above technical objectives, the present invention adopts the following technical solutions:

[0007] The first aspect of the present invention provides a copper-carbon quantum dot nanozyme composition, comprising the following components:

[0008] Poria cocos and divalent copper salt.

[0009] In one or more embodiments, the divalent copper salt includes one of copper sulfate, copper chloride, copper nitrate, or copper carbonate, preferably copper sulfate.

[0010] In one or more embodiments, the Poria cocos includes one of Poria cocos or Polyporus umbellatus of the genus Poria cocos, preferably Poria cocos of the genus Poria cocos.

[0011] In one or more embodiments, the copper-carbon quantum dot nanozyme is spherical and has a particle size of about 7.21 nm.

[0012] The second aspect of the present invention provides a method for preparing the copper-carbon quantum dot nanozyme described in the first aspect, comprising: mixing Poria cocos, divalent copper salt and water, and heating the mixture to react to obtain the copper-carbon quantum dot nanozyme.

[0013] In one or more embodiments, after the heating reaction is completed, the supernatant is filtered and dialyzed in ultrapure water, filtered again, and freeze-dried to obtain the copper-carbon quantum dot nanozyme.

[0014] Preferably, the pore size of the filter membrane used in the double filtration method is 0.22 μm.

[0015] Preferably, the molecular weight cut-off of the dialysis membrane used for dialysis is 450-550 Da, preferably 500 Da.

[0016] In one or more embodiments, the pulverized Poria cocos is sieved through a 90-120 mesh sieve, preferably a 100 mesh sieve.

[0017] In one or more embodiments, the molar ratio of the mass of Poria cocos to the divalent copper salt is: 1g: 1.5-2.5×10 -4 mmol, preferably 1g:2×10 -4 mmol.

[0018] In one or more embodiments, the water is ultrapure water.

[0019] In one or more embodiments, the concentration of the divalent copper salt in water is 7.5 to 12.5×10 -4 mM, preferably 1.0×10 -3 mM.

[0020] In one or more embodiments, the temperature of the heating reaction is 170-200° C., preferably 180° C.; the time of the heating reaction is 2-5 hours, preferably 3 hours.

[0021] The third aspect of the present invention provides an application of the copper-carbon quantum dot nanozyme described in the first aspect, and constructs a colorimetric sensor based on the copper-carbon quantum dot nanozyme described in the first aspect for detecting glyphosate.

[0022] A fourth aspect of the present invention provides a method for detecting glyphosate, comprising:

[0023] (1) placing a gradient concentration of glyphosate and the copper-carbon quantum dot nanozyme described in the first aspect in a buffer solution, then adding a color developer 3,3',5,5'-tetramethylbenzidine (TMB) and hydrogen peroxide, and after incubation, taking an image of the mixed solution after incubation, and then obtaining the RGB value of the mixed solution image to establish a standard curve between the glyphosate concentration and the RGB value;

[0024] (2) Obtain the concentration of glyphosate in the sample to be tested based on the standard curve between glyphosate concentration and RGB value.

[0025] In one or more embodiments, the gradient concentration is 0.02-2.8 mM.

[0026] In one or more embodiments, the buffer solution is a NaAc-HAc buffer solution.

[0027] In one or more embodiments, the concentration of the copper-carbon quantum dot nanozyme in the buffer solution is 1 to 20 μg mL -1 , preferably 10 μg mL -1 .

[0028] In one or more embodiments, the incubation temperature is 35-38° C., preferably 37° C.; the incubation time is 20-30 min, preferably 20 min.

[0029] The beneficial effects of the present invention are:

[0030] (1) In the present invention, Poria cocos and Cu 2+ As raw materials, copper-carbon quantum dots (CuCDs) with excellent peroxidase activity were prepared by a one-pot hydrothermal method, which can be used for sensitive, accurate and selective detection of glyphosate. 2+ Combined with oxygen-rich and nitrogen-rich components in Poria cocos, redox reactions occurred to obtain Cu 2+ / Cu + CuCDs, Cu 2+ Doping changes the electron cloud distribution of CDs, enhances the electron transport capacity of CuCDs, and synergizes with CDs to produce three types of ROS (·OH, ·O2 - and 1 O2), thereby exerting excellent peroxidase activity. Glyphosate occupies the active sites by chelating with the hydroxyl, amino, carbonyl, carboxyl and Cu on the surface of CuCDs, poisoning the active sites and selectively inhibiting the peroxidase activity of CuCDs, providing a powerful choice for simple, rapid, sensitive and selective detection of glyphosate.

[0031] (2) The CuCDs in the present invention have excellent specificity for glyphosate detection, and also have a wider linear range (5-120 μM) and a lower detection limit (2.66 μM). BRIEF DESCRIPTION OF THE DRAWINGS

[0032] The accompanying drawings in the specification, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0033] Figure 1 Schematic diagram of the synthesis and detection of glyphosate by CuCDs nanozymes;

[0034] Figure 2 A is a TEM image of CuCDs (inset: high-resolution TEM and size distribution histogram); B is the FT-IR spectrum of CuCDs and CDs; C is the XPS measurement spectrum of CuCDs and CDs; D is the high-resolution XPS spectrum of Cu2p; E is the high-resolution XPS spectrum of O1s of CuCDs and CDs; F is the high-resolution XPS spectrum of N1s of CuCDs and CDs;

[0035] Figure 3 A is the TEM image of CDs (inset: high-resolution TEM and size distribution histogram); B is the XRD spectra of CuCDs and CDs; C is the high-resolution XPS spectra of C1s of CDs and CuCDs;

[0036] Figure 4 UV-visible (A) and fluorescence (B) spectra of CuCDs and CDs;

[0037] Figure 5 A is the UV-visible spectra of different reaction systems (the inset is the picture of the response reaction system taken under daylight); B is the UV-visible spectra of CDs, CuCDs, CDs-Cu, and Cu and the corresponding pictures taken under fluorescent light; C is the assay experiment of the catalytic activity of CDs and CuCDs enzymes; D is the effect of Na2EDTA on the activity of CDs and CuCDs-type peroxidases; kinetic experiments with TMB (E) and H2O2 (F) as substrates;

[0038] Figure 6 The middle is the peroxidase-like activity under different temperatures (A), different pH conditions (B), different TMB concentrations (C) and different hydrogen peroxide concentrations (D);

[0039] Figure 7 Cu 2+ Effects of concentration, (B) reaction time, and (C) reaction temperature on the activity of synthesized CuCDs-like peroxidases;

[0040] Figure 8 Peroxidase-like activity of CuCDs under (A) 365 nm UV irradiation for 120 min and (B) 4 °C storage for 6 months;

[0041] Fig. 9 Steady-state kinetic analysis of CDs with (A) TMB and (B) H2O2 as substrates at pH 4.0;

[0042] Fig.10is the relative activity in the CuCDs+H2O2+IPA / p-BQ / NaN3 (1 mM) system;

[0043] Fig.11 ·OH, ·O2 in (A, B, C)DMPO / TEMP+CuCDs+H2O2 system - and EPR spectra of 1O2; (D) CuCDs (200 μg mL -1 ) UV absorption spectra of Cyt C (50 μM) before and after;

[0044] Fig.12 Cyclic voltammetric current response (A) and electrochemical impedance curve (B) of CuCDs and CDs;

[0045] Fig.13 are the pesticides used in the experiment and their corresponding structures;

[0046] Fig.14 The UV-visible spectra of the CuCDs+TMB+H2O2 system after incubation with (A) different pesticides and (B) different concentrations of glyphosate; (C) UV-visible and fluorescence spectra of CuCDs / glyphosate; (D) FT-IR spectra of glyphosate and CuCDs / glyphosate, (E) reaction time curve of TMB colorimetric reaction catalyzed by CuCDs and CuCDs / glyphosate at pH 4.0; wherein 1 to 11 in (A) are 1. Parathion, 2. Phosemet, 3. MP, 4. Carbendazim, 5. Thiram, 6. Fipronil, 7. Dichlorvos, 8. Carbaryl, 9. Quinalphos, 10. TM and 11. Glyphosate, respectively;

[0047] Fig.15 (A) Effect of glyphosate addition order on enzyme catalytic activity; glyphosate on (B) ·OH, (C) ·O2 - and (D) 1 O2 response; (B, C, D respectively use 30mM H2O2, 150μMRiboflavin+15μM Met UV light, 30mM NaClO+30mM H2O2 to produce ·OH, ·O2 - and 1 O2; the concentrations of the capture agents were 1 mM TA, 150 μM NBT, and 0.25 mM DBPF);

[0048] Fig.16(A) UV-visible spectra of CDs+TMB+H2O2 system after co-incubation with different pesticides and corresponding pictures UV-visible spectra of different reaction systems at pH 4.0; (B) Linearity of CDs response to glyphosate (the inset is the corresponding picture taken under fluorescent light); 1 to 11 in (A) are 1. Parathion, 2. Phosemet, 3. MP, 4. Carbendazim, 5. Thiram, 6. Fipronil, 7. Dichlorvos, 8. Carbaryl, 9. Quinalphos, 10. TM and 11. Glyphosate, respectively;

[0049] Fig.17 The R value changes of CuCDs+glyphosate (60μM)+TMB+H2O2 reaction system at different (A) incubation time and (B) different CuCDs concentrations;

[0050] Fig.18 (A) shows the linear relationship between R value and glyphosate concentration; (B) shows the R value of glyphosate (60 μM) in the presence of different interfering substances (1000 μM) (the inset is the corresponding picture); 1 to 19 in (B) are 1.K + 2.Na + 3.Mg 2+ 4.Ca 2+ ,5.L-Pro, 6.L-Leu, 7.L-Val, 8.L-Ser, 9.TM, 10.Quinalphos, 11.Thiram, 12.Dichlorvos, 13.Phosemet, 14.MP, 15.Parathion, 16.Fipronil, 17.Carbaryl, 18.Carbaryl, 19.Glyphosate. DETAILED DESCRIPTION

[0051] It should be noted that the following detailed descriptions are exemplary and are intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used in the present invention have the same meanings as those commonly understood by those skilled in the art to which the present invention belongs.

[0052] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit exemplary embodiments according to the present invention. As used herein, unless the context clearly indicates otherwise, the singular form is also intended to include the plural form. In addition, it should be understood that when the terms "comprising" and / or "including" are used in this specification, it indicates the presence of features, steps, operations, devices, components and / or combinations thereof.

[0053] In order to enable those skilled in the art to more clearly understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below in conjunction with specific embodiments.

[0054] Materials and Equipment

[0055] Pesticides Glyphosate (98%), methyl thiophanate (TM, 99%), Quinalphos (99%), Thiram (97%), Dichlorvos (1 mg mL -1 ), phosmet (Phosemet, 99%), methyl parathion (MP, 1 mg mL -1 ), Parathion (1 mg mL -1 ), Fipronil (Fipronil 99%), Carbaryl 99%), Carbendazim (Carbendazim, 99%), 1,3-propanesultone (PS, 99%), and 4-sulfophenyl isothiocyanate sodium salt (SPI, 99%) were purchased from Aladdin Reagent Company (Shanghai, China). CuSO4·5H2O (99%), sodium borohydride (NaBH4, 98%), triethylamine (99%), 1,4-dioxane (99%), anhydrous ethanol, isopropyl alcohol (IPA, 99%), p-benzoquinone (p-BQ, 99%), sodium azide (NaN3, 99%), 5,5-dimethyl-1-pyrrolidine N-oxide (DMPO, BR, 97%), 2,2′,6,6′-tetramethylpiperidine (TEMP, GC, 98%), acetonitrile (HPLC), and formic acid (HPLC) were purchased from Sinopharm Group Chemical Reagent Co., Ltd. (Beijing, China). Dimethyl sulfoxide (DMSO, 99%), sodium hydroxide (97%), HCl (36-8%, GR), NaCl (99%), KCl (99%), MgCl2 (98%), CaCl2 (96%), FeCl3 (98%), K3[Fe(CN)6] (99%), Na2EDTA·2H2O (99%), and H2O2 (30%) were purchased from MacLean Reagent Company (Shanghai, China). L-Proline (L-Proline 99%, BR), L-Leucine (L-Leucine 99%, BR), L-Valine (L-Valine 99%, BR), L-Serine (L-Serine 99%, BR), 3,3',5,5'-tetramethylbenzidine (TMB, 99%, BR), and cytochrome c from horse heart (Cyt c, 95%, BR) were purchased from Shanghai Yuanye Biotechnology Co., Ltd. (Shanghai, China). Ultrapure water (18.25 MΩ cm) was prepared by a molecular water purification system (Millipore, USA).

[0056] The morphology and size were characterized by a JEOL / JEM-F200 transmission electron microscope (TEM, JEOL, Japan) with an accelerating voltage of 200 kV and a JEOL / JSM-7610Fplus scanning electron microscope (SEM, JEOL, Japan) with an accelerating voltage of 15 kV. X-ray photoelectron spectroscopy (XPS) was measured by K-Alpha (Thermo Fisher Scientific, USA). X-ray diffraction (XRD) was measured on Empyrean (PANalytical, Netherlands). UV-visible absorption spectra were performed using a UV-2600 UV-visible spectrophotometer (Shimadzu, Japan). Fourier transform infrared spectroscopy (FT-IR) was measured by Spectrum Two (PerkinElmer, USA). Fluorescence spectra were recorded by LS-55 fluorescence spectrometer (PerkinElmer, USA) with excitation and emission voltages of 700 V and a slit width of 10 nm. Thermo Forma 3111 humidified incubator (Thermo Fisher) was selected for reaction incubation. A handheld homogenizer S10 (TUOHE, China) was used to pretreat the actual samples. A Bruker EMX PLUS electron spin resonance spectrometer (Bruker, Germany) was used to collect electron paramagnetic resonance (EPR) signal data. The colorimetric images were captured by a smartphone (Xiaomi 11, China). Electrochemical detection was performed on a CHI660E electrochemical workstation (Shanghai Chenhua, China).

[0057] Example 1

[0058] The dried Poria cocos was crushed and passed through a 100-mesh sieve. The crushed Poria cocos (2.5 g), copper sulfate pentahydrate solution (20 μL, 25 mM) and ultrapure water (50 mL) were mixed and added to a Teflon-lined autoclave and reacted at 180°C for 3 hours. The obtained supernatant was filtered through a 0.22 μm membrane and dialyzed in ultrapure water for 3 days (the molecular weight cutoff of the dialysis membrane used for dialysis was 500 Da). After filtering again (0.22 μm), the copper-carbon quantum dots CuCDs were obtained by freeze drying and stored at 4°C for later use.

[0059] Comparative Example 1

[0060] The dried Poria cocos was crushed and passed through a 100-mesh sieve. The crushed Poria cocos (2.5 g) was mixed with ultrapure water (50 mL), added to a Teflon-lined autoclave, and reacted at 180°C for 3 hours. The obtained supernatant was filtered through a 0.22 μm membrane and dialyzed in ultrapure water for 3 days (the molecular weight cutoff of the dialysis membrane used for dialysis was 500 Da). After filtering again (0.22 μm), the carbon quantum dots CDs were freeze-dried and stored at 4°C for later use.

[0061] Example 2

[0062] The structures and compositions of CuCDs and CDs were determined by transmission electron microscopy (TEM), X-ray diffraction (XRD), Fourier transform infrared (FTIR) and X-ray photoelectron spectroscopy (XPS). Figure 2 shown.

[0063] TEM images show that the spherical CuCDs are well dispersed and have a narrow size distribution (average diameter 7.21 nm) ( Figure 2 A). CDs have similar morphology and size (average diameter of 6.28 nm) ( Figure 3 A). High-resolution TEM of CuCDs and CDs shows the same 0.21 nm ordered lattice planes ( Figure 2 A and illustration in 3A), belongs to sp 2 -(100) characteristic plane of graphite carbon. Therefore, it can be considered that CuCDs and CDs have the same crystal core. This inference can also be verified by the broad diffraction peak with a central position of about 23.4° observed in the XRD patterns of CDs and CuCDs ( Figure 3 B) FT-IR then reveals the distribution of surface functional groups. Figure 2 B, 3243, 2923, 1700, 1517, 1391 and 1021cm -1 The bands are attributed to the stretching vibrations of NH / OH, CH, C=O, C=C / C=N, CN and COC groups. Compared with CDs, the NH / OH peak position in CuCDs has a blue shift (3294 cm -1 →3243cm -1 ), and the relative content also decreased, indicating that Cu atoms on the surface of CDs mainly participate in the synthesis by complexing with NH / OH. XPS was used to further analyze the surface state and composition of CuCDs. The XPS full scan spectrum of CuCDs showed the presence of C1s (61.5%), N1s (2.0%), O1s (36.4%) and Cu2p (0.1%) orbitals, corresponding to the peaks of 286.1, 401.1, 533.1 and 977.1 eV, respectively ( Figure 2 C). In addition, the XPS high-resolution fine spectrum clearly shows the existence of different valence states of Cu and different binding sites of Cu with oxygen and nitrogen atoms. In the high-resolution Cu2p spectrum of CuCDs ( Figure 2 D), Cu + 2p 3 / 2 (932.7eV), Cu 2+ 2p 3 / 2 (936.1eV), Cu+ 2p 1 / 2 (941.3eV) and Cu 2+ 2p 1 / 2 (945.9eV) has two pairs of characteristic peaks, indicating that Cu + and Cu 2+ The existence of Cu + The appearance of indicates that some Cu 2+ In addition, the two satellite peaks at 953.5 and 956.1 eV show Cu-N and Cu-O structures, respectively. The high-resolution O1s spectrum of CuCDs has four characteristic peaks at 533.4, 531.6, 532.5 and 530.6, which come from C-OH, OC=O, C=O and Cu-O bonds ( Figure 2 E). In addition, the high-resolution N1s spectrum of CuCDs consists of four characteristic peaks located at 404.0, 40.1.2, 399.9, and 397.9 eV, which can be attributed to -NH2, C=N, CN, and Cu-N( Figure 5-2 F). Compared with the high-resolution O1s (533.6, 532.5 and 531.6 eV for C-OH, OC=O and C=O bonds) and N1s (404.1, 401.9 and 400.3 eV for -NH2, C=N and CN) spectra of CDs ( Figure 2 E and 2F), the binding energy in CuCDs is lower because Cu coordinates with oxygen-rich and nitrogen-rich functional groups on the surface of CDs, changing the distribution of surface electrons. By comparing the UV-visible and fluorescence spectra of CDs ( Figure 4 ), the absorption of CuCDs n→π* transition at 272 nm blue-shifted, while the fluorescence emission at 445 nm decreased significantly by 28%, indicating that the metal ligand covalently changed the electron distribution, confirming the occurrence of redox reaction between Cu and CDs.

[0064] Example 3

[0065] 3.1 Study on the peroxidase-like activity of CuCDs nanozymes:

[0066] The peroxidase-like catalytic activity of CDs and CuCDs was studied using TMB as a chromogenic substrate. CDs or CuCDs (10 μL, 2 mg mL -1 ), TMB (10 μL, 10 mM) and H2O2 (10 μL, 100 mM) were mixed with NaAc-HAc buffer system (170 μL), incubated at 37°C for 20 min, and the UV-visible spectrum and absorbance of oxTMB at 652 nm were recorded at room temperature.

[0067] The steady-state kinetic study was the same as above. The concentration of TMB (0.5 mM) or H2O2 (0.5 mM) was fixed, and different concentrations of H2O2 or TMB were added. The absorbance at 652 nm was measured by time scan mode at different time intervals. The double reciprocal curves of TMB and H2O2 were drawn according to the Michaelis-Menten equation.

[0068]

[0069] Where v and vmax are the initial reaction rate and maximum reaction rate, respectively, Km is the Michaelis constant, and [S] is the substrate concentration.

[0070] exist Figure 5 In A, the CuCDs+TMB+H2O2 system exhibits characteristic absorption enhancement for oxTMB at 652nm and presents an obvious blue color (see Figure 5 A illustration). In contrast, a blue signal also appeared in the CuCDs+TMB solution, indicating that it has very weak oxidase-like activity and strong peroxidase activity. It is worth noting that CDs itself also has a certain catalytic activity ( Figure 5 B), whose enzyme catalytic activity is 50% of that of CuCDs ( Figure 5 B and C). In contrast, CDs-Cu (by making Cu 2+ The activity of CuCDs was lower, at 21% of that of CuCDs, while Cu (at a concentration equivalent to that in CuCDs) showed low-efficiency peroxidase-like activity ( Figure 5 B) When Na2EDTA, a ligand with strong interaction with metal ions and functional groups, was introduced, the catalytic activities of CDs and CuCDs were significantly reduced ( Figure 5 D). Therefore, it is reasonable to speculate that the excellent peroxidase-like activity of CuCDs may come from the synergistic effect between Cu and CDs.

[0071] Next, the effects of temperature, pH, and substrate concentration on the peroxidase-like activity of CuCDs were evaluated. Figure 6 As shown in Figure 2, when the optimum temperature and pH were 37°C and pH 4.0, respectively, the catalytic activity was highest after adding 0.5 mM TMB and 5 mM H2O2, which were selected as the optimal concentrations. Subsequently, the synthesis parameters of CuCDs were optimized. 2+ When the concentration was 10 μM and the reaction was carried out at 180°C for 3 h, CuCDs with high peroxidase-like activity at pH 4 were obtained. Figure 7 Next, the effects of UV irradiation and storage time on CuCDs-type peroxidase were evaluated ( Figure 8). The results showed that the peroxidase-like activity of CuCDs was almost unchanged after continuous UV irradiation for 120 min or storage for 6 months at 4°C. Therefore, the excellent peroxidase-like activity and high stability of CuCDs can be used in biosensor analysis to ensure efficient and stable results.

[0072] To further explore the peroxidase-like catalytic properties of CuCDs, steady-state kinetic experiments were carried out at pH 4 with different concentrations of TMB and H2O2 as substrates. The kinetic data of CuCDs conformed to the concentration-dependent patterns of the Michaelis-Menten and Lineweaver-Burk models ( Figure 5 E and 5F). When TMB was used as substrate, the Km and vmax values ​​were 0.34 mM and 1.16 × 10 -6 M.s -1 When H2O2 was used as substrate, Km and vmax were 2.46 mM and 1.05 × 10 -6 M.s -1 Compared with the enzyme-like activity of CDs ( Fig. 9 ), the Km of CuCDs for TMB is smaller than that of CDs, indicating that Cu doping improves the affinity of CDs for TMB, further indicating that the superior catalytic performance of CuCDs may come from the synergistic effect between CDs and Cu.

[0073] 3.2 Peroxidase-like catalytic mechanism of CuCDs nanozymes

[0074] In order to explore the catalytic mechanism of the peroxidase-like activity of CuCDs, a reactive oxygen species (ROS) quenching experiment was first used to determine which types of ROS were involved in the catalytic reaction. Isopropyl alcohol (IPA), p-benzoquinone (p-BQ) and sodium azide (NaN3) were used as ·OH, ·O2 - and 1 O2 scavenger. Fig.10 As shown in the figure, compared with the blank control group, the addition of any of the three scavengers resulted in a significant decrease in the absorption at 652 nm. This indicates that all three ROS are produced during the CuCDs-catalyzed oxidation of hydrogen peroxide. Next, in order to more accurately identify the types of ROS, an EPR experiment was carried out. As shown in the EPR spectrum ( Fig.11 As shown in A, B, and C, in the presence of capture agents (DMPO or TEMP), different ROS signals appeared in CuCDs+H2O2, confirming that ·OH, O2- and 1 O2. On the other hand, cytochrome C (Cyt C) is a common electron transfer agent in the redox process and is used here to detect the electron accepting ability of CuCDs. Fig.11In D, the two absorption peaks at 525 and 548 nm belong to the characteristic absorption of the reduced state of Cyt C. When CuCDs are added to the system, as expected, these two absorption peaks disappear, and a new absorption peak appears at 530 nm, indicating that electrons are transferred from Cyt C to CuCDs, which is consistent with the results reported in the literature. In addition, the conductivity of the material was monitored by electrochemical methods, and it was found that CuCDs had a higher current response at 0.46 V than CDs, and a smaller Nyquist radius ( Fig.12 ), indicating that the resistance of the electron transfer process is smaller, Cu 2+ The doping of improves the electron transfer efficiency of CDs as nanozymes.

[0075] Considering the conjugated framework of CuCDs, a possible catalytic mechanism was proposed. The substrate TMB and H2O2 bind to the surface hydroxyl, amino, carboxyl and carbonyl groups of CuCDs through hydrogen bonding. Then, the electron-deficient structure on CuCDs obtains an electron from TMB to generate oxTMB and reduced CuCDs. The obtained electron forms p-π conjugation with the large π system in CuCDs and is quickly and evenly distributed to form a short-lived intermediate (reduced CDs). After that, the electron is captured by Cu and transferred to the neighboring H2O2, while CuCDs returns to its original structure. Generally, Cu(Ⅱ) has good Fenton reaction activity and can catalyze H2O2 to generate ·OOH, which quickly recombines and splits to produce ·O2 - , ·OOH / ·O2 - Further oxidation by Cu(Ⅱ) produces 1 O2. Therefore, in the presence of hydrogen peroxide, multiple ROS were generated based on the synergistic effect of Cu and CDs, and high peroxidase-like activity was exhibited.

[0076] Example 4

[0077] CuCDs nanoenzyme colorimetric detection of glyphosate and its mechanism:

[0078] Interestingly, in the presence of CuCDs and a range of different pesticides ( Fig.13 ) coexisted, the inhibitory effect of other pesticides on the 652nm signal output was negligible, and only when it coexisted with glyphosate did it show a significant weakening ( Fig.14 A), and as the concentration of glyphosate increases (1-140 μM), the absorbance gradually decreases ( Fig.14 B), indicating that the proposed strategy can achieve specific detection of glyphosate. In order to reveal its intrinsic mechanism, the mechanism of glyphosate selectively inhibiting CuCDs nanozyme was studied.

[0079] In general, the inhibition mechanism of nanozymes mainly includes the synergistic effect of the analyte, poisoning of the active site, ROS consumption and oxTMB reduction, and the latter two are obviously based on the reducibility of the analyte to achieve the purpose of detection. Fig.15 As shown in A, after glyphosate was mixed with CuCDs and TMB, H2O2 was added to start the reaction, and the activity of the nanozyme was inhibited; however, when the order of adding reagents was adjusted and glyphosate was introduced into the CuCDs+TMB+H2O2 system after the catalytic reaction was completed, there was no obvious difference in the UV-visible spectrum. This phenomenon shows that glyphosate can directly interact with the nanozyme, rather than promoting the reduction of oxTMB. The effect of glyphosate on the activity of the nanozyme is not based on redox reactions. Terephthalic acid (TA) is a commonly used ·OH capture agent, which combines with it to generate a strong fluorescent product hydroxyterephthalic acid (HOTA). NBT (nitro blue tetrazolium) in ·O2 - When present, a blue-purple product is produced, while 1 O2 has a specific fluorescence quenching effect on DBPF. After adding different capture agents and glyphosate into the system, the UV-visible / fluorescence spectra were measured respectively. Fig.15 BD shows that with the increase of glyphosate concentration, the fluorescence of TA+·OH gradually decreases, and NBT+·O2 - The absorbance of glyphosate gradually decreased, indicating that glyphosate has certain ·OH and ·O2 - Scavenging effect. After being added to the DBPF+1O2 system, the fluorescence recovered to a certain extent, indicating that glyphosate and 1O2 also have an interaction relationship. Next, in order to clarify the interaction relationship between glyphosate and CuCDs, a CuCDs / glyphosate composite material was prepared (CuCDs and glyphosate were stirred at room temperature for 24 hours, dialyzed for 3 days, and freeze-dried after membrane). Compared with CuCDs, in the UV-visible spectrum, the n→π* transition of CuCDs / glyphosate blue-shifted to 260nm, the fluorescence spectrum red-shifted to 500nm, and the fluorescence quenching was 81% ( Figure 4 , 14C); FT-IR spectrum ( Fig.14 D) The NH / OH and CH absorption peaks of CuCDs and CuCDs / glyphosate remain consistent. However, the stretching vibration peak of P=O at 1334 cm-1 of CuCDs / glyphosate complex is red-shifted to 1340 cm-1. -1 , CuCDs at 1659cm -1 The stretching vibration peak of -CONH2- is red-shifted to 1619 cm -1 , at 1019cm -1The COC vibration peak at the position of CuCDs was significantly reduced. This indicates that glyphosate and CuCDs have undergone complexation. In addition, compared with CuCDs, CuCDs / glyphosate has almost lost all its catalytic activity ( Fig.14 E). This indicates that active site poisoning is the main reason for the inhibition of nanozyme activity. Glyphosate changes the electron cloud distribution state of CuCDs by binding to the active site, thus leading to a decrease in enzyme-like activity.

[0080] It is worth mentioning that a similar high pesticide selectivity was found in the CDs+H2O2+TMB system ( Fig.16 ), and also confirmed from another aspect that in addition to Cu 2+ The surface functional groups of CDs also provide specific binding sites for glyphosate as active sites. Most studies on Cu-doped nanomaterials show that Cu 2+ / Cu + It is an active site. The presence of metal active sites provides a general strategy for nanoenzyme-based detection of glyphosate. In this sense, some molecules with chelating ability can also partially inhibit catalytic activity, which can well explain why the sensor is affected by non-target pesticides. However, the difference of the present invention is that CDs and Cu work synergistically to provide multiple specific binding sites for the binding of glyphosate, achieving highly selective and sensitive detection.

[0081] Example 5

[0082] Colorimetric detection of glyphosate based on CuCDs:

[0083] When detecting glyphosate, different doses of glyphosate (10.0μL, 0.02-2.8mM), CuCDs (2.0μL, 1.0mgmL-1), and NaAc-HAc buffer system (168μL) were premixed, and TMB (10μL, 10mM) and H2O2 (10μL, 100mM) were added in sequence. After incubation at 37°C for 20min, the UV-visible spectrum and absorbance of oxTMB at 652nm were recorded, and photos were taken with a smartphone under fluorescent light to obtain the RGB values ​​of the image (red, green, and blue channels). Finally, a linear equation between the RGB value and the glyphosate concentration was established.

[0084] In order to achieve accurate on-site colorimetric detection and achieve optimal performance, the incubation time (20 min) and the concentration of CuCDs (10 μg mL-1) were studied and optimized. Fig.17 ). Under the optimal conditions, the color of the system gradually changes from blue to colorless as the concentration of glyphosate increases. Fig.18 A illustration, by recording the brightness value RGB value of the solution, a linear relationship between glyphosate concentration and RGB value was established ( Fig.18 A), the linear range is 1.00-130μM (y=1.5560x+15.3345, R2=0.9921), and the detection limit (LOD) of glyphosate by colorimetry is 0.56μM. It has a wider linear range (5-120μM) and a lower detection limit (2.66μM) than CDs alone for glyphosate detection ( Fig.16 B); Compared with the previously reported fluorescence and colorimetric detection methods (Table 1), the CuCDs-based glyphosate detection has excellent specific detection performance. Repeated analysis of 5 samples with the same glyphosate concentration obtained accurate and high-precision detection results (relative standard deviation (RSD) was 4.89%). Therefore, the constructed glyphosate colorimetric detection platform has good stability, high selectivity, good repeatability, high sensitivity, and is suitable for on-site detection. The stability and repeatability of glyphosate (20, 60, 100 μM) detection were further studied. The results showed that the relative standard deviations of intra-day (5-day parallel detection) and inter-day (5-day span) changes were 0.98-3.54% and 0.74-5.83%, respectively, with good precision.

[0085] In addition, in order to reduce pests and weeds and other multiple purposes, the combined use of pesticides is of great significance in actual agricultural activities. Therefore, the sensor should be able to accurately and selectively detect glyphosate when it coexists with other pesticides. Fig.18 As shown in B, the impact of common pesticides (methyl thiophanate, quinalphos, thiram, dichlorvos, phosmet, methyl parathion, parathion, fipronil, carbaryl, and carbendazim) on glyphosate detection is negligible. 2+ The presence of responsive chemicals such as thiram, quinalphos and methyl thiophanate will not cause significant deviations in the results, indicating that the method has good selectivity. In addition, the sensor shows high anti-interference ability to common metal ions and small molecules such as amino acids ( Fig.18 B). In general, the colorimetric method established based on the synergistic effect of CuCDs nanoenzyme surface groups and Cu is not only suitable for rapid on-site detection, but also achieves direct, highly selective and efficient detection of glyphosate.

[0086] Table 1 Comparison of CuCDs-based and other reported glyphosate detection methods

[0087]

[0088]

[0089] Experimental Example 1

[0090] Food samples (i.e., potatoes and eggplants) were purchased from a local supermarket (Changsha, China) and used for actual sample analysis. Fresh sample pieces (1.0 kg) were sprayed with different concentrations of glyphosate, mixed with ultrapure water (2 L), extracted with ultrasound for 30 min, and centrifuged (8000 r min-1) for 10 min. The supernatant was filtered with a 0.22 μm filter membrane, vacuum dried and concentrated, and re-dissolved in ultrapure water (1.0 mL). Finally, the extract (10 μL) was added to the reaction system, incubated at 37 °C for 20 min, and the image was taken under a fixed light source to obtain the image, and the RGB value was recorded to determine the glyphosate content.

[0091] The results are shown in Table 2. No glyphosate was detected in potatoes and eggplants. After spraying different concentrations of glyphosate, the spike recovery was 95.11-103.7%, RSD < 8.44%, and the results were basically consistent with the HPLC-MS analysis results, verifying that the method has good reliability, accuracy and precision. In summary, this method based on the inhibition of peroxidase activity by CuCDs provides a promising direction for the sensitive, accurate and specific detection of glyphosate in actual samples, and provides a valuable solution for ensuring food safety.

[0092] Table 2 Glyphosate spike detection results based on CuCDs nanoparticle enzyme activity inhibition method and HPLC-MS method

[0093]

[0094]

[0095] a means not detected.

[0096] In summary, the present invention has established a colorimetric sensing system based on the activity of CuCDs-type peroxidases. The pesticide glyphosate inhibits the activity of the enzyme by directly binding to the active site of the enzyme on CuCDs, thereby achieving direct, sensitive and specific detection. The source of the enzyme activity and the response mechanism of glyphosate were systematically analyzed, and it was clarified that the hydroxyl, amino, carbonyl and carboxyl groups on the surface of CuCDs synergistically act with Cu as active sites to produce ·OH, ·O2 - and 1 O2 exerts nanoenzyme activity, while glyphosate selectively binds to CuCDs, resulting in active site binding poisoning, and realizes the mechanism of specific detection by consuming ROS free radicals. The present invention not only demonstrates the great potential of metal-doped CDs in the development of nanoenzyme materials, but more importantly, clarifies the relationship between the chemical structure of CuCDs and nanoenzyme activity and analytical detection performance, which is of great significance to promote the development of peroxidase nanozymes with high catalytic activity and target molecule specificity.

Claims

1. A copper-carbon quantum dot nanozyme composition, characterized in that: Includes the following components: Poria cocos and divalent copper salt.

2. The composition according to claim 1, characterized in that The divalent copper salt includes one of copper sulfate, copper chloride, copper nitrate, or copper carbonate, preferably copper sulfate; Or, the Poria cocos includes one of Poria cocos or Polyporus umbellatus of the genus Poria cocos, preferably Poria cocos of the genus Poria cocos.

3. The method for preparing the copper-carbon quantum dot nanozyme according to any one of claims 1 to 2, characterized in that: include: After mixing Poria cocos, divalent copper salt and water, heating reaction is performed to obtain copper-carbon quantum dot nanozyme.

4. The preparation method according to claim 3, characterized in that: After the heating reaction is completed, the supernatant is filtered and dialyzed in ultrapure water, filtered again, and freeze-dried to obtain the copper-carbon quantum dot nanozyme; Preferably, the pore size of the filter membrane used in the double filtration method is 0.22 μm. Preferably, the molecular weight cut-off of the dialysis membrane used for dialysis is 450-550 Da, preferably 500 Da.

5. The preparation method according to claim 3, characterized in that: After the Poria cocos is crushed, it is passed through a 90-120 mesh sieve, preferably a 100 mesh sieve; Or, the molar ratio of the mass of Poria cocos to the divalent copper salt is: 1g: 1.5~2.5×10 -4 mmol, preferably 1g:2×10 - 4 mmol.

6. The preparation method according to claim 3, wherein the concentration of the divalent copper salt in water is 7.5 to 12.5×10 -4 mM, preferably 1.0×10 -3 mM; Alternatively, the heating reaction temperature is 170-200° C., preferably 180° C.; the heating reaction time is 2-5 h, preferably 3 h.

7. The use of the copper-carbon quantum dot nanozyme according to any one of claims 1 to 2, characterized in that: A colorimetric sensor is constructed based on the copper-carbon quantum dot nanozyme according to any one of claims 1 to 2 for detecting glyphosate.

8. A method for detecting glyphosate, characterized in that: include: (1) placing a gradient concentration of glyphosate and the copper-carbon quantum dot nanozyme according to any one of claims 1 to 2 in a buffer solution, then adding a color developer 3,3',5,5'-tetramethylbenzidine and hydrogen peroxide, incubating, photographing and obtaining an image of the mixed solution after incubation, then obtaining the RGB value of the mixed solution image, and establishing a standard curve between the glyphosate concentration and the RGB value; (2) Obtain the concentration of glyphosate in the sample to be tested based on the standard curve between glyphosate concentration and RGB value.

9. The method according to claim 8, characterized in that In one or more embodiments, the gradient concentration is 0.02 to 2.8 mM; Alternatively, the buffer solution is a NaAc-HAc buffer solution.

10. The method according to claim 8, characterized in that The concentration of copper-carbon quantum dot nanozymes in the buffer solution was 1-20 μg mL -1 , preferably 10 μg mL -1 ; Alternatively, the incubation temperature is 35-38° C., preferably 37° C.; the incubation time is 20-30 min, preferably 20 min.

Images