Nano enzyme as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of nano materials, and particularly relates to a nano enzyme as well as a preparation method and application thereof. The nano enzyme is of a box-shaped structure composed of iron oxide and cobalt oxide, and the box-shaped structure is of a double-wall structure. The nano enzyme has peroxidase activity, good stability and high catalytic activity, and can be used as a mimic enzyme of natural peroxidase; and moreover, the compound formed based on the nano-enzyme and the pathogenic bacterium antibody is used for detecting pathogenic bacteria, the accuracy is high, the sensitivity is strong, and the pathogenic bacteria with the concentration of 100-106 CFU / mL can be accurately detected.

Description

Technical Field

[0001] The present invention belongs to the technical field of nanomaterials, and specifically relates to a nanozyme and a preparation method and application thereof. Background Art

[0002] Pathogenic bacteria (such as Brucella, Legionella pneumophila or Yersinia pestis) are a type of bacteria that can cause disease in humans or other organisms. These bacteria enter the host through various pathways, grow and multiply in the body, and produce toxins or other harmful substances, thereby causing symptoms of infection or poisoning. Early clinical diagnosis can effectively reduce the harm of pathogenic bacteria, which is of great significance to improving treatment effects and shortening the course of treatment.

[0003] At present, the gold standard for clinical identification of pathogenic bacteria is the blood culture method, which is long, time-consuming and labor-intensive. Although there are technologies such as serum agglutination test (SAT) and polymerase chain reaction (PCR) as auxiliary supplements, they also have problems such as low specificity, poor sensitivity and high cost to varying degrees. It is necessary to develop new technologies for the detection of pathogenic bacteria that are convenient, sensitive and low-cost.

[0004] Prior art can also realize specific capture and detection of bacteria by combining pathogenic bacteria recognition elements such as antibodies with enzymes, based on which a more convenient, low-cost, specific and sensitive new method for pathogenic bacteria detection can be designed. Then, the enzyme currently combined with pathogenic bacteria recognition elements such as antibodies is basically a natural enzyme, and its stability, heat resistance or acid and alkali resistance are relatively poor. As one of the natural enzyme alternative enzymes, nanozymes (generally obtained by modifying catalytic groups with chemically inert nanomaterials, having both the unique properties of nanomaterials and the high catalytic activity of natural enzymes) have higher stability, heat resistance, acid and alkali resistance, and low cost, easy modification, and have the potential to detect pathogenic bacteria after combining with pathogenic bacteria recognition elements such as antibodies. Summary of the invention

[0005] The present invention develops a nanozyme, which is a cobalt-iron oxide double-wall hollow nanobox (Co3O4 / Co-Fe oxide double-shelled nanoboxes, DSNBs), which is prepared by ZIF-67 nanocubes (NCs) to obtain ZIF-67@Co-Fe PBA yolk shell nanocubes, and then heat-treated; based on the nanozyme combined with antibodies, pathogenic bacteria can be detected.

[0006] In order to achieve the above object, the present invention can adopt the following technical solutions:

[0007] On one hand, the present invention provides a nanozyme, wherein the nanozyme is a box-like structure composed of iron oxide and cobalt oxide, and the box-like structure has a double wall.

[0008] Preferably, the nanozyme satisfies one or more of the following conditions: (a) the iron oxide is ferroferric oxide; (b) the cobalt oxide is tricobalt tetraoxide.

[0009] On the other hand, the present invention provides a method for preparing the nanozyme of the present invention, comprising: (1) mixing a ZIF-67 nanocube dispersion and a K3[Fe(CN)6] solution to obtain ZIF-67@Co-FePBA yolk shell nanocubes; and (2) heat-treating the ZIF-67@Co-FePBA yolk shell nanocubes to obtain the nanozyme.

[0010] Preferably, the above preparation method satisfies one or more of the following conditions: (i) the preparation method of ZIF-67 nanocubes comprises: mixing Co(NO3)2·6H2O, hexadecyltrimethylammonium bromide and 2-methylimidazole to obtain ZIF-67 nanocubes; (ii) the dispersant of the ZIF-67 nanocube dispersion is ethanol; (iii) the K3[Fe(CN)6] solution is an aqueous solution; (iv) the mass ratio of ZIF-67 nanocubes and K3[Fe(CN)6] is (20-30):50; (v) the heat treatment temperature is 300℃-400℃.

[0011] In another aspect, the present invention provides a nanozyme-antibody complex, comprising an antibody and the nanozyme of the present invention.

[0012] Preferably, the above-mentioned antibody is an antibody that specifically recognizes pathogenic bacteria.

[0013] On the other hand, the present invention provides a method for preparing the nanozyme-antibody complex of the present invention, comprising: mixing the antibody and the nanozyme, reacting, and enriching to obtain the nanozyme-antibody complex; wherein the antibody is an antibody with an initial concentration of 3.5 mg / mL-4 mg / mL diluted 1000-4000 times.

[0014] In another aspect, the present invention provides an enzyme-linked immunosorbent assay reagent or kit for detecting pathogenic bacteria, comprising the nanozyme-antibody complex of the present invention.

[0015] In another aspect, the present invention provides a method for detecting pathogenic bacteria for non-diagnostic purposes, comprising: (1) mixing pathogenic bacteria and the nanozyme-antibody complex of the present invention to obtain reactant 1; (2) mixing reactant 1 with TMB colorimetric solution to obtain reactant 2; (3) testing the absorbance of reactant 2; and (4) calculating the concentration of pathogenic bacteria based on a standard curve of absorbance and pathogenic bacteria concentration.

[0016] Preferably, the above method satisfies one or more of the following conditions: (a) in step (2), the pH in reactant 1 is adjusted to 3-6 before reacting; (b) in step (2), the temperature of the mixed reaction is 40°C-60°C; (c) the concentration of the nanozyme in the nanozyme-antibody complex is 0.05 mg / mL-0.06 mg / mL.

[0017] The beneficial effects of the present invention include:

[0018] (1) The nanozyme provided by the present invention has peroxidase activity, good stability, high catalytic activity, and can be used as a mimetic enzyme of natural peroxidase.

[0019] (2) Based on the complex formed by the nanozyme and the pathogenic bacteria antibody in the present invention, the pathogenic bacteria are detected with high accuracy and strong sensitivity, and the concentration of 100 CFU / mL-10 6 CFU / mL of pathogenic bacteria. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 Transmission electron microscopy and X-ray diffraction characterization results of NCs;

[0021] Figure 2 Transmission electron microscopy and X-ray diffraction characterization results of YSNCs;

[0022] Figure 3 The results of transmission electron microscopy and X-ray diffraction characterization of DSNBs;

[0023] Figure 4 is the absorption spectrum of nanozyme;

[0024] Figure 5 The absorbance spectra of the substrate catalyzed by the nanozyme under different pH conditions;

[0025] Figure 6 The absorbance spectra of the substrate catalyzed by the nanozyme under different temperature conditions;

[0026] Figure 7 The absorbance spectrum of the substrate catalyzed by the nanozyme under different concentration conditions;

[0027] Figure 8 It is the specific test result of Brucella antibody ELISA;

[0028] Fig. 9 The absorbance spectra of the substrate catalyzed by the complex composed of Brucella antibody and nanozyme at different concentrations;

[0029] Fig.10 The calibration curve for the detection of Brucella by nanozyme-Brucella antibody;

[0030] Fig.11 Absorbance spectra of nanozyme-Brucella antibody detection of different bacteria. DETAILED DESCRIPTION

[0031] The examples are provided to better illustrate the present invention, but the present invention is not limited to the examples. Therefore, those skilled in the art may make non-essential improvements and adjustments to the implementation scheme according to the above invention content, which still fall within the protection scope of the present invention.

[0032] The terms used herein are only used to describe specific embodiments and are not intended to limit the present disclosure. Unless the context has a significantly different meaning, expressions in the singular include expressions in the plural. As used herein, it should be understood that terms such as "include", "have", "include" are intended to indicate the presence of features, numbers, operations, components, parts, elements, materials or combinations. The terms of the present invention are disclosed in the specification, and are not intended to exclude the possibility that one or more other features, numbers, operations, components, parts, elements, materials or combinations thereof may exist or may be added. As used herein, " / " may be interpreted as "and" or "or", depending on the circumstances.

[0033] An embodiment of the present invention provides a nanozyme, wherein the nanozyme is a box-like structure composed of iron oxide and cobalt oxide, and the box-like structure has a double wall.

[0034] It should be noted that the nanozyme in the present invention is a cobalt-iron oxide double-walled hollow nanobox (Co3O4 / Co-Feoxide double-shelled nanoboxes, DSNBs), which is prepared by ZIF-67 nanocubes (NCs) to obtain ZIF-67@Co-Fe PBA yolk shell nanocubes, and then heat treated; it has peroxidase activity, good stability, high catalytic activity, and can be used as a mimetic enzyme of natural peroxidase.

[0035] In some specific examples, the nanozyme satisfies one or more of the following conditions: (a) the iron oxide is ferroferric oxide; (b) the cobalt oxide is tricobalt tetraoxide.

[0036] It should be noted that the iron oxide in the present invention may preferably be ferrosoferric oxide, and the cobalt oxide may preferably be cobaltous oxide; the enzymes selected from the above oxides have better catalytic activity.

[0037] The embodiment of the present invention also provides a method for preparing the nanozyme of the present invention, comprising: (1) mixing a ZIF-67 nanocube dispersion and a K3[Fe(CN)6] solution to obtain ZIF-67@Co-FePBA yolk shell nanocubes; (2) heat treating the ZIF-67@Co-FePBA yolk shell nanocubes to obtain the nanozyme.

[0038] In some specific examples, the above preparation method satisfies one or more of the following conditions:

[0039] (i) A method for preparing ZIF-67 nanocubes comprises: mixing Co(NO3)2·6H2O, hexadecyltrimethylammonium bromide and 2-methylimidazole to prepare ZIF-67 nanocubes; specifically, the ZIF-67 nanocubes in the present invention can be existing commercially available ones, or can be prepared according to the above scheme, preferably prepared according to the above method, and the activity of the prepared enzyme is better;

[0040] (ii) the dispersant of the ZIF-67 nanocube dispersion is ethanol; specifically, ZIF-67 nanocubes are generally dispersed using an organic solvent, such as ethanol;

[0041] (iii) K3[Fe(CN)6] solution is an aqueous solution;

[0042] (iv) the mass ratio of ZIF-67 nanocubes to K3[Fe(CN)6] is (20-30):50, such as 22:50, 25:50 or 27:50;

[0043] (v) The heat treatment temperature is 300°C-400°C, such as 330°C, 350°C or 380°C. In addition, the heat treatment time can be more than 2 hours, such as 2 hours, 3 hours or 5 hours.

[0044] An embodiment of the present invention also provides a nanozyme-antibody complex, comprising an antibody and the nanozyme of the present invention.

[0045] It should be noted that the nanozyme in the present invention has peroxidase activity that catalyzes H2O2 to oxidize TMB to develop a blue color, so by combining it with an antibody, the substance specifically recognized by the antibody, such as an antigen, can be detected through the antibody's specific recognition ability and the fluorescence changes of the enzyme and the color developer.

[0046] In some specific examples, the above antibodies are antibodies that specifically recognize pathogenic bacteria.

[0047] It should be noted that the antibody in the present invention can be an antibody that specifically recognizes pathogenic bacteria, such as Brucella, Legionella pneumophila or Yersinia pestis and other bacterial antibodies; the antibody can be homemade or commercially available.

[0048] An embodiment of the present invention also provides a method for preparing the nanozyme-antibody complex of the present invention, comprising: mixing the antibody and the nanozyme, reacting, and enriching to obtain the nanozyme-antibody complex; wherein the antibody is an antibody with an initial concentration of 3.5 mg / mL-4 mg / mL diluted 1000-4000 times.

[0049] It should be noted that the diluent for diluting the above-mentioned antibody is PBS (0.1M, pH=7.2); in addition, when the antibody is diluted 1000-4000 times, the antibody obtained can ensure the peroxidase activity of the nanozyme-Brucella monoclonal antibody, so the concentration of the antibody is 1:1000-1:4000.

[0050] An embodiment of the present invention also provides an enzyme-linked immunosorbent assay reagent or kit for detecting pathogenic bacteria, comprising the nanozyme-antibody complex of the present invention.

[0051] It should be noted that based on the specific recognition ability of the antibody in the nanozyme-antibody complex and the catalytic activity of the nanozyme and the color developer, the nanozyme-antibody complex can be prepared into an enzyme-linked immunosorbent assay reagent or a kit with other auxiliary reagents to achieve the detection of pathogenic bacteria; other auxiliary reagents include color developers or blocking buffers, etc.; in addition, the form of the kit is also a form known in the art.

[0052] An embodiment of the present invention also provides a method for detecting pathogenic bacteria for non-diagnostic purposes, comprising: (1) mixing pathogenic bacteria and the nanozyme-antibody complex of the present invention to obtain reactant 1; (2) mixing reactant 1 with TMB colorimetric solution to obtain reactant 2; (3) testing the absorbance of reactant 2; (4) calculating the concentration of pathogenic bacteria based on a standard curve of absorbance and pathogenic bacteria concentration.

[0053] It should be noted that based on the nanozyme-antibody complex of the present invention, quantitative detection of pathogenic bacteria can be achieved. In addition to diagnosing diseases through the detection of pathogenic bacteria, it can also be used to detect the content of pathogenic bacteria in the environment, such as Brucella in water.

[0054] In some specific examples, the above method satisfies one or more of the following conditions:

[0055] (a) In step (2), the pH of reactant 1 is adjusted to 3-6 before reacting; specifically, the nanozyme of the present invention has a higher catalytic activity at a pH of 3-6, especially at a pH of 4, the catalytic activity is higher than that at other pHs;

[0056] (b) In step (2), the temperature of the mixed reaction is 40°C-60°C, such as 45°C, 50°C or 55°C; specifically, the nanozyme of the present invention has a higher catalytic activity at 40°C-60°C, especially at 50°C, the catalytic activity is higher than other temperatures;

[0057] (c) The concentration of nanozyme in the nanozyme-antibody complex is 0.05 mg / mL-0.06 mg / mL.

[0058] It should be noted that in the above preparation method, it is preferred that conditions (a) and (b) are satisfied simultaneously, that is, the catalytic activity is highest when the pH is 5 and the temperature is 50°C.

[0059] In order to better understand the present invention, the content of the present invention is further explained below in conjunction with specific examples, but the content of the present invention is not limited to the following examples.

[0060] In the following examples, Brucella (Bovine Brucella abortus, vaccine strain 104M) was provided by Beijing Institute of Biology, Staphylococcus aureus (ATCC25923) and Acinetobacter baumannii (ATCC17978) were from National Engineering Technology Research Center for Immunobiological Products, and Staphylococcus epidermidis was provided by Banan District People's Hospital of Chongqing; the preparation of TSA culture medium includes: 30g of tryptone soy broth culture medium, 15g of agar powder, ultrapure water added to 1L, and autoclaved at 121°C for 25min; TBST buffer is 1×PBS+0.5% Tween-20 by volume; the diluent is 1×PBS+0.1% Tween-20 by volume, 1mg / mL BSA; the blocking buffer is PBSTBA, 1×PBS+0.05% Tween-20 by volume+1% BSA by mass.

[0061] 1. Preparation and characterization of nanozymes

[0062] (A) Preparation of ZIF-67 nanocubes (NCs)

[0063] 1 mol Co(NO3)2·6H2O was dissolved in 10 mL of an aqueous solution containing 5 mg of hexadecyltrimethylammonium bromide (CTAB); the solution was then quickly injected into 70 mL of an aqueous solution containing 0.05 mol of 2-methylimidazole (2-MIM), and stirred at room temperature for 40 minutes. The product was collected by centrifugation and washed several times with ethanol to obtain ZIF-67NCs; the ZIF-67NCs were characterized by transmission electron microscopy and X-ray diffraction. The results are shown in Figure 1 .

[0064] (II) Preparation of ZIF-67@Co-FePBA yolk shell nanocubes (YSNCs)

[0065] 22 mg of the ZIF-67NCs prepared above were dispersed in 15 mL of ethanol to obtain a ZIF-67NCs dispersion; 50 mg of K3[Fe(CN)6] was dissolved in 5 mL of H2O to form a transparent solution, namely, a K3[Fe(CN)6] aqueous solution; then the K3[Fe(CN)6] aqueous solution was quickly injected into the ZIF-67NCs dispersion and stirred continuously at room temperature for at least 2 hours; then, the product was collected by centrifugation and washed three times with water and ethanol to obtain YSNCs; the YSNCs were characterized by transmission electron microscopy and X-ray diffraction. The results are shown in FIG. Figure 2 .

[0066] (III) Preparation of Cobalt-Iron Oxide Double-Wall Hollow Nanoboxes (DSNBs)

[0067] The YSNCs prepared above were heat treated at 350℃ in air for 2h to obtain DSNBs. The DSNBs were characterized by transmission electron microscopy and X-ray diffraction. Figure 3 .

[0068] 2. Nanozyme DSNBs enzyme activity related tests

[0069] (I) Verification of peroxidase activity of nanozyme DSNBs

[0070] 20 μL of the chromogenic substrate TMB (3,3',5,5'-tetramethylbenzidine) aqueous solution (0.5 mM) and 20 μL of H2O2 (1 mM) aqueous solution and 100 μL of the aqueous dispersion solution (0.05 mg / mL) of the materials prepared in each step of Example 1 were respectively added to 1.86 mL of acetic acid-sodium acetate buffer solution (0.1 M, pH = 4), and TMB+H2O2 and TMB+DSNBs were used as controls, respectively. The reaction was carried out at 37°C for 3 min, and the absorption spectrum in the range of 500 nm to 800 nm was measured.

[0071] The test results are as follows Figure 4 As shown by Figure 4 It can be seen that the nanozyme DSNBs of the present invention has peroxidase activity that catalyzes H2O2 to oxidize TMB to produce a blue color, and has a maximum absorbance at 652 nm.

[0072] (II) Optimization of acidity conditions of nanozyme DSNBs

[0073] 20 μL of chromogenic substrate TMB (3,3',5,5'-tetramethylbenzidine) solution (0.5 mM) and 20 μL of H2O2 (1 mM) solution and 100 μL of DSNBs nanozyme aqueous dispersion solution (0.05 mg / mL) were added to 1.86 mL of acetic acid-sodium acetate buffer solution (0.1 M) with pH = 3, 4, 5, 6, 7 and 8, respectively, reacted at 37°C for 3 min, and the absorbance at 652 nm was measured.

[0074] Test results such as Figure 5 As shown, the results show that the nanozyme DSNBs of the present invention has the highest catalytic activity under the acidity condition of pH=4.

[0075] (III) Optimization of nanozyme catalytic temperature conditions

[0076] 20 μL of chromogenic substrate TMB (3,3',5,5'-tetramethylbenzidine) solution (0.5 mM) and 20 μL of H2O2 (1 mM) solution and 100 μL of DSNBs nanozyme dispersion solution (0.05 mg / mL) were added to 1.86 mL of acetic acid-sodium acetate buffer solution (0.1 M, pH = 4), respectively, and reacted at 20°C, 30°C, 40°C, 50°C, and 60°C for 3 min, and the absorbance at 652 nm was measured. The results are shown in Figure 6 .

[0077] Test results such as Figure 6 As shown, the results show that the nanozyme DSNBs of the present invention has the highest catalytic activity at a temperature of 50°C.

[0078] (IV) Optimization of nanozyme concentration conditions

[0079] 20 μL of chromogenic substrate TMB (3,3',5,5'-tetramethylbenzidine) solution (0.5 mM) and 20 μL of H2O2 (1 mM) solution were added to 1.86 mL of acetic acid-sodium acetate buffer solution (0.1 M, pH = 4), and 100 μL of 0.01 mg / mL, 0.02 mg / mL, 0.03 mg / mL, 0.04 mg / mL, 0.05 mg / mL and 0.06 mg / mL DSNBs nanozyme dispersion solutions were added, respectively. The reaction was carried out at 50 ° C for 3 min, and the absorbance at 652 nm was measured.

[0080] Test results such as Figure 7 As shown, the results show that the catalytic activity of the nanozyme DSNBs of the present invention increases with increasing concentration and tends to be stable at 0.05 mg / mL, which is selected as the optimal concentration condition for detecting pathogens.

[0081] 3. Preparation and effect verification of nanozyme-brucella antibody complex

[0082] (I) Brucella culture and dilution

[0083] A disposable inoculation loop was used to dip the frozen stock solution of Brucella on TSA culture medium to make four-zone lines, and cultured at 37°C for 72 hours; a single colony of Brucella was picked and placed in a conical flask containing 20 mL of TSB culture medium, and cultured at 37°C and 220 rpm for 32 hours to activate the first live bacterial solution; 200 μL of the first live bacterial solution was drawn into a conical flask containing 20 mL of TSB culture medium, placed in a constant temperature culture oscillator, and cultured at 37°C and 220 rpm for 8 hours to perform secondary activation to obtain a second live bacterial solution; the second live bacterial solution was centrifuged at 5000 rpm for 30 minutes, the supernatant was discarded, the precipitate was collected, resuspended with PBS (0.01 mol / L) and centrifuged again, washed 3 times, blown and evenly collected in a centrifuge tube.

[0084] The spectrophotometer (U-3900, HITACHI, Japan) was used for detection. PBS was used as a blank control and the wavelength was zeroed at 600 nm. The two-live bacterial solution was evenly resuspended and PBS (0.01 mol / L) was added in small amounts several times to adjust the OD600 value to 1. At this time, the bacterial solution concentration was 1.45×10 10 CFU / mL; diluted with PBS (0.01 mol / L) to the required concentration when used.

[0085] (II) ELISA verification of the specificity of Brucella antibodies (NOVUS, USA, NB100-73111, monoclonal antibody)

[0086] In a 96-well plate, except for the last row, add Brucella solution (10 8CFU / mL, 70 μL / well), overnight at 4°C; operate a plate washer to wash the 96-well plate with PBST buffer (1×PBS + 0.05% Tween-20 by volume) as the plate washer, add 300 μL / well of blocking buffer after washing, incubate at 37°C for 2h, and then wash the 96-well plate with PBST buffer; dilute Brucella antibody (NOVUS, USA, NB100-73111, monoclonal antibody) with 0.01 mol / L PBS at a ratio of 1:125 and mix well. 100 μL PBS was added to each well, and the diluted antibody was added to the first row at 200 μL / well, and 100 μL / well was added to the last row of detection wells. Starting from the first row, 1:2 dilution was performed in sequence, and the dilution was performed in sequence to the second to last row. 100 μL of liquid was discarded, incubated at 37°C for 1 hour, and the plate was washed with PBST; 100 μL (1:10000) goat anti-mouse IgG2a (HRP) diluted in PBST buffer was added to each well, incubated at 37°C for 40 minutes, and then the plate was washed with PBST; 100 μL TMB ELISA Substrate colorimetric solution was added to each well, and the 96-well plate was placed in a dark place to avoid light for 10 minutes, and 100 μL 450nm TMB ELISA Substrate stop solution was added to each well to stop the reaction, and the absorbance was detected at 450nm.

[0087] Test results such as Figure 8 As shown, the absorbance of the Brucella group was significantly higher than that of the PBS control group, and the absorbance increased with the increase of antibody concentration, indicating that there was a concentration-dependent binding ability between the Brucella antibody (NOVUS, USA, NB100-73111, monoclonal antibody) and Brucella.

[0088] (III) Preparation of Nanozyme-Brucella Antibody Complex

[0089] 10 μL of Brucella antibodies (NB100-73111, NOVUS, USA, monoclonal antibody, initial concentration of 3.9 mg / mL) (1:125, 1:250, 1:500, 1:1000, 1:2000 and 1:4000) at different dilutions (0.1 M PBS, pH = 7.2) were incubated with 100 μL of the above-prepared nanozyme DSNBs (0.05 mg / mL) at 37 ° C for 24 h, and after centrifugal enrichment, they were dispersed into 1 mL centrifuge tubes to obtain nanozyme-Brucella antibody complexes with different ratios.

[0090] In 1.86 mL of acetic acid-sodium acetate buffer solution (0.1 M, pH = 4), add 20 μL of TMB solution (0.5 mM) and 20 μL of H2O2 (1 mM) solution, and then add 100 μL of the above-mentioned nanozyme-Brucella antibody complex to react in a 50°C water bath for 3 min, and then measure the absorbance at 652 nm.

[0091] Test results such as Fig. 9 As shown, the results showed that the absorbance produced by the substrate catalyzed by the nanozyme-Brucella antibody complex decreased with the increase of antibody concentration, indicating that the bound antibody can significantly affect the peroxidase activity of the nanozyme-Brucella monoclonal antibody complex. When the concentration of Brucella monoclonal antibody is 1:1000-1:4000, the peroxidase activity of the nanozyme-Brucella monoclonal antibody can be guaranteed. In order to ensure the specific capture of Brucella by the complex, an antibody concentration of 1:1000 was selected to prepare the complex.

[0092] (IV) Quantitative detection of Brucella by enzyme-linked immunosorbent assay using nanozyme-Brucella antibody complex

[0093] 100 μL of Brucella bacteria solution of different concentrations (concentration of 10 2 CFU / mL, 10 3 CFU / mL, 10 4 CFU / mL, 10 5 CFU / mL and 10 6 CFU / mL), incubate at 4°C overnight. After the incubation is completed, operate the plate washer to wash the 96-well plate with PBST, then add 300 μL / well of blocking buffer, incubate at 37°C for 2h, and then wash the 96-well plate with PBST buffer; add 100 μL of nanozyme-Brucella antibody complex solution (nanozyme 0.05 mg / mL, antibody 1:1000) to the bacterial liquid wells respectively, incubate at 37°C for 2h, wash 5 times with washing solution, and then add 100 μL TMB (0.5 mM) color development solution to each well, react at 50°C for 3min, use the single antibody group as the blank control, measure the absorbance at 652nm and draw a calibration curve.

[0094] The results are as follows Fig.10 As shown, the linear range is 100 CFU / mL-10 6 CFU / mL, indicating that this method can accurately detect 100CFU / mL-10 6 Brucella in CFU / mL concentration range.

[0095] (V) Specificity of nanozyme-Brucella antibody complex in detecting Brucella

[0096] Brucella bacterial solution (10 8 CFU / mL), Staphylococcus aureus liquid (10 8 CFU / mL), Staphylococcus epidermidis liquid (10 8 CFU / mL), Acinetobacter baumannii liquid (10 8 CFU / mL) and Escherichia coli (10 8 CFU / mL) bacterial solution, incubate at 4°C overnight. After incubation, operate the plate washer to wash the 96-well plate with PBST buffer, then add 300 μL / well of blocking buffer, incubate at 37°C for 2h, and then wash the 96-well plate with PBST buffer; add 100 μL of nanozyme-Brucella antibody complex solution (nanozyme 0.05 mg / mL, antibody concentration is diluted 1:1000 (initial concentration is 3.9 mg / mL)) to the bacterial solution wells respectively, incubate at 37°C for 2h, wash 5 times with washing solution, then add 100 μL TMB (0.5 mM) color development solution to each well, react at 50°C for 3min, and measure the absorbance at 652nm.

[0097] The results are as follows Fig.11 As shown, the results showed that the nanozyme-Brucella antibody complex has high specificity for Brucella and can effectively avoid interference from negative bacteria.

[0098] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the purpose and scope of the technical solution of the present invention, which should be covered by the scope of the claims of the present invention.

Claims

1. A nanozyme, characterized in that: Nanozyme is a box-like structure composed of iron oxide and cobalt oxide, and the box-like structure has a double wall.

2. The nanozyme according to claim 1, characterized in that Nanozymes meet one or more of the following conditions: (a) the iron oxide is ferroferric oxide; (b) The cobalt oxide is tricobalt tetroxide.

3. The method for preparing the nanozyme according to claim 1 or 2, characterized in that: include: (1) ZIF-67 nanocube dispersion and K3[Fe(CN)6] solution were mixed to obtain ZIF-67@Co-FePBA yolk shell nanocubes; (2) Nanozymes were obtained by heat treatment of ZIF-67@Co-FePBA yolk shell nanocubes.

4. The preparation method according to claim 4, characterized in that: The preparation method meets one or more of the following conditions: (i) A method for preparing ZIF-67 nanocubes includes: mixing Co(NO3)2·6H2O, hexadecyltrimethylammonium bromide and 2-methylimidazole to prepare ZIF-67 nanocubes; (ii) The dispersant of ZIF-67 nanocube dispersion is ethanol; (iii) K3[Fe(CN)6] solution is an aqueous solution; (iv) the mass ratio of ZIF-67 nanocubes and K3[Fe(CN)6] is (20-30):50; (v) The heat treatment temperature is 300°C-400°C.

5. A nanozyme-antibody complex, characterized in that Comprising an antibody and the nanozyme according to claim 1 or 2.

6. The nanozyme-antibody complex according to claim 5, characterized in that Antibodies are antibodies that specifically recognize pathogenic bacteria.

7. The method for preparing the nanozyme-antibody complex according to claim 5 or 6, characterized in that: include: The antibody and nanozyme are mixed, reacted, and enriched to obtain a nanozyme-antibody complex; wherein the antibody is obtained by diluting the initial concentration of 3.9 mg / mL by 1000-4000 times.

8. An enzyme-linked immunosorbent assay reagent or kit for detecting pathogenic bacteria, characterized in that: Comprising the nanozyme-antibody complex described in claim 5 or 6.

9. A method for detecting pathogenic bacteria for non-diagnostic purposes, characterized in that: include: (1) mixing pathogenic bacteria and the nanozyme-antibody complex according to claim 5 or 6 to obtain a reactant 1; (2) reactant 1 is mixed with TMB color developing solution to obtain reactant 2; (3) the absorbance of reactant 2 is tested; (4) the concentration of pathogenic bacteria is calculated based on a standard curve of absorbance and pathogenic bacteria concentration.

10. The method according to claim 9, characterized in that The method meets one or more of the following conditions: (a) In step (2), the pH of the reactant 1 is adjusted to 3-6 before reacting; (b) In step (2), the temperature of the mixed reaction is 40°C-60°C; (c) The concentration of nanozyme in the nanozyme-antibody complex is 0.05 mg / mL-0.06 mg / mL.

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