CN114195179A - Method for non-diagnosis purpose double-antibody sandwich enzyme-linked immunosorbent assay of bacillus cereus - Google Patents

Method for non-diagnosis purpose double-antibody sandwich enzyme-linked immunosorbent assay of bacillus cereus Download PDF

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CN114195179A
CN114195179A CN202111243750.8A CN202111243750A CN114195179A CN 114195179 A CN114195179 A CN 114195179A CN 202111243750 A CN202111243750 A CN 202111243750A CN 114195179 A CN114195179 A CN 114195179A
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enzyme
antibody
nanoparticles
bacillus cereus
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CN114195179B (en
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徐振林
孟静南
刘英菊
沈兴
王涓
王弘
雷红涛
沈玉栋
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South China Agricultural University
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Abstract

The invention discloses a method for non-diagnosis-purpose double-resistance sandwich enzyme-linked immunoassay of bacillus cereus, which utilizes synthesized Cu/CeO2Nanoparticles as mimetic enzymes, instead of HRP, were labeled (conjugated) with secondary antibodies, which had horseradish peroxidase (HRP) activity. Furthermore, monoclonal antibodies against b.cereus were used as capture antibodies (coating antibodies), polyclonal antibodies against b.cereus were used as detection antibodies, Cu/CeO2The nanoparticle is used as a mimic enzyme labeled secondary antibody, and a sandwich ELISA analysis method of B.cereus is established, has the advantages of high sensitivity, good specificity, low cost and simple operation, and is used for rapid, high-flux and low-cost detection of the B.cereus in food.

Description

Method for non-diagnosis purpose double-antibody sandwich enzyme-linked immunosorbent assay of bacillus cereus
Technical Field
The invention relates to the technical field of detection of bacillus cereus, in particular to a method for non-diagnosis-purpose double-resistance sandwich enzyme-linked immunodetection of bacillus cereus.
Background
Bacillus cereus (b. cereus) is widely present in nature, a opportunistic pathogen that causes food poisoning with two distinct symptoms, diarrhea and vomiting. Cereus is well adapted to different environments, such as soil, plants, insects and the intestinal tract of mammals. Because of these habits, it is often separated from the food. The contamination rate of 3 kinds of foods such as milk, cooked food, vegetables and the like is reported to be higher than 30%. Food poisoning events caused by bacillus cereus seriously threaten food safety and even seriously threaten human life safety.
Currently, methods for detecting b.cereus include a culture method, a PCR method, and an immunoassay method. The culture method is a 'gold standard' method identified by B.cereus and comprises culture separation, selective enrichment, biochemical identification and serological identification, but the detection time generally needs 2-3 days. The PCR method aims at the specific fragment DNA of the genus B.cereus and has the characteristics of high sensitivity and short detection time. It often does not eliminate the complicated procedures of enrichment, cell disruption and nucleic acid extraction. And for the b. cereus population, false positives may result due to high conservation and similarity of 16S rRNA gene sequences.
Although the detection method of food-borne pathogenic bacteria is continuously developed, enzyme-linked immunosorbent assay (ELISA) is a common method for detecting microorganisms at present by virtue of simplicity, specificity, sensitivity and high flux without a large-scale instrument. It is noteworthy, however, that the ability of conventional ELISA to convert target concentrations to color changes depends on the native enzyme, e.g., HRP or ALP. However, their use is hampered by the high cost and stringent storage conditions. The nano enzyme has the potential possibility of replacing natural enzyme due to the characteristics of low cost, convenient preparation, good stability and the like.
Disclosure of Invention
The invention aims to overcome the defects in the prior art and provides a method for detecting bacillus cereus by non-diagnostic double-resistance sandwich enzyme-linked immunosorbent assay.
The first purpose of the invention is to provide Cu/CeO2And (3) nanoparticles.
The second purpose of the invention is to provide Cu/CeO2A method for preparing nanoparticles.
The third purpose of the invention is to provide Cu/CeO prepared by the preparation method2And (3) nanoparticles.
The fourth object of the invention is the Cu/CeO2The application of the nano-particles in preparing enzyme linked immunosorbent assay kits or carrying out enzyme linked immune reaction.
The fifth purpose of the invention is to provide an enzyme-labeled secondary antibody.
It is a sixth object of the present invention to provide any of the above Cu/CeO2The application of the nano-particles or the simulated enzyme-labeled secondary antibody in constructing an enzyme-linked immunosorbent assay detection method and/or an enzyme-linked immunosorbent assay kit.
It is a seventh object of the present invention to provide a method for the non-diagnostic purpose double-antibody sandwich enzyme-linked immunodetection of Bacillus cereus.
The eighth purpose of the invention is to provide a kit for double-antibody sandwich enzyme-linked immunoassay of bacillus cereus.
The invention uses Bacillus cereus B.cereus (ATCC 14579, presented by Guangdong institute of microbiology, Guangdong province) to immunize a BALB/c mouse with age of 8 weeks and a New Zealand white rabbit with age of 2.5kg respectively, and obtains monoclonal and polyclonal antibodies of the Bacillus cereus B.cereus after 5 times of normal immunization. Simultaneous synthesis of Cu/CeO2The nanoparticles serve as a mimic enzyme, which is labeled (coupled) with a secondary antibody instead of HRP, which has horseradish peroxidase (HRP) activity. Using monoclonal antibody of B.cereus as capture antibody, polyclonal antibody of B.cereus as detection antibody, Cu/CeO2The nanoparticles are used as mimic enzyme labeled secondary antibody, a sandwich ELISA analysis method of B.cereus is established, and LOD is 1.7 multiplied by 102CFU/mL，R2＝0.993。
The invention firstly claims a Cu/CeO2Nanoparticles of the Cu/CeO2The nanoparticles were prepared as follows: ce (NO)3)3·6H2O、CuCl2·2H2Regulating the pH value of the glycol solution of O and PVP by acetic acid, and then carrying out hydrothermal reaction to obtain a precursor; and calcining the precursor to obtain the catalyst.
Preferably, Ce (NO) is calculated by mass3)3·6H2O、CuCl2·2H2The dosage ratio of O to PVP is 300-500: 1 to 20: 100 to 600.
More preferably, Ce (NO)3)3·6H2O、CuCl2·2H2The dosage ratio of O and PVP is 434: 10: 300.
preferably, the pH of the reaction system is 2.0-6.0.
More preferably, the pH of the reaction system is 4.0.
More preferably, the hydrothermal reaction is carried out at 120-250 ℃ for 4-15 h.
Further preferably, the hydrothermal reaction is carried out at 160 ℃ for 8 h.
More preferably, the calcining condition is 200-500 ℃ for 1-6 h.
Further preferably, the hydrothermal reaction is carried out at 300 ℃ for 2 h.
Further claimed is a Cu/CeO2Method for preparing nanoparticles, Ce (NO)3)3·6H2O、CuCl2·2H2Carrying out hydrothermal reaction on the O and PVP glycol solution to obtain a precursor; and calcining the precursor to obtain the catalyst.
Also claimed is Cu/CeO prepared by the preparation method2And (3) nanoparticles.
The invention also claims the Cu/CeO2The application of the nano-particles in preparing an enzyme linked immunosorbent assay kit or carrying out enzyme linked immunosorbent assay.
The invention also claims an enzyme-labeled secondary antibody, which is prepared from the Cu/CeO2The nanoparticles are coupled to a secondary antibody. The Cu/CeO2Nanoparticles as mimetic enzymes, which replace HRP with secondary antibody labels (conjugated), have horseradish peroxidase (HRP) activity
Preferably, the secondary antibody is goat anti-rabbit IgG.
Any of the Cu/CeO is also claimed2The application of the nano-particles or the simulated enzyme-labeled secondary antibody in constructing an enzyme-linked immunosorbent assay detection method and/or an enzyme-linked immunosorbent assay kit.
The simulated enzyme labeled secondary antibody is used as an enzyme-labeled secondary antibody, the monoclonal antibody against the bacillus cereus is used as a coating antibody, and the polyclonal antibody against the bacillus cereus is used as a detection antibody.
Preferably, it is TMB and H2O2The mixed solution of (2) is a color developing solution.
More preferably, the method comprises the following steps:
the monoclonal antibody coats the ELISA plate;
washing the enzyme label plate;
sealing redundant sites in the holes of the enzyme label plate;
washing and drying the ELISA plate;
loading a sample to be detected on an enzyme label plate, and incubating;
washing the enzyme label plate;
loading the polyclonal antibody on an ELISA plate, and incubating;
washing the enzyme label plate;
the enzyme-labeled secondary antibody is applied to an enzyme-labeled plate and incubated;
washing the enzyme label plate;
loading the enzyme label plate with a developing solution to perform a developing reaction;
the absorbance of the sample at 652nm was measured.
More preferably, the monoclonal antibody coated ELISA plate is coated with 1.25-10.0 mug/mL of monoclonal antibody, 50-150 mug/hole, and fully reacts at 25-40 ℃.
Further preferably, the monoclonal antibody coated ELISA plate is coated with 2.5. mu.g/mL of the monoclonal antibody, 100. mu.L/well, and fully reacted at 4 ℃.
More preferably, the sample loading amount of the sample to be detected is 50-150 mu L/hole.
Even more preferably, the sample to be tested is loaded in an amount of 100. mu.L/well.
More preferably, after the sample to be detected is loaded on the ELISA plate, the incubation condition is that incubation is carried out for 30-60 min at 25-40 ℃.
Further preferably, the incubation condition is incubation for 40min at 37 ℃ after the sample to be detected is loaded on the ELISA plate.
More preferably, the loading amount of the polyclonal antibody is 50-150 μ L/well, wherein the loading amount is 0.5-16 μ g/mL.
Even more preferably, the loading of polyclonal antibody at 2.0. mu.g/mL is 100. mu.L/well.
More preferably, after the polyclonal antibody is loaded on the ELISA plate, the incubation condition is that the incubation is carried out for 30-60 min at 25-40 ℃.
Further preferably, the incubation condition is 37 ℃ for 40min after the polyclonal antibody is loaded on the ELISA plate.
More preferably, the sample loading amount of the enzyme-labeled secondary antibody is 50-150 mu L/hole, wherein the sample loading amount is 1-10 mu g/mL.
More preferably, the amount of the enzyme-labeled secondary antibody to be loaded at 5. mu.g/mL is 100. mu.L/well.
More preferably, after the enzyme-labeled secondary antibody is loaded on the enzyme-labeled plate, incubation is performed for 30-60 min at 25-40 ℃.
Further preferably, after the enzyme-labeled secondary antibody is applied to the enzyme-labeled plate, the incubation is performed at 37 ℃ for 30 min.
More preferably, the sample loading amount of the color developing solution is 50-150 mu L/hole.
More preferably, the amount of the developing solution to be applied is 100. mu.L/well.
More preferably, after the enzyme label plate is loaded with the developing solution, incubation is performed for 0-14 min at 5-45 ℃.
Further preferably, the incubation condition is 35 ℃ for 8min after the enzyme label plate is loaded with the developing solution.
More preferably, the drying ELISA plate is formed by inverting the ELISA plate and drying the plate for 0.5-2 hours at 25-40 ℃.
Further preferably, the dry ELISA plate is prepared by inverting the ELISA plate and drying at 37 deg.C for 1 h.
More preferably, the washing ELISA plate is washed by PBST for 1-5 times and 100-350 mu L/hole, and then the ELISA plate is thrown or patted dry.
Further preferably, the washing ELISA plate is washed by PBST for 2 times at 300 muL/hole, and then the ELISA plate is dried.
More preferably, the temperature of the color reaction is 5-45 ℃.
Further preferably, the temperature of the color reaction is 35 ℃.
More preferably, the pH of the color developing solution is 2-8.
Further preferably, the color developing solution has a pH of 4.
More preferably, the concentration of TMB in the color developing solution is 5-45 mM.
Further preferably, the concentration of TMB in the color developing solution is 25 mM.
More preferably, H in the developing solution2O2The concentration of (B) is 2.5 to 20 mM.
Further excellenceOptionally, H in the color development liquid2O2Is 10 mM.
More preferably, the solvent of the color developing solution is PBS buffer.
More preferably, the reaction time for color development is 0 to 14 min.
Further preferably, the reaction time for color development is 8 min.
Preferably, the monoclonal antibody is prepared as a bacillus cereus immunized mouse.
Preferably, the polyclonal antibody is prepared as a bacillus cereus immune rabbit.
Preferably, the coating concentration of the coating antibody is 1.25-10.0 mug/mL, and the concentration of the detection antibody is 0.5-16 mug/mL.
Preferably, the coating antibody is coated at a concentration of 2.5. mu.g/mL and the detection antibody is at a concentration of 2.0. mu.g/mL.
Also claimed is a kit for double-antibody sandwich enzyme-linked immunodetection of Bacillus cereus, which contains the simulated enzyme-labeled secondary antibody, the monoclonal antibody resisting the Bacillus cereus and the polyclonal antibody resisting the Bacillus cereus.
Preferably, the monoclonal antibody against Bacillus cereus is immobilized on a solid support.
Preferably, the color developing solution also contains color developing solution which is TMB and H2O2The mixed solution of (1).
More preferably, the pH of the color developing solution is 2-8.
Further preferably, the color developing solution has a pH of 4.
More preferably, the concentration of TMB in the color developing solution is 5-40 mM.
Further preferably, the concentration of TMB in the color developing solution is 25 mM.
More preferably, H in the developing solution2O2The concentration of (B) is 2.5 to 20 mM.
Further preferably, H in the developing solution2O2Is 10 mM.
More preferably, the solvent of the color developing solution is PBS buffer.
Compared with the prior art, the invention has the following beneficial effects:
the invention utilizes the synthesis of Cu/CeO2Nanoparticles as mimetic enzymes, instead of HRP, were labeled (conjugated) with secondary antibodies, which had horseradish peroxidase (HRP) activity. Furthermore, monoclonal antibodies against b.cereus were used as capture antibodies (coating antibodies), polyclonal antibodies against b.cereus were used as detection antibodies, Cu/CeO2The nanoparticle is used as a mimic enzyme labeled secondary antibody, and a sandwich ELISA analysis method of B.cereus is established, has the advantages of high sensitivity, good specificity, low cost and simple operation, and is used for rapid, high-flux and low-cost detection of the B.cereus in food.
Drawings
FIG. 1 shows antibody specificity.
FIG. 2 shows Cu/CeO2And (5) characterizing the result.
FIG. 3 shows Cu/CeO2Synthesis conditions for Cu/CeO2Influence of nanoparticle preparation.
FIG. 4 shows Cu/CeO2Steady state kinetic analysis of catalytic TMB.
FIG. 5 is a graph of ambient conditions vs. Cu/CeO2Influence of catalytic properties.
Fig. 6 is a standard curve of bacillus cereus based on a mimetic enzyme double antibody sandwich method.
Detailed Description
The present invention will be described in further detail with reference to the drawings and specific examples, which are provided for illustration only and are not intended to limit the scope of the present invention. The test methods used in the following examples are all conventional methods unless otherwise specified; the materials, reagents and the like used are, unless otherwise specified, commercially available reagents and materials.
Example 1 preparation of Bacillus cereus B.cereus antibodies
First, experiment method
1. Preparation of immunogens
After centrifugation and washing of the culture broth of bacillus cereus (ATCC 14579, presented by the institute of microbiology, guangdong province), it was inactivated with 4% (m/m) paraformaldehyde and fixed for 1h, washed with sterile physiological saline, resuspended in sterile physiological saline, and left at-80 ℃ until use.
2. Animal immunization protocol
(1) Experimental animals: 4 BALB/c mice 6-8 weeks old and 1 new Zealand white rabbit 2.5-3.0 kg were selected for immunization.
(2) Emulsification: emulsifying the immunogen and adjuvant in equal volume, wherein the immunogen of the immunized mouse and rabbit respectively contains 10 percent8And 109The individual cells, the first immunization, were emulsified with complete adjuvant and the booster immunization with incomplete adjuvant. After complete emulsification, abdominal cavity and subcutaneous multi-point injection are adopted.
(3) The immunization method comprises the following steps: mice and rabbits were immunized every two weeks according to a specific immunization protocol.
(4) And (3) detecting the serum titer: on day 7 after the completion of the third immunization, the titer of the antiserum was measured by the indirect ELISA method. Wherein, the blood is taken from the mouse through the broken tail, and the blood is taken from the rabbit through the ear vein.
The specific steps of the serum titer detection are as follows:
1) using coating liquid to treat inactivated Bacillus cereus B.cereus with the ratio of 107CFU/mL cells were diluted and plated on 96-well microtiter plates at 100. mu.L/well overnight at 37 ℃. The next day the elisa plate was removed, washed twice with PBST, and blotted dry on absorbent paper.
2) After thorough washing, the redundant sites are sealed by blocking liquid at 200 mu L/hole, and the cells are placed in an incubator at 37 ℃ for incubation for 2h and then taken out and dried for later use.
3) The mouse antiserum or the rabbit antiserum is added into an enzyme label plate through gradient dilution, meanwhile, negative control is carried out, 100 mu L/hole is incubated at 37 ℃ for 40min, and then washing and patting are carried out for 5 times.
4) HRP goat anti-mouse or goat anti-rabbit IgG at a ratio of 1:5000 was added to the ELISA plate at 100. mu.L/well, incubated at 37 ℃ for 30min, and washed and patted dry 5 times.
5) The color developing solution (TMB: h2O21:1, v/v) was added to the microplate at 100 μ L/well and incubated at 37 ℃ for 10 min. 50 μ L of 10% (v/v) H was added2SO4Terminating the reaction and using the enzymeThe absorbance at 450nm was measured by a standard meter. Wherein the color developing solutions TMB and H2O2The concentration of both is 2.5mM, and both are prepared by sodium citrate buffer solution with pH 3.50.01M, and the buffer solution is stored at 4 ℃ before use, and is mixed uniformly in a volume of 1:1 when in use.
(5) Preparing an antibody:
1) polyclonal antibodies: and (3) injecting 10% chloral hydrate into the abdominal cavity of the rabbit for anesthesia 7 days after the last boosting immunization is finished, and taking the whole blood of the rabbit to prepare rabbit antiserum.
2) Monoclonal antibodies: the intraperitoneal injection of mice with the best titer is about 10 days after the last booster immunization8And (4) carrying out impact immunization on the individual cells without any adjuvant. Spleen cells were selected for fusion with murine myeloma cells. Hybridoma cells with good specificity and high titer are screened by an indirect ELISA method (including positive and negative screening), and the screened cells are subjected to 5 rounds of limiting dilution to obtain the best antibody-producing single cell strain. And (3) carrying out clonal amplification on the obtained single cells, and injecting the single cells into the abdominal cavity of a BALB/c mouse with the age of 10-12 weeks to generate ascites.
(6) Purification and preservation of antibodies: purifying the rabbit antiserum and ascites by adopting an octanoic acid-saturated ammonium sulfate method, dialyzing to obtain a polyclonal antibody (16mg/mL) and a monoclonal antibody (10mg/mL) of bacillus cereus, measuring the concentration, and subpackaging and storing at-20 ℃.
Second, experimental results
From the seventh day after the third booster immunization, the titer test was carried out using mouse antiserum and rabbit antiserum, and the results are shown in Table 1, which shows that the titer increased with the increase in the number of immunizations. Polyclonal antibody is prepared by using rabbit antiserum, and monoclonal antibody is prepared by generating hybridoma secreting monoclonal antibody after spleen cells of mouse 2 and myeloma cells of mouse are fused.
TABLE 1 results of immunotiter
Figure BDA0003320197900000071
Example 2 Bacillus cereus B.cereus antibody specificity
First, experiment method
Inactivated bacillus cereus (10) was diluted with coating solution7CFU/mL) and strains of other species (10)8CFU/mL) (B.thuringiensis. Methicilin-reactive S.aureus, E.coli O157: H7, L.monocytogenes, V.parahaemolyticus, B.thuringiensis, B.licheniformis, B.megaterium, B.mycoides, Methicilin-reactive S.aureus, S.enteridis, S.typhiurium and S.pullorum) were then coated on 96-well plates at 100. mu.L/well overnight at 37 ℃. The next day the elisa plate was removed, washed twice with PBST, and blotted dry on absorbent paper.
The rest steps are the same as the antibody titer determination step.
Second, experimental results
The antibody dilution with the absorbance of about 1.0-1.5 is selected, the specificity of the monoclonal antibody is observed, and the result shows that when the monoclonal antibody is diluted by 32 times, the monoclonal antibody only reacts to B.cereus, and the absorbance decreases along with the increase of the dilution, which indicates that the prepared monoclonal antibody has high specificity, and the specific details are shown in figure 1.
EXAMPLE 3 monoclonal and polyclonal antibody concentrations
First, experiment method
The concentrations of the coating antibody (monoclonal antibody prepared in example 1) and the detection antibody (polyclonal antibody prepared in example 1) were optimized by a checkerboard assay, with the following specific experimental steps:
1) the coated antibody (monoclonal antibody prepared in example 1) was diluted with the coating solution to a concentration of 10, 5, 2.5, 1.25. mu.g/mL, coated on a 96-well plate, 100. mu.L/well, and left overnight at 4 ℃. The next day the elisa plate was removed, washed twice with PBST, and blotted dry on absorbent paper.
2) After sufficient washing, the redundant sites are sealed by blocking liquid at 200 mu L/hole, incubated at 37 ℃ for 2h, taken out and dried for later use.
3) The samples were released to a concentration of 10 using phosphate buffer pH 7.40.01M7CFU/mL was added to the microplate, while negative control was performed, 100. mu.L/well, incubated at 37 ℃ for 40min, and washed 5 times and patted dry.
4) The detection antibody (polyclonal antibody prepared in example 1) was diluted with phosphate buffer at pH 7.40.01M to concentrations of 16, 8, 4, 2,1, 0.5, 0. mu.g/mL and 100. mu.L/well, incubated at 37 ℃ for 40min, and washed 5 times and patted dry.
5) Goat anti-mouse or goat anti-rabbit IgG-HRP was diluted at 1:5000 using phosphate-Tween 20 buffer pH 7.40.01M, added to the microplate at 100. mu.L/well, incubated at 37 ℃ for 30min, and washed 5 times and patted dry.
6) The color developing solution (TMB: h2O21:1, v/v) was added to the microplate at 100 μ L/well and incubated at 37 ℃ for 10 min. 50 μ L of 10% (v/v) H was added2SO4The reaction was terminated and the absorbance at 450nm was measured using a microplate reader. Wherein the color developing solutions TMB and H2O2The concentration of both is 2.5mM, and both are prepared by sodium citrate buffer solution with pH 3.50.01M, and the buffer solution is stored at 4 ℃ before use, and is mixed uniformly in a volume of 1:1 when in use.
Since the natural enzyme (goat anti-mouse or goat anti-rabbit IgG) requires strong acid to stop the reaction, the TMB + is changed into TMB2+The color changed from blue to yellow, with a maximum absorbance at 450 nm.
Second, experimental results
As shown in Table 2, 4 pairs of pairs with P/N values > 8 were obtained, and the concentrations of the coating antibody (monoclonal antibody prepared in example 1) and the detection antibody (polyclonal antibody prepared in example 1) were 10, 5, 2.5 and 1.25. mu.g/mL, respectively, and 2.0. mu.g/mL, respectively. Wherein the maximum P/N values were obtained when the concentrations of the coating antibody (monoclonal antibody prepared in example 1) and the detection antibody (polyclonal antibody prepared in example 1) were 2.5. mu.g/mL and 2.0. mu.g/mL, respectively.
Table 2 checkerboard measurements:
Figure BDA0003320197900000091
EXAMPLE 4Cu/CeO2Synthesis and characterization of nanoparticles
First, experiment method
434mg of Ce (NO)3)3·6H2O，10mg CuCl2·2H2O, adding 20mL of ethylene glycol (the dosage of PVP is 100-200 mg, 300mg and 400-600 mg) into 300mg of PVP, adjusting the pH value of a reaction system to be 4.0 by acetic acid, synthesizing a precursor through hydrothermal reaction (160 ℃, 8h), and further calcining at 300 ℃ for 2h to obtain a finished product Cu/CeO2Nanoparticles.
Cu/CeO2The surface functional groups are characterized by instruments such as a Scanning Electron Microscope (SEM), a Transmission Electron Microscope (TEM), an X-ray energy spectrum analysis (EDS), X-ray diffraction (XRD), a Fourier transform infrared absorption spectrometer (FTIR), an ultraviolet-visible spectrophotometry (UV-Vis) and the like.
Second, experimental results
Cu/CeO2The characterization results of the nanoparticle finished product are shown in FIG. 3, and FIG. 2A shows Cu/CeO2Is a nano particle with the diameter of 200nm and regular and uniform shape, and the surface is rough. TEM (FIG. 2B) shows Cu/CeO2Is a hollow structure.
The characteristic peak of Cu in the EDS spectrum is obvious (figure 2C), which shows that the Cu element is successfully doped in CeO2In (1).
EDS elemental distribution plots (fig. 2D to 2G) show a uniform distribution of elements including Cu, Ce, and O.
Cu/CeO2XRD of the nanoparticles (fig. 2H) clearly showed bragg reflection angles at 28.5, 33.1, 47.5, 56.3 ° matching the (111), (200), (220), (311) crystallographic planes, which are comparable to CeO2The body centered cubic structures of (JCPDS No. 34-0394). In addition, diffraction peaks of the (222), (400) and (331) crystal planes are also evident.
In the FTIR spectrum (FIG. 2I), at 3446cm-1And 1061cm-1The absorption peak is respectively attributed to-OH stretching vibration and bending vibration, and 1618cm-1And 1382cm-1The absorption peaks at (A) are C ═ O and Ce-O-Ce stretching vibrations, respectively. Thus, FTIR spectroscopy indicated that the concentration of Cu/CeO2The surface of the nanoparticles has the presence of-COOH.
EXAMPLE 5 amount of PVP and pH of reaction System to Cu/CeO2Nano-particlesInfluence of particles
Firstly, the pH value of a reaction system is adjusted to Cu/CeO2Influence of nanoparticles
1. Experimental methods
434mg of Ce (NO)3)3·6H2O，10mg CuCl2·2H2O, adding 20mL of ethylene glycol into 300mg of PVP, adjusting the pH value of a reaction system to be 2.0, 4.0 and 6.0 by using acetic acid, synthesizing a precursor through a hydrothermal reaction (160 ℃, 8h), and further calcining at 300 ℃ for 2h to obtain a finished product Cu/CeO2And (3) nanoparticles.
2. Experimental methods
Cu/CeO2The result of synthesizing the final product of nanoparticles is shown in FIG. 3A, and when the system pH is 4, synthesized Cu/CeO2The nanoparticles are uniform in size and small in diameter (200 nm).
Second, the dosage of PVP to Cu/CeO2Influence of nanoparticles
1. Experimental methods
434mg of Ce (NO)3)3·6H2O，10mg CuCl2·2H2O, adding 20mL of ethylene glycol (the dosage of PVP is 100-200 mg, 300mg and 400-600 mg) into a certain amount of PVP, adjusting the pH value of a reaction system to be 4.0 by acetic acid, synthesizing a precursor through hydrothermal reaction (160 ℃, 8h), and further calcining at 300 ℃ for 2h to obtain a finished product Cu/CeO2And (3) nanoparticles.
2. Experimental methods
Cu/CeO2The results of the synthesized final product of nanoparticles are shown in FIG. 3B, and when PVP was added in an amount of 300mg, Cu/CeO was synthesized2The nanoparticles are complete and uniform.
EXAMPLE 6Cu/CeO2Peroxidase Activity assay of nanoparticles
First, experiment method
200 μ L1 mg/mL of Cu/CeO prepared in example 4 was monitored over 10min in a 0.01M Tris-HCl buffer at pH 4.02The nanoparticles catalyze different concentrations of 500. mu.L of TMB (0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4 and 0.45mM) and 500. mu.L of 0.1mM H2O2The change in absorbance at 652nm was produced by MichaCalculation of V from the elis-Menten equationmaxObtaining the concentration of the substrate TMB and the maximum reaction speed VmaxThe equation of the double derivatives to finally obtain the Mie constant Km。Cu/CeO2Peroxidase activity of nanoparticles by Cu/CeO2Mie's constant K of catalysis of substrate TMB by nanoparticlesmIs evaluated, wherein KmCalculated by the Michaelis-Menten equation as follows.
Figure BDA0003320197900000111
Wherein: kmValue is the Michaelis constant, VmaxIs the reaction rate when the enzyme is saturated with the substrate, [ S ]]Is the substrate concentration.
Second, experimental results
The results are shown in FIG. 4, where the steady state kinetics experiment shows Cu/CeO2Peroxidase-catalyzed processes of nanoparticles. The substrate concentration and initial velocity in the curves (FIGS. 4A and 4B) show a typical Michaelis-Menten reaction. Cu/CeO based on Lineweaver-Burk plot and with TMB as substrate2Mie constant K of nanoparticlesm0.33 mM, lower than in literature (Zhu, H., Quan, Z., Hou, H., Cai, Y., Liu, W.,&horseradish peroxidase enzyme constant (K.sub.21.068.) among Liu, Y. (2020). Acolorometric immunological based on cobalt hydroxide nanoparticles as oxidase microorganisms for detection of ochratoxin A.Analytica Chimica Acta,1132,101-109.10.1016/j.aca.2020.07.068m0.43mM), indicating Cu/CeO2The nanoparticles have a higher affinity for the substrate of TMB.
EXAMPLE 7 Secondary antibodies with Cu/CeO2Coupling of nanoparticles
First, experiment method
10mg/mL of Cu/CeO prepared in example 42The nanoparticles were ultrasonically dispersed in 1mL Tris-HCl (0.01mol/L, pH 7.4) buffer, followed by activation of Cu/CeO with 0.4mol/L EDC and 0.1mol/L NHS2The carboxyl groups on the nanoparticles were coupled with commercial 1mg/mL goat anti-mouse IgG and the excess sites were blocked with 50. mu.L of 10% (m/m) BSA. Finish sealingAfter completion, excess unbound antibody and BSA were removed by centrifugation (6000rpm, 10min) and resuspended in 1mL Tris-HCl buffer for use.
Second, experimental results
The product was measured by UV-Vis and the results are shown in FIG. 2J. Cu/CeO2Carboxyl on the surface of the nanoparticle can be directly used for loading Ab2(commercial goat anti-mouse IgG) without modification. In FIG. 3J, Ab2Has an absorption peak at 280 nm. Cu/CeO2The nanoparticles peak at about 370 nm. Ab2(commercial goat anti-mouse IgG) with Cu/CeO2After coupling of the nanoparticles, Cu/CeO was detected2Absorption peaks of nanoparticles and proteins, confirmation of Ab2Successfully modified in Cu/CeO2Preparing Cu/CeO on the nano particles2-Ab2。
EXAMPLE 8Cu/CeO2Optimization of nanoparticle catalytic conditions
In the following experiments, the buffer system was 0.01mol/L PBS, Cu/CeO2The concentration is 5mM, the reaction system is 200 muL, 20 muL Cu/CeO2，50μL H2O2And 50. mu.L of TMB, 80. mu.L of PBS buffer, in which H2O2And TMB were both formulated in PBS buffer.
First, reaction temperature vs. Cu/CeO2Influence of catalytic Activity
1. Experimental methods
20μL Cu/CeO280 μ L of pH 6.0 PBS buffer, 50 μ L of 10mM H2O2And 50. mu.L of 10mM TMB, which were placed at 5, 10, 15, 20, 25, 30, 35, 40 ℃ for reaction for 10min, 3 in each group, and the absorption intensity at 652nm was measured at the end of the reaction.
2. Results of the experiment
The results are shown in FIG. 5A, Cu/CeO with increasing temperature2The absorption intensity of the color generated by catalyzing TMB at 652nm is gradually enhanced, the color reaches the maximum value after 35 ℃, and the absorption intensity does not rise any more when the temperature is continuously increased, so that the Cu/CeO2The temperature at which the TMB is catalyzed is preferably 35 ℃.
II, pH of reaction system to Cu/CeO2Influence of catalytic Activity
1. Experimental methods
20μL Cu/CeO280 μ L of PBS buffer at different pH (2, 3, 4, 5, 6, 7, 8), 50 μ L of 10mM H2O2And 50. mu.L of 10mM TMB, reacted at 35 ℃ for 10min, 3 in each set, and the absorption intensity at 652nm was measured at the end of the reaction.
2. Results of the experiment
The experimental result is shown in FIG. 5B, when the pH of the reaction system is 2-4, the Cu/CeO2The absorption intensity of the color generated by the catalytic TMB at 652nm is gradually increased and reaches the maximum value after the pH value is 4.0, and the absorption intensity begins to drop suddenly when the pH value is continuously increased, so that the Cu/CeO2The preferred reaction system for catalyzing TMB has a pH of 4.
III, TMB concentration vs. Cu/CeO2Influence of catalytic Activity
1. Experimental methods
20μL Cu/CeO280 μ L of pH 4 PBS buffer, 50 μ L of 10mM H2O2And 50. mu.L of TMB at different concentrations (5, 10, 15, 20, 25, 30, 35, 40mM) were placed at 35 ℃ for 10min, 3 in parallel in each group, and the absorption intensity at 652nm was measured at the end of the reaction.
2. Results of the experiment
The results of the experiment are shown in FIG. 5C, with increasing TMB concentration, Cu/CeO2The absorption intensity of the color generated by the catalytic TMB at 652nm is gradually increased and reaches the maximum value at 25mM, and the absorption intensity does not rise any more when the concentration is continuously increased, so that the Cu/CeO2A preferred reaction system for catalyzing TMB is a TMB concentration of 25 mM.
Fourth, reaction time for Cu/CeO2Influence of catalytic Activity
1. Experimental methods
20μL Cu/CeO280 μ L of pH 4 PBS buffer, 50 μ L of 10mM H2O2And 50. mu.L of 25mM TMB, reacted at 35 ℃ for various times (0, 2, 4, 6, 8, 10, 12, 14min), 3 in each group, and the absorption intensity at 652nm was measured at the end of the reaction.
2. Results of the experiment
The results of the experiment are shown in FIG. 5D, which follow the reactionIncrease in size, Cu/CeO2The absorption intensity of the color generated by the catalytic TMB at 652nm is gradually enhanced and reaches the maximum value at 8min, and the absorption intensity does not rise any more when the reaction is continued, so that the Cu/CeO2The preferred reaction time for catalysis of TMB is 8 min.
In summary, Cu/CeO2The reaction for catalyzing TMB is greatly influenced by external conditions, the obtained optimal reaction temperature is 35 ℃, the pH value is 4, the concentration of TMB is 25mM, and the reaction time is 8 min.
Example 9 determination of Bacillus cereus B.Cereus Standard Curve based on the enzyme mimetic double antibody Sandwich method
First, experiment method
1. Coating: the microplate was coated with 2.5. mu.g/mL of the monoclonal antibody prepared in example 1 at 100. mu.L/well overnight at 4 ℃.
2. Washing: the microplate was washed 2 times with PBST at 300. mu.L/well and then spin-dried.
3. And (3) sealing: the excess sites were blocked by incubation with 3% (mass fraction) of skimmed milk powder at 200. mu.L/well for 2h at 37 ℃.
4. Drying: and (3) forcibly throwing off the liquid in the ELISA plate, patting the ELISA plate on absorbent paper, and inversely drying the ELISA plate at 37 ℃ for 1 h.
5. Sample preparation: add 100. mu.L of sample per well (Bacillus cereus from 10 with PBS)8CFU/mL was initially diluted 3-fold for a total of 13 gradients, and used as a test sample, with an additional PBS blank control) and incubated at 37 ℃ for 40 min.
6. Washing: the microplate was washed 5 times with PBST, 300. mu.L/well, and patted dry on absorbent paper.
7. Detecting an antibody: 2.0. mu.g/mL of the polyclonal antibody prepared in example 1 was added to the microplate at 100. mu.L/well and incubated at 37 ℃ for 40 min.
8. Washing: the same as step 6.
9. Simulating an enzyme-labeled secondary antibody: the Cu/CeO prepared in example 72 -Ab2Add 5. mu.g/mL 100. mu.L/well of enzyme standard and incubate at 37 ℃ for 30 min.
10. Washing: the same as step 6.
11. Display deviceColor: Cu/CeO obtained on the basis of example 82The optimal condition for catalyzing the TMB reaction is to prepare the required color developing solution. The color developing solution comprises two parts: are respectively TMB and H2O2Solutions, both prepared separately with phosphate buffer pH 4.00.01M, were provided at final concentrations of 25mM and 10mM, respectively. Storing at 4 deg.C before use, mixing the two solutions at a volume of 1:1, adding into enzyme labeling plate at 100 μ L/well, incubating at 35 deg.C for 8min, and measuring the absorption intensity at 652nm after reaction.
12. And (3) determination: the absorbance of the sample at 652nm was measured.
Because, the mimic enzyme is used as a catalyst to catalyze TMB and H2O2In the reaction, the termination by strong acid can lead to the complete decomposition of the mimic enzyme, and simultaneously generate black substances, which influence the determination. The absorbance at 652nm was therefore determined.
Second, experimental results
The linear range of bacillus cereus obtained by using a double-antibody sandwich method based on mimic enzyme is 3.2 multiplied by 102To 1X 105CFU/mL，R2Please refer to fig. 6 specifically for 0.993.
Bacillus cereus B.based on mimic enzyme double antibody sandwich method LOD ═ 1.7 × 102CFU/mL (LOD is the mean absorbance of blank plus 3 times the standard deviation corresponding to antibody concentration).
Example 10 method for determining bacillus cereus b.based on mimetic enzyme double antibody sandwich method
Detection method
1. Coating: the microplate was coated with 2.5. mu.g/mL of the monoclonal antibody prepared in example 1 at 100. mu.L/well overnight at 4 ℃.
2. Washing: the microplate was washed 2 times with PBST at 300. mu.L/well and then spin-dried.
3. And (3) sealing: the excess sites were blocked by incubation with 3% (mass fraction) of skimmed milk powder at 200. mu.L/well for 2h at 37 ℃.
4. Drying: and (3) forcibly throwing off the liquid in the ELISA plate, patting the ELISA plate on absorbent paper, and inversely drying the ELISA plate at 37 ℃ for 1 h.
5. Sample preparation: mu.L of the sample to be tested was added to each well and incubated at 37 ℃ for 40 min.
6. Washing: the microplate was washed 5 times with PBST, 300. mu.L/well, and patted dry on absorbent paper.
7. Detecting an antibody: 2.0. mu.g/mL of the polyclonal antibody prepared in example 1 was added to the microplate at 100. mu.L/well and incubated at 37 ℃ for 40 min.
8. Washing: the same as step 6.
9. Simulating an enzyme-labeled secondary antibody: the Cu/CeO prepared in example 42 -Ab2Add 5. mu.g/mL 100. mu.L/well of enzyme standard and incubate at 37 ℃ for 30 min.
10. Washing: the same as step 6.
11. Color development: the color developing solution comprises two parts: are respectively TMB and H2O2Solutions, both prepared separately with phosphate buffer pH 4.00.01M, were 25mM and 10mM final concentrations, respectively. The enzyme-linked immunosorbent assay solution is stored at 4 ℃ before use, the two solutions are uniformly mixed in a volume of 1:1 when in use, added into an ELISA plate at a concentration of 100 mu L/hole, and incubated for 8min at 35 ℃.
12. And (3) determination: the absorbance of the sample at 652nm was measured.
Second, result judgment
The sample was judged to be positive by generating an absorbance at 652 nm. If the concentration of Bacillus cereus B.cereus is less than 1.7X 102CFU/mL, since the captured samples were so small as not to cause sufficient signal change, was judged negative.
Example 11 kit for determining bacillus cereus b.cereus based on mimic enzyme double-antibody sandwich method
A, make up
Monoclonal prepared in example 1, microplate, washing solution (PBST), 3% skim milk powder, polyclonal antibody prepared in example 1, Cu/CeO prepared in example 72-Ab2Color developing solution (TMB and H)2O210mM PBS at 25mM and 10mM solutions pH 4.0, respectively).
Second, use method
The same as in example 10.
Comparative example method for determining bacillus cereus by double-antibody sandwich method based on mimic enzyme
Detection method
The same as example 10, but multiple antibodies are used as coating antibodies to coat the ELISA plate, and monoclonal antibodies are used as detection antibodies to add into the ELISA plate for incubation.
Second, result judgment
No absorbance at 652nm, and could not be used for detecting B.cereus.
It should be noted that the above embodiments are only used for illustrating the technical solutions of the present invention, and not for limiting the scope of the present invention, and those skilled in the art can make other variations or modifications based on the above description and ideas, and it is not necessary or exhaustive to all embodiments. Any modification, equivalent replacement, and improvement made within the spirit and principle of the present invention should be included in the protection scope of the claims of the present invention.

Claims (10)

1. Cu/CeO2Nanoparticles, characterized in that the Cu/CeO2The nanoparticles were prepared as follows: ce (NO)3)3·6H2O、CuCl2·2H2Carrying out hydrothermal reaction on the O and PVP glycol solution to obtain a precursor; and calcining the precursor to obtain the catalyst.
2. Cu/CeO according to claim 12Nanoparticles, characterized in that, calculated on the mass, Ce (NO)3)3·6H2O、CuCl2·2H2The dosage ratio of O to PVP is 300-500: 1-20: 200 to 500.
3. Cu/CeO2A method for producing nanoparticles, characterized in that Ce (NO)3)3·6H2O、CuCl2·2H2Carrying out hydrothermal reaction on the O and PVP glycol solution to obtain a precursor; and calcining the precursor to obtain the catalyst.
4. Cu/CeO prepared by the preparation method of claim 32And (3) nanoparticles.
5. Cu/CeO according to claim 42The application of the nano-particles in preparing an enzyme linked immunosorbent assay kit or carrying out enzyme linked immunosorbent assay.
6. A simulated enzyme-labeled secondary antibody, characterized in that the enzyme-labeled secondary antibody is the Cu/CeO antibody of any one of claims 1 or 42The nanoparticles were coupled to a secondary antibody.
7. Cu/CeO according to any one of claims 1 or 42The application of the nano-particles or the simulated enzyme-labeled secondary antibody of claim 5 in constructing an enzyme-linked immunosorbent assay detection method and/or an enzyme-linked immunosorbent assay kit.
8. A method for the non-diagnostic purpose double-antibody sandwich enzyme-linked immunoassay of Bacillus cereus is characterized in that the simulated enzyme-labeled secondary antibody of claim 6 is used as an enzyme-labeled secondary antibody, a monoclonal antibody against the Bacillus cereus is used as a coating antibody, and a polyclonal antibody against the Bacillus cereus is used as a detection antibody.
9. The method of claim 8, wherein the coating antibody is coated at a concentration of 1.25 to 10.0 μ g/mL and the detection antibody is at a concentration of 0.5 to 16 μ g/mL.
10. A kit for double-antibody sandwich enzyme-linked immunodetection of Bacillus cereus is characterized by comprising the simulated enzyme-labeled secondary antibody, the monoclonal antibody resisting the Bacillus cereus and the polyclonal antibody resisting the Bacillus cereus, which are disclosed by claim 6.
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