CN118858624B - A method and application of detecting Salmonella typhimurium based on cobalt-platinum nanoalloy pseudoenzyme - Google Patents
A method and application of detecting Salmonella typhimurium based on cobalt-platinum nanoalloy pseudoenzyme Download PDFInfo
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- 241000293869 Salmonella enterica subsp. enterica serovar Typhimurium Species 0.000 title claims abstract description 45
- 238000000034 method Methods 0.000 title claims abstract description 31
- GUBSQCSIIDQXLB-UHFFFAOYSA-N cobalt platinum Chemical compound [Co].[Pt].[Pt].[Pt] GUBSQCSIIDQXLB-UHFFFAOYSA-N 0.000 title claims abstract description 11
- 238000001514 detection method Methods 0.000 claims abstract description 26
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56911—Bacteria
- G01N33/56916—Enterobacteria, e.g. shigella, salmonella, klebsiella, serratia
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- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
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Abstract
一种基于钴铂纳米合金拟酶检测鼠伤寒沙门氏菌的方法与应用，包括以下步骤：制备CoPt NPs；采用TECP活化巯基修饰的Apt对CoPt NPs表面进行功能化获得CoPtNPs‑Apt探针；将探针与单克隆抗体进行筛选配对，获得一对适配体/抗体；将CoPt NPs‑Apt探针通过Apt/鼠伤寒杆菌/抗体的三明治型识别固定在孔板上，CoPtNPs和CoPt NPs‑Apt探针通过高效的过氧化物酶样活性来促进无色四甲基联苯胺TMB转化为蓝色氧化产物oxTMB；当待测样本中有鼠伤寒沙门氏菌时，由无色向蓝色的变化，实现对沙门氏菌的检测。该方法可实现稳定、灵敏、特异的食品样品中的鼠伤寒沙门氏菌的检测。
A method and application of detecting Salmonella typhimurium based on cobalt-platinum nano-alloy pseudo-enzyme, comprising the following steps: preparing CoPt NPs; using TECP activated thiol-modified Apt to functionalize the surface of CoPt NPs to obtain CoPtNPs-Apt probes; screening and pairing the probes with monoclonal antibodies to obtain a pair of aptamers/antibodies; immobilizing the CoPt NPs-Apt probes on a well plate through Apt/Salmonella typhimurium/antibody sandwich recognition, and CoPtNPs and CoPt NPs-Apt probes promote the conversion of colorless tetramethylbenzidine TMB into a blue oxidation product oxTMB through efficient peroxidase-like activity; when there is Salmonella typhimurium in the sample to be tested, the color changes from colorless to blue to achieve detection of Salmonella. This method can achieve stable, sensitive and specific detection of Salmonella typhimurium in food samples.
Description
Technical Field
The invention belongs to the technical field of biological medicine, and particularly relates to a method for detecting salmonella typhimurium based on cobalt-platinum nano alloy pseudoenzyme and application thereof.
Background
Salmonella is a pathogenic genus that poses a major threat to human health by causing food borne infections. More than 2000 serotypes have been identified. Among them, salmonella typhimurium is a major serotype, which is closely related to the occurrence of human food-borne diseases due to its high virulence. Salmonella typhimurium is transmitted through contaminated foods and through the oral and fecal pathways to infect healthy people and cause secondary infections. Salmonella cannot be detected in food such as milk powder, cheese and the like according to the regulations of the disease control center in China. Therefore, in order to ensure food safety, a stable, sensitive and specific salmonella typhimurium detection method is needed.
The enzyme-linked immunosorbent assay (ELISA) is a technology for detecting salmonella typhimurium based on the basic principle of specific antigen-antibody binding reaction, the detection concentration of salmonella typhimurium is determined by the intensity of a chromogenic reaction formed by the oxidation catalysis of a labeled natural enzyme by a substrate, and the reaction determines the stability and sensitivity of detection. However, these natural enzymes have three disadvantages of (1) being mainly composed of proteins produced by living cells, which are complicated in preparation process, (2) high in cost of extraction equipment, which results in an increase in price, and (3) poor stability, which requires strict storage conditions. Thus, nanoezymes have been investigated as viable alternatives to natural enzymes.
The nano-enzyme is a nano-material with catalytic activity similar to that of natural enzyme, and has the advantages of easy purification, high cost efficiency, high stability, strong environment restoration capability and the like. Most nanoenzymes are typically prepared using large amounts of expensive precious metals, such as pure platinum nanoenzymes, gold platinum nanoenzymes, etc., which are expensive. Part of the nano-enzymes are prepared from Fe 3O4 materials, but the preparation time is long, the procedure is complex, the catalytic effect of the nano-enzymes is limited, and the detection application of the nano-enzymes is limited. The cobalt-platinum nano-alloy pseudoenzyme (CoPtNPs) consists of a cobalt core and a platinum shell, the cobalt cost is lower, and the catalytic efficiency is obviously higher than that of the biotinylated peroxidase (HRP). Therefore, there is an urgent need to establish a method for detecting salmonella typhimurium based on the immunoadsorption technology (NLISA) of CoPtNPs materials.
Disclosure of Invention
The invention aims to provide a method for detecting salmonella typhimurium based on cobalt-platinum nano-alloy pseudoenzyme and application thereof, and the method can realize stable, sensitive and specific detection of salmonella typhimurium in food samples.
In order to achieve the aim, the technical scheme adopted by the invention is that the method for detecting salmonella typhimurium based on cobalt-platinum nano alloy pseudoenzyme comprises the following steps:
(1) CoPtNPs with ultrahigh catalytic activity and biocompatibility is prepared by a one-step method;
(2) Performing functionalization on the surface of CoPtNPs by adopting a tris (2-carboxyethyl) phosphine activated sulfhydryl modified salmonella DNA aptamer to obtain a CoPtNPs-Apt probe;
(3) Screening and pairing CoPt NPs-Apt probes and monoclonal antibodies to obtain a pair of aptamer/antibodies capable of realizing sandwich type reaction of Apt/typhimurium/antibodies;
(4) The method comprises the steps of fixing CoPtNPs-Apt probes on a pore plate through sandwich recognition of Apt/typhimurium bacillus/antibody, enabling CoPt NPs and CoPtNPs-Apt probes to promote conversion of colorless tetramethylbenzidine TMB into a blue oxidation product oxTMB through efficient peroxidase-like activity, enabling a sample to be detected to change from colorless to blue when salmonella typhimurium exists in the sample to be detected, enabling the sample to be detected to change into yellow after H 2SO4 is added, and achieving colorimetric and quantitative detection of salmonella typhimurium.
In the step (1), the CoPtNPs is synthesized by a one-pot method by adopting a high-temperature water phase method, and the specific preparation method comprises the steps of sequentially adding platinum diacetylacetone, cobalt acetate tetrahydrate, polyvinylpyrrolidone, tetraethylene glycol trisulfide and acetaldehyde with the mass ratio of 10:7:80:10 into a reaction vessel, mixing, and reacting for 0.5-1 h at the temperature of 280-320 ℃ to obtain CoPtNPs with cobalt as a core and platinum as a shell.
Further, in the step (2), the nucleotide sequence of the salmonella DNA aptamer is shown as SEQ ID NO. 1 and SEQ ID NO. 2.
Further, the specific step of the step (2) is that after the thiol-modified salmonella DNA aptamer is mixed and activated with tris (2-carboxyethyl) phosphine, the mixture is added into CoPtNPs solution, the mixture is mixed and centrifuged under magnetic stirring, unbound thiol-modified salmonella DNA aptamer is removed, and the obtained precipitate is washed by deionized water and then is redispersed in PBS solution to obtain the CoPt NPs-Apt probe.
Further, in the step (3), the amino acid sequence of the monoclonal antibody is shown as SEQ ID NO. 3.
In order to achieve the above purpose, the invention also provides application of the method in a kit for immunodetection of salmonella typhimurium, wherein the kit comprises CoPtNPs prepared by the method, and the detection sensitivity is 3×10 3 CFU/mL.
Compared with the prior art, the invention has the following beneficial effects:
(1) The catalytic efficiency of CoPtNPs consisting of cobalt core and platinum shell is obviously higher than that of HRP biological enzyme, the method has strong stability and high sensitivity for detecting salmonella typhimurium, the cobalt material cost is low, and the problems of poor stability and high biological enzyme cost in the existing detection method are solved;
(2) The structure and the composite elements of the CoPt NPs in the invention lead the CoPt NPs to have higher catalytic activity and storage stability, and a nano enzyme-linked immunosorbent technique (NLISA) is successfully developed by utilizing CoPtNPs with high catalytic activity and biocompatibility so as to realize the sensitivity, specificity and stability detection of the salmonella typhimurium, the NLISA has lower detection limit on the detection of the salmonella typhimurium, the lowest detection sensitivity can reach 3X 10 3 CFU/mL, and the method can be used for the detection in actual food samples;
(3) The NLISA method based on CoPtNPs can promote naked eye detection of salmonella typhimurium, and is convenient for equipment-free detection.
Drawings
FIG. 1 is an SEM image of CoPtNPs of the preparation of an example;
FIG. 2 is a graph showing the characterization of catalytic activity of CoPtNPs pairs of Tetramethylbenzidine (TMB) prepared in the examples, (a) CoPtNPs catalytic rate curves for TMB in 20 minutes at different concentrations of H 2O2, (b) CoPtNPs catalytic rate curves for TMB in 1 minute at different concentrations of H 2O2, and (c) a double reciprocal linear plot of the Mies curve for H 2O2 for CoPtNPs;
FIG. 3 is a graph showing the stability of catalytic TMB after storage at normal temperature of CoPtNPs-Apt prepared in the examples;
FIG. 4 is a denaturing gel electrophoresis of a monoclonal antibody against Salmonella;
FIG. 5 is a schematic diagram of the principle of CoPtNPs-based NLISA detection of Salmonella typhimurium;
FIG. 6 is a graph showing the results of two aptamers paired with four antibodies to detect Salmonella in the examples;
FIG. 7 is a graph showing the results of NLISA detection of various concentrations of Salmonella typhimurium in the examples.
Detailed Description
The invention is described in further detail below with reference to the drawings and the specific examples.
All starting materials reagents in the examples of the present application are commercially available or prepared according to conventional methods well known to those skilled in the art.
The PBS solution used in the following examples was prepared by weighing 137mmol/LNaCl, 2.7mmol/LKCl, 10mmpl/LNa 2HPO4、1.8mmol/LKH2PO4 in 800mL of water, adjusting pH to 7.4, and then fixing volume to 1L.
The DNA aptamer (Apt) sequences used in the examples below were all synthesized by Shanghai.
Examples
A method for detecting salmonella typhimurium based on cobalt-platinum nano-alloy pseudoenzyme comprises the following steps:
(1) CoPtNPs with ultrahigh catalytic activity and biocompatibility is prepared by a one-step method, wherein CoPtNPs is synthesized by a one-pot method by adopting a high-temperature aqueous phase method, and the preparation method comprises the steps of adding 10mg of platinum acetylacetonate, 7mg of cobalt acetate tetrahydrate, luo Wantong mg of polyethylene glycol and 10mg of tetraethylene glycol into a 100mL three-necked flask, magnetically stirring, mixing at 500rpm, and degassing under mild condition under N 2 to form a clear solution, rapidly injecting 1mL of anhydrous acetaldehyde under the superposition of N 2, heating the solution to 280 ℃ at the speed of 10 ℃ per min, keeping the temperature at 280 ℃ for 1h, and cooling to room temperature. And adding 100-200 mL of acetone into the reaction solution, centrifuging at 8000rpm for 10-20 min, and centrifuging with deionized water (14000 rpm, 10 min) for 2 times. The product was redispersed in deionized water for further use. The electron microscope characterization diagram of the prepared nano enzyme material is shown in figure 1, and the material is shown in the figure, wherein platinum is taken as a shell, and cobalt is taken as a core, so that the peroxide catalytic capability of the material is improved.
Evaluation of catalytic performance of CoPtNPs prepared 50. Mu.L TMB (0.5 mM) and 50. Mu.L H 2O2 (50. Mu.M, 100. Mu.M, 200. Mu.M, 300. Mu.M, 500. Mu.M) at different concentrations were added to 50. Mu.L sodium acetate-acetic acid buffer (0.1M, pH=4.0). These mixtures were then transferred to 96-well plates and placed in an oven at 50 ℃ for 10min for temperature equilibration. Next, 30. Mu.L of CoPt NPs solution (8.5X10 -14 M) was added to each well and immediately placed in a microplate reader to record absorbance at 652nm wavelength over different time periods. As shown in FIG. 2, the CoPt NPs nanoenzyme has a Michaelis constant Km for hydrogen peroxide equal to 23.67mM, a maximum catalytic rate Vmax equal to 5.38X10 -6 M/S, a maximum catalytic performance Kcat equal to 6.33X10 7/S by dividing Vmax by the mass concentration of the substance, and excellent catalytic performance.
Evaluation of storage stability of prepared CoPt NPs were stored in aqueous solution (8.5X10 -8 M), the date of storage was recorded, 5. Mu.L of single component TMB solution was periodically added to 100. Mu.L, these mixtures were transferred to 96 well plates and placed in an oven at 50℃for 10min for temperature equilibration, immediately placed in a microplate reader, and absorbance at 652nm wavelength was recorded for different time periods. As a result, coPtNPs retained about 91.8% of the original peroxidase activity after 5 months of storage at 4℃as shown in FIG. 3. Furthermore, coPtNPs maintained 77.3% of the initial peroxidase activity over 9 months of storage at 4 ℃, indicating that CoPtNPs nm enzyme exhibited good storage stability.
(2) The method comprises the steps of mixing 300 mu L of sulfhydryl Apt (100 mu M) with 300 mu L of TCEP (tri (2-carboxyethyl) phosphine (100 mu M) for activating for 30min, adding into 300 mu L CoPtNPs solution (8.5X10 -8 M) and mixing for 3h under magnetic stirring, centrifuging at 14000rpm for 10min, and removing unbound sulfhydryl Apt. The precipitate (CoPtNPs-Apt)) was washed 2 times with deionized water and dispersed in a 300 μ LPBS solution to obtain a CoPtNPs-Apt probe.
The nucleotide sequence of the salmonella DNA aptamer is shown as SEQ ID NO.1 and SEQ ID NO. 2;
Apt1:5’-C6-SH-AGTAATGCCCGGTAGTTATTCAAAGATGAGTAG GAAAAGA-3’;(SEQ ID NO:1)
Apt2:5’-C6-SH-TATGGCGGCGTCACCCGACGGGGACTTGACATTATGACAG-3’;(SEQ ID NO:2)
(3) Screening and pairing CoPt NPs-Apt probes and monoclonal antibodies to obtain a pair of aptamer/antibodies capable of realizing sandwich type reaction of Apt/typhimurium/antibodies;
A schematic of monoclonal antibody detection is shown in FIG. 4. The antibody numbers were 4 monoclonal antibodies 4H10F4 (antibody 1), 6H7H6G4 (antibody 2), 8H7G2 (antibody 3), and 5F11G8 (antibody 4), respectively. First, 100. Mu.L of a monoclonal antibody at a concentration of 10. Mu.g/ml was added to a 96-well microplate and incubated at 37℃for 1 hour. Subsequently, 200. Mu.L of 5% BSA was added to each well for blocking for 1 hour. Then, a salmonella typhimurium solution of a certain concentration was added and incubated at 37 ℃ for 30min, and salmonella typhimurium was adsorbed by the labeled antibody on a 96-well microplate. After washing, 100. Mu.L of CoPtNPs-Apt (10. Mu.g/mL) is added into 96 wells, 100. Mu.L of LTMB is added after washing for reaction for 10min, finally 50. Mu.L of LH 2SO4 is added into each well to stop reaction and measure OD450, the negative measured value is subtracted from the positive measured value to obtain the best Apt and monoclonal antibody number for preparing NLISA, and the best NLISA detection result for salmonella typhimurium can be obtained by matching the antibody 2 with the Apt2 as shown in FIG. 6.
The preparation of the salmonella typhimurium monoclonal antibody comprises the following steps:
monoclonal antibodies were prepared by the subject group, and the antibody numbers were 4 monoclonal antibodies in total of 4H10F4 (antibody 1), 6H7H6G4 (antibody 2), 8H7G2 (antibody 3), and 5F11G8 (antibody 4), respectively. The monoclonal antibody preparation steps comprise:
1) 200 μg of Salmonella typhimurium flagellin (Sigma, SRP 8029) was mixed thoroughly with an equal volume of Freund's complete adjuvant and injected intraperitoneally or subcutaneously at multiple points. Then, every 2-4 weeks, 100 mug of salmonella typhimurium flagellin is mixed with Freund's incomplete adjuvant in equal volume, and the mixture is injected into abdominal cavity or subcutaneous multipoint for enhancing immunity. 2-3, after the tail is broken, blood is taken out, the immune titer is measured, after the titer reaches 1:100000, the blood is taken out of the eye sockets of the mice for preservation after sprint immunization, and spleen is taken for fusion.
2) After the mice are killed by cervical vertebra, the mice are soaked in 75% alcohol, and the ultra-clean bench aseptically shears off the abdominal fur, so that the sterility of the peritoneum is ensured, and the spleens are taken out. Placed in a screen mesh in a dish containing 20mL of serum-free 1640 medium. Two elbow droppers, one for fixing spleen and one for scraping and pressing spleen, extruding spleen cells to prepare spleen cell suspension, and transferring into a centrifuge tube. Centrifuging at 1000rpm for 4-10min, and removing supernatant. New 10mL serum-free 1640 medium was added and the cell was washed 4-5 times repeatedly. Finally, the cells were suspended in 5mL serum-free medium to prepare component I. Counting SP2/0 myeloma cells, centrifuging to remove supernatant, wherein the ratio of SP2/20 to the number of spleen cells is 1:5-1:10. Cells were suspended in serum-free 1640 medium (10 mL) and centrifuged 2-3 times to wash the cells. 1000rpm for 4-10min. Finally, the cells were suspended in 5mL serum-free medium to prepare fraction II. Component I was mixed with component II and PEG1450 was added to induce fusion. Plating was performed with 96-well plates.
3) Detection screening protein antigens were diluted to 2. Mu.g/mL with coating solution, added to an ELISA plate at 200 ng/well, blocked with 5% BSA at 4℃overnight, and cell culture supernatants (at least 4 days without changing the solution) were added to the ultra-clean bench at 80-100. Mu.L per well, plus positive control (serum of immunized mice), negative control (SP 2/0 supernatant or negative mouse serum). The reactivity with antigen is measured, and the clone hole with high positive value and OD450 value greater than 1 is selected for subcloning.
4) After the cells in the positive wells were blown and mixed uniformly, 100. Mu.L of the cells were removed, and the cells were added to a small tube containing 400. Mu.L of the culture medium and mixed uniformly, and the cells were counted. And (5) gradient dilution. 180 cells were added to 7mL of medium and plated onto 96-well plates for culturing. Paving out the monoclonal as much as possible, culturing for 3-5 days, selecting the monoclonal, and timely supplementing liquid. The supernatant from each well Shan Xibao clone was removed and tested for screening. And (3) subcloning positive subcloning with high half-selected OD, and continuing subcloning to finally obtain the monoclonal antibody specifically and sensitively identified for salmonella typhimurium. The gel results after antibody purification are shown in FIG. 4, and four antibodies were obtained with light chains around 28kDa and heavy chains at 50kDa. The invention carries out protein amino acid sequencing on the light chain variable region of the antibody 2, and the sequencing result is shown as SEQ ID NO. 3: CHLQSWSPYRTFA.
(4) As shown in FIG. 5, coPt NPs-Apt probes are immobilized on an orifice plate through sandwich recognition of Apt/Salmonella typhimurium/antibody, coPtNPs and CoPtNPs-Apt probes promote the conversion of colorless tetramethylbenzidine TMB into a blue oxidation product oxTMB through efficient peroxidase-like activity, and when salmonella typhimurium exists in a sample to be detected, the change from colorless to blue is realized to detect salmonella. The specific sensitivity analysis detection steps for detecting salmonella typhimurium are as follows:
Salmonella typhimurium (10-fold dilution) was detected in PBS and skimmed milk and diluted to a series of concentration gradients (0、103CFU/mL、3×103CFU/mL、104CFU/mL、3×104CFU/mL、105CFU/mL、3×105CFU/mL、106CFU/mL).. Mu.L of monoclonal antibody (monoclonal No. 6H7H6G 4) at a concentration of 10. Mu.g/ml was first added to 96-well microwell plates, incubated at 37℃for 1H, and each well was blocked with 5% BSA 200. Mu.L for 1H to prepare detection microwell plates which were storable for more than half a year at-20 ℃. The diluted salmonella typhimurium solution is incubated for 40min at 37 ℃,100 mu L of CoPtNPs-Apt (10 mu g/mL) is added into 96 holes after washing, 100 mu L of TMB is added after washing for reaction for 10min to observe the color development condition, and finally 50 mu L H 2SO4 is added into each hole to terminate the determination of OD450, so that the sensitive quantitative detection is realized, the OD450 signal is shown as figure 7, the signal value obtained by the determination of the method is increased along with the increment of the salmonella typhimurium concentration, and the lowest detection sensitivity can reach 3X 10 3 CFU/mL.
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| CN1318152A (en) * | 1998-09-17 | 2001-10-17 | 莱比锡试验诊断有限公司 | Salmonella antigen formulation and kit for detecting salmonella antibodies |
| CN103558388A (en) * | 2013-10-24 | 2014-02-05 | 江南大学 | Double-antibody sandwich method for detecting salmonella typhimurium in food based on monoclonal antibodies |
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