A method and application of detecting Salmonella typhimurium based on cobalt-platinum nanoalloy pseudoenzyme

Abstract

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

[0001] The present invention belongs to the technical field of biomedicine, and specifically relates to a method and application of detecting Salmonella typhimurium based on cobalt-platinum nano-alloy pseudo-enzyme. Background Art

[0002] Salmonella is a genus of pathogenic bacteria that poses a major threat to human health by causing foodborne infectious diseases. More than 2,000 serotypes have been identified. Among them, Salmonella Typhimurium is a major serotype that is closely related to the occurrence of human foodborne diseases due to its high virulence. Salmonella Typhimurium infects healthy people through contaminated food and through oral and fecal transmission and causes secondary infections. According to the regulations of my country's Center for Disease Control and Prevention, Salmonella must not be detected in foods such as milk powder and cheese. Therefore, in order to ensure food safety, a stable, sensitive and specific method for detecting Salmonella Typhimurium is needed.

[0003] Enzyme-linked immunosorbent assay (ELISA) is a technique 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 the color reaction, which is formed by the oxidation catalysis of the labeled natural enzyme through the substrate. This reaction determines the stability and sensitivity of the detection. However, these natural enzymes have three shortcomings: (1) they are mainly composed of proteins produced by living cells, and their preparation process is complicated; (2) the extraction equipment is expensive, resulting in price increases; (3) they are poorly stable and require strict storage conditions. Therefore, nanozymes have been studied as a viable alternative to natural enzymes.

[0004] Nanozymes are a class of nanomaterials with catalytic activity similar to that of natural enzymes, with advantages such as easy purification, high cost-effectiveness, high stability and strong environmental recovery ability. Most nanozymes are usually prepared using a large amount of expensive precious metals, such as pure platinum nanozymes, gold-platinum nanozymes, etc., which are expensive. Some nanozymes are prepared using Fe3O4 materials, but the preparation time is long and the procedures are complicated, which limits their catalytic effect and their detection applications. Cobalt-platinum nanoalloy pseudoenzymes (CoPtNPs) are composed of a cobalt core and a platinum shell. The cost of cobalt is low, and its catalytic efficiency is significantly higher than that of biotinylated peroxidase (HRP). Therefore, it is urgent to establish a method for detecting Salmonella typhimurium using immunoadsorption technology (NLISA) based on CoPtNPs materials. Summary of the invention

[0005] The purpose of the present invention is to provide a method and application of detecting Salmonella typhimurium based on cobalt-platinum nanoalloy pseudoenzyme, which can realize stable, sensitive and specific detection of Salmonella typhimurium in food samples.

[0006] To achieve the above object, the technical solution adopted by the present invention is: a method for detecting Salmonella typhimurium based on cobalt-platinum nanoalloy pseudoenzyme, comprising the following steps:

[0007] (1) One-step preparation of CoPtNPs with ultrahigh catalytic activity and biocompatibility;

[0008] (2) The surface of CoPtNPs was functionalized with a thiol-modified Salmonella DNA aptamer activated by tris(2-carboxyethyl)phosphine to obtain a CoPtNPs-Apt probe;

[0009] (3) Screening and pairing the CoPt NPs-Apt probe with a monoclonal antibody to obtain a pair of aptamers / antibodies that can achieve a sandwich reaction of Apt / Salmonella typhimurium / antibody;

[0010] (4) The CoPtNPs-Apt probe was fixed on the well plate through the sandwich recognition of Apt / Salmonella typhimurium / antibody. The CoPt NPs and CoPtNPs-Apt probe promoted the conversion of colorless tetramethylbenzidine TMB into the blue oxidation product oxTMB through efficient peroxidase-like activity. When Salmonella typhimurium was present in the sample to be tested, the sample changed from colorless to blue, and turned to yellow after being terminated by adding H2SO4, thus realizing the colorimetric and quantitative detection of Salmonella typhimurium.

[0011] Furthermore, in step (1), the CoPtNPs are synthesized in one step by a high-temperature aqueous phase method. The specific preparation method is: diacetylacetonate platinum, cobalt acetate tetrahydrate, polyvinylpyrrolidone, triethylene glycol and acetaldehyde in a mass ratio of 10:7:80:10 are added to a reaction container in sequence and mixed, and reacted at a temperature of 280 to 320°C for 0.5 to 1h to obtain CoPtNPs with cobalt as the core and platinum as the shell.

[0012] Furthermore, in step (2), the nucleotide sequences of the Salmonella DNA aptamer are shown in SEQ ID NO: 1 and SEQ ID NO: 2.

[0013] Furthermore, the specific steps of step (2) are: after the thiol-modified Salmonella DNA aptamer is mixed and activated with tri(2-carboxyethyl)phosphine, it is added to the CoPtNPs solution, mixed and centrifuged under magnetic stirring, unbound thiol-modified Salmonella DNA aptamer is removed, and the obtained precipitate is washed with deionized water and then redispersed in a PBS solution to obtain a CoPt NPs-Apt probe.

[0014] Furthermore, in step (3), the amino acid sequence of the monoclonal antibody is shown in SEQ ID NO:3.

[0015] To achieve the above object, the present invention also provides the use of the above method in a kit for immunodetection of Salmonella typhimurium, wherein the kit comprises CoPtNPs prepared by the above method, and the detection sensitivity is 3×10 3 CFU / mL.

[0016] Compared with the prior art, the present invention has the following beneficial effects:

[0017] (1) The catalytic efficiency of CoPtNPs composed of a cobalt core and a platinum shell in the present invention is significantly higher than that of HRP bioenzyme. This method has strong stability and high sensitivity for detecting Salmonella typhimurium, and the cost of cobalt materials is low, which solves the problems of poor stability and high cost of bioenzymes in existing detection methods;

[0018] (2) The structure and composite elements of CoPt NPs in the present invention make them have high catalytic activity and storage stability. Using CoPt NPs with high catalytic activity and biocompatibility, a nano-enzyme-linked immunosorbent assay (NLISA) was successfully developed to achieve sensitive, specific and stable detection of Salmonella typhimurium. The NLISA has a low detection limit for the detection of Salmonella typhimurium, with a minimum detection sensitivity of 3×10 3 CFU / mL, wide linear range, and can be used for detection in actual food samples;

[0019] (3) The CoPtNPs-based NLISA method of the present invention can promote the naked eye detection of Salmonella typhimurium and facilitate equipment-free detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 is a SEM image of CoPtNPs prepared in Example;

[0021] Figure 2 The catalytic activity characterization curves of the CoPtNPs prepared in the example to tetramethylbenzidine (TMB), (a) catalytic rate curve of CoPtNPs to TMB under different H2O2 concentrations within 20 minutes, (b) catalytic rate curve of CoPtNPs to TMB under different H2O2 concentrations within 1 minute, (c) Michaelis-Menten curve double reciprocal linear plot of CoPtNPs to H2O2;

[0022] Figure 3 This is a graph showing the stability of CoPtNPs-Apt prepared in the example after being stored at room temperature in catalyzing TMB;

[0023] Figure 4 It is the denaturing gel electrophoresis diagram of monoclonal antibodies against Salmonella;

[0024] Figure 5Schematic diagram of the principle of NLISA based on CoPtNPs for detecting Salmonella typhimurium;

[0025] Figure 6 This is a graph showing the results of detecting Salmonella by pairing two aptamers with four antibodies in the embodiment;

[0026] Figure 7 This is a graph showing the results of NLISA detecting different concentrations of Salmonella typhimurium in the example. DETAILED DESCRIPTION

[0027] The present invention is further described in detail below with reference to the accompanying drawings and specific embodiments.

[0028] All raw materials and reagents in the examples of the present application were purchased on the market or prepared according to conventional methods well known to those skilled in the art.

[0029] The preparation process of the PBS solution used in the following examples is as follows: weigh 137mmol / LNaCl, 2.7mmol / LKCl, 10mmol / LNa2HPO4, and 1.8mmol / LKH2PO4, dissolve in 800mL water, adjust the pH to 7.4, and then make up to 1L.

[0030] The DNA aptamer (Apt) sequences used in the following examples were all synthesized by Shanghai Bioengineering.

[0031] Example

[0032] A method for detecting Salmonella typhimurium based on cobalt-platinum nanoalloy pseudoenzyme comprises the following steps:

[0033] (1) One-step preparation of CoPtNPs with ultra-high catalytic activity and biocompatibility; the CoPtNPs are synthesized in one pot using a high-temperature aqueous phase method. The specific preparation method is as follows: 10 mg of acetylacetonate platinum, 7 mg of cobalt acetate tetrahydrate, 80 mg of polyvinylpyrrolidone and 10 mg of tetraethylene glycol are added to a 100 mL three-necked flask and magnetically stirred, mixed at a speed of 500 rpm, and degassed under gentle N2 to form a clear solution; under N2 superposition, 1 mL of anhydrous acetaldehyde is quickly injected, and then the solution is heated to 280°C at a rate of 10°C / min, and maintained at 280°C for 1 hour, and then cooled to room temperature. The precipitate is used as the product, 100-200 mL of acetone is added to the reaction solution, centrifuged at 8000 rpm for 10-20 min, and centrifuged twice with deionized water (14000 rpm, 10 min). The product is redispersed in deionized water for further use. The electron microscopy characterization of the prepared nanozyme material is shown in the figure below. Figure 1 As shown in the figure, it can be seen that the material has platinum as the shell and cobalt as the core, thereby improving its peroxide catalytic ability.

[0034] Evaluation of the catalytic performance of the prepared CoPtNPs: 50 μL of TMB (0.5 mM) and 50 μL of H2O2 of different concentrations (50 μM, 100 μM, 200 μM, 300 μM, 500 μM) were added to 50 μL of sodium acetate-acetate buffer (0.1 M, pH = 4.0). Then, the mixture was transferred to a 96-well plate and placed in an oven at 50 ° C for temperature equilibration for 10 min. Then, 30 μL of CoPt NPs solution (8.5 × 10 -14 M), and immediately placed in a microplate reader to record the absorbance at 652 nm at different time periods. Figure 2 As shown in Figure 2, the Michaelis constant Km of CoPt NPs nanozyme for hydrogen peroxide is 23.67 mM, and the maximum catalytic rate Vmax is 5.38 × 10 -6 M / S, the catalytic rate is Vmax divided by the molar concentration, and the maximum catalytic performance Kcat is equal to 6.33×10 7 / S, excellent catalytic performance.

[0035] Evaluation of the storage stability of the prepared CoPt NPs: The prepared CoPt NPs were stored in an aqueous solution (8.5×10 -8 M), record the storage date, take 5 μL regularly and add it to 100 μL of single-component TMB solution, transfer these mixtures to a 96-well plate, and place them in a 50°C oven for temperature equilibrium for 10 minutes, then immediately place them in a microplate reader and record the absorbance at 652 nm at different time periods. The results are shown in Figure 3 As shown, CoPtNPs retained approximately 91.8% of the initial peroxidase activity after being stored at 4°C for 5 months. In addition, after 9 months of storage at 4°C, CoPtNPs retained 77.3% of the initial peroxidase activity, indicating that CoPtNPs nanozymes exhibited good storage stability.

[0036] (2) The surface of CoPt NPs was functionalized with a thiol-modified Salmonella DNA aptamer (Apt) activated by tri(2-carboxyethyl)phosphine to obtain a CoPt NPs-Apt probe. The specific steps were as follows: 300 μL of thiol Apt (100 μM) was mixed with 300 μL of TCEP (100 μM) for activation for 30 min, and then added to 300 μL of CoPt NPs solution (8.5×10 -8M) Mixed under magnetic stirring for 3 h, centrifuged at 14000 rpm for 10 min to remove unbound thiol Apt. The precipitate (CoPtNPs-Apt)) was washed twice with deionized water and dispersed in 300 μL PBS solution to obtain CoPtNPs-Apt probe.

[0037] The nucleotide sequences of the Salmonella DNA aptamer are shown in SEQ ID NO: 1 and SEQ ID NO: 2;

[0038] Apt1:5'-C6-SH-AGTAATGCCCGGTAGTTATTCAAAGATGAGTAG GAAAAGA-3'; (SEQ IDNO:1)

[0039] Apt2:5'-C6-SH-TATGGCGGCGTCACCCGACGGGGACTTGACATTATGACAG-3'; (SEQ ID NO: 2)

[0040] (3) Screening and pairing the CoPt NPs-Apt probe with a monoclonal antibody to obtain a pair of aptamers / antibodies that can achieve a sandwich reaction of Apt / Salmonella typhimurium / antibody;

[0041] Schematic diagram of monoclonal antibody detection Figure 4 As shown. The antibody numbers are 4H10F4 (antibody 1), 6H7H6G4 (antibody 2), 8H7G2 (antibody 3), and 5F11G8 (antibody 4), a total of 4 monoclonal antibodies. First, 100 μL of monoclonal antibody at a concentration of 10 μg / ml was added to a 96-well microplate and incubated at 37°C for 1 hour. Subsequently, 200 μL of 5% BSA was added to each well for blocking for 1 hour. Then, a certain concentration of Salmonella typhimurium solution was added and incubated at 37°C for 30 minutes, and Salmonella typhimurium was adsorbed by the labeled antibody on the 96-well microplate. After washing, 100 μL of CoPtNPs-Apt (10 μg / mL) was added to the 96-well plate. After washing, 100 μL of TMB was added to react for 10 min to observe the color development. Finally, 50 μL of H2SO4 was added to each well to terminate the reaction and measure OD450. The optimal Apt and monoclonal antibody number for preparing NLISA was obtained by deducting the negative value from the positive value. The measurement results are shown in the figure. Figure 6 As shown, antibody 2 combined with Apt2 can obtain the best NLISA detection results for Salmonella typhimurium.

[0042] Preparation of the Salmonella typhimurium monoclonal antibody:

[0043] The monoclonal antibodies were prepared by the research team, and the antibody numbers were 4H10F4 (antibody 1), 6H7H6G4 (antibody 2), 8H7G2 (antibody 3), and 5F11G8 (antibody 4), a total of 4 monoclonal antibodies. The steps for preparing monoclonal antibodies include:

[0044] 1) Take 200μg of Salmonella typhimurium flagellin (Sigma, SRP8029) and mix it with an equal volume of Freund's complete adjuvant, and inject it intraperitoneally or subcutaneously at multiple points. After that, every 2-4 weeks, mix 100μg of Salmonella typhimurium flagellin with an equal volume of Freund's incomplete adjuvant, and inject it intraperitoneally or subcutaneously at multiple points for booster immunization. After 2-3 immunizations, cut off the tail to collect blood and determine the immune titer. When the titer reaches 1:100000, after sprint immunization, collect blood from the mouse's eye sockets and keep it, and take the spleen for fusion.

[0045] 2) After the mice were killed by cervical dislocation, they were soaked in 75% alcohol, and the abdominal fur was cut aseptically on a clean bench to ensure the sterility of the peritoneum and remove the spleen. Place it in a mesh in a plate containing 20mL of serum-free 1640 medium. Use two elbow droppers, one to fix the spleen and the other to scrape and press the spleen, squeeze out the spleen cells, make a spleen cell suspension, and transfer it into a centrifuge tube. Centrifuge at 1000rpm for 4-10min, and aspirate the supernatant. Add 10mL of new serum-free 1640 medium, repeat 4-5 times to wash the cells. Finally, suspend the cells with 5mL of serum-free medium to prepare component I. Take SP2 / 0 myeloma cells and count them. The ratio of SP2 / 20 to spleen cells is between 1:5-1:10. Centrifuge and remove the supernatant. Add serum-free 1640 medium (10mL) to suspend the cells, and then centrifuge 2-3 times to wash the cells. 1000rpm 4-10min. Finally, the cells were suspended in 5 mL of serum-free medium to prepare component II. Component I was mixed with component II, and PEG1450 was added to induce fusion. The cells were plated and cultured in a 96-well plate.

[0046] 3) Detection and screening: dilute the protein antigen to 2 μg / mL with coating solution, add 200 ng / well to the enzyme-linked reaction plate, incubate at 4°C overnight and block with 5% BSA, add 80-100 μL of cell culture supernatant (without changing the solution for at least 4 days) to each well in the clean bench, set up a positive control (immunized mouse serum) and a negative control (SP2 / 0 supernatant or negative mouse serum). Determine its reactivity with the antigen, and for clones with high positive values, the OD450 value is greater than 1, and the strain is selected for subcloning.

[0047] 4) Mix the cells in the positive wells by blowing and taking out 100 μL, add it to a small test tube containing 400 μL of culture medium, mix it, and count the cells. Gradient dilution. Take 180 cells and add them to 7 mL of culture medium, spread them on a 96-well plate for culture. Try to spread out single clones, culture them for 3-5 days to select single clones, and replenish the fluid when appropriate. Take out the supernatant from each single cell clone well, and test and screen. Select the positive subclones with high OD, continue subcloning, and finally obtain monoclonal antibodies that are specific and sensitive to Salmonella typhimurium. The gel results after antibody purification are as follows: Figure 4 As shown, the light chains of the four antibodies obtained are around 28 kDa, and the heavy chains are around 50 kDa. The present invention performs protein amino acid sequencing on the light chain variable region of antibody 2, and the sequencing result is shown in SEQ ID NO: 3: CHLQSWSPYRTFA.

[0048] (4) Figure 5 As shown, the CoPt NPs-Apt probe is fixed on the well plate through the sandwich recognition of Apt / Salmonella typhimurium / antibody. The CoPtNPs and CoPtNPs-Apt probe promote the conversion of colorless tetramethylbenzidine TMB into the 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, realizing the detection of Salmonella. The specific sensitivity analysis detection steps for detecting Salmonella typhimurium are as follows:

[0049] S. typhimurium was tested in PBS and skim milk (10-fold dilution) and diluted into a series of concentration gradients (0, 10 3 CFU / mL, 3×10 3 CFU / mL, 10 4 CFU / mL, 3×10 4 CFU / mL, 10 5 CFU / mL, 3×10 5 CFU / mL, 10 6 CFU / mL). First, add 100 μL of monoclonal antibody (monoclonal number 6H7H6G4) with a concentration of 10 μg / ml to a 96-well microplate, incubate at 37°C for 1 hour, add 200 μL of 5% BSA to each well for blocking for 1 hour, and prepare a detection microplate, which can be stored at -20°C for more than half a year. Incubate the diluted Salmonella typhimurium solution at 37°C for 40 minutes, wash, add 100 μL of CoPtNPs-Apt (10 μg / mL) to the 96 wells, wash, add 100 μL of TMB to react for 10 minutes to observe the color development, and finally add 50 μL of H2SO4 to each well to terminate the OD450 determination, thereby achieving sensitive quantitative detection. The OD450 signal is as follows: Figure 7As shown in Figure 2, the signal value obtained by this method increases with the increase of the concentration of Salmonella typhimurium, and the minimum detection sensitivity can reach 3×10 3 CFU / mL.

Claims

1. A method for detecting Salmonella typhimurium based on cobalt-platinum nanoalloy pseudoenzyme, characterized in that: The following steps are involved: (1) One-step preparation of CoPt NPs with ultrahigh catalytic activity and biocompatibility; the CoPt NPs are synthesized in one step by a high-temperature aqueous phase method, and the specific preparation method is as follows: platinum diacetylacetonate, cobalt acetate tetrahydrate, polyvinylpyrrolidone, tetraethylene glycol and acetaldehyde in a mass ratio of 10:7:80:10 are sequentially added into a reaction container and mixed, and reacted at a temperature of 280-320°C for 0.5-1h to obtain CoPt NPs with cobalt as the core and platinum as the shell; (2) The surface of CoPt NPs was functionalized with a thiol-modified Salmonella DNA aptamer activated by tri(2-carboxyethyl)phosphine to obtain a CoPt NPs-Apt probe; the specific steps are as follows: the thiol-modified Salmonella DNA aptamer was mixed and activated with tri(2-carboxyethyl)phosphine, and then added to a CoPt NPs solution, and the mixture was mixed and centrifuged under magnetic stirring to remove unbound thiol-modified Salmonella DNA aptamer, and the obtained precipitate was washed with deionized water and then redispersed in a PBS solution to obtain a CoPt NPs-Apt probe; (3) Screening and pairing the CoPt NPs-Apt probe with a monoclonal antibody to obtain a pair of aptamers / antibodies that can achieve a sandwich reaction of Apt / Salmonella typhimurium / antibody; (4) The CoPt NPs-Apt probe was fixed on the well plate through the sandwich recognition of Apt / Salmonella typhimurium / antibody. The CoPt NPs and CoPt NPs-Apt probe promoted the conversion of colorless tetramethylbenzidine TMB into the blue oxidation product oxTMB through efficient peroxidase-like activity. When Salmonella typhimurium was present in the sample to be tested, the sample changed from colorless to blue, and turned to yellow after being terminated by adding H2SO4, thus realizing the colorimetric and quantitative detection of Salmonella typhimurium.

2. The method for detecting Salmonella typhimurium based on cobalt-platinum nanoalloy pseudoenzyme according to claim 1, characterized in that: In step (2), the nucleotide sequences of the Salmonella DNA aptamer are shown in SEQ ID NO: 1 and SEQ ID NO:

2.

3. The method for detecting Salmonella typhimurium based on cobalt-platinum nanoalloy pseudoenzyme according to claim 1, characterized in that: In step (3), the amino acid sequence of the monoclonal antibody is shown in SEQ ID NO:

3.

4. Use of the method according to any one of claims 1 to 3 in a kit for immunodetection of Salmonella typhimurium, wherein the kit comprises the CoPt NPs prepared in claim 1, and the detection sensitivity is 3×10 3 CFU / mL.

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