CN113804633B - Based on Au-Fe 3 O 4 Preparation method and application of salmonella recognition immune probe of nano material - Google Patents

Based on Au-Fe 3 O 4 Preparation method and application of salmonella recognition immune probe of nano material Download PDF

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CN113804633B
CN113804633B CN202111078082.8A CN202111078082A CN113804633B CN 113804633 B CN113804633 B CN 113804633B CN 202111078082 A CN202111078082 A CN 202111078082A CN 113804633 B CN113804633 B CN 113804633B
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曾景斌
赵田雨
张雨
温聪颖
刘宏玉
崔淑华
唐仕明
崔炳文
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China University of Petroleum East China
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Abstract

The invention discloses a method based on Au-Fe 3 O 4 Preparation method and application of nano-material salmonella recognition immune probe, wherein the salmonella recognition immune probe is Au-Fe 3 O 4 Heterodimer recognition probes are gold nanoparticles and Fe 3 O 4 The nano particles are combined and covalently coupled with a salmonella monoclonal antibody, so that the recognition, capture and detection integration of salmonella can be realized; and also can utilize Au-Fe 3 O 4 The heterodimer recognition probe realizes qualitative and semi-quantitative detection of salmonella by a magnetic capture and colorimetric method, has high detection speed and high sensitivity, and can be used for detecting salmonella in milk and fetal bovine serum.

Description

Based on Au-Fe 3 O 4 Preparation method and application of salmonella recognition immune probe of nano material
Technical Field
The invention belongs to the technical field of salmonella detectionDomain, in particular to a Au-Fe based 3 O 4 A preparation method and application of a salmonella recognition immune probe of a nano material.
Background
Food safety issues are frequent in the world today, and more consumers and related departments are beginning to pay attention to food safety issues. It is estimated that six hundred million people worldwide suffer from unsafe food consumption and result in nearly forty-two thousand deaths each year, and low and medium income countries lose billion dollars in productivity and medical costs each year due to contaminated food. According to statistics of a food-borne disease outbreak database, various pathogens exist in food, including escherichia coli, salmonella, pseudomonas aeruginosa, staphylococcus aureus and the like, and the pathogens all have different degrees of threat to human health by taking food as a medium. Among them, salmonella bacteria are gram-negative bacteria, have a wide host range in nature, and are easily transmitted from animal to animal, from animal to human, and from human to human by direct or indirect routes, and have been considered as one of the most important causes of food-borne diseases worldwide. The limit control of salmonella in the current national food safety standard in China is aimed at instant pre-packaged foods: the limit requirements of salmonella in 11 instant pre-packaged foods such as meat products, aquatic products, instant egg products and the like in GB 29921-2013 are as follows: n=5, c=0, m=0 CFU (n: number of samples taken for the same batch of product; c: number of samples exceeding the maximum allowable value of m; m: limit value of acceptable level of microbial indicator).
For salmonella detection, the conventional plate colony counting and identifying method is sensitive, but has long time, complex operation and strict detection environment. Other detection methods proposed in recent years, for example: although Polymerase Chain Reaction (PCR) analysis and enzyme-linked immunosorbent assay (ELISA) improve detection efficiency and specific recognition capability to a certain extent, the problems of enrichment before detection, large-scale instrument for detection and the like still exist. Therefore, the method is difficult to realize real-time and rapid detection of salmonella.
Since food-borne pathogens are usually present in complex biological matrices at very low doses, the capture enrichment prior to detection helps to increase the sensitivity of the detection. The magnetic separation is realized by mixing the magnetic nano material connected with the specific recognition target bioaffinity molecules (antibodies and aptamers) with the complex sample containing the target, so that the magnetic material is combined with the target and the magnetic field is utilized to further achieve the purposes of separation and enrichment. Iron oxide-based Magnetic Nanoparticles (MNPs) are widely used in bacterial detection platforms due to their ability to be manipulated by magnetic fields, large specific surface area, fast kinetics in solution, etc. (Congying Wen, et al ACS appl. Mater. Interfaces,9 (2017), 9416-9425;S.Zhan,et al.J.Hanzard.Mater,274 (2014), 115-123). Although these methods can achieve rapid magnetic response and magnetic separation, magnetic nanomaterials often need to be used in combination with other methods to ultimately achieve pathogen detection.
The colorimetric method has the advantages of easy naked eye observation of color, low cost, simple operation and the like, and is more suitable for real-time and rapid detection of salmonella. In the existing salmonella colorimetric method, precious metal nano materials (gold nano particles, silver nano particles and the like) modified by antibodies are mainly used for detection, nanoparticle aggregates are formed after salmonella is combined with the precious metal nano materials, so that the absorption intensity or the absorption peak position of the salmonella is changed in an ultraviolet spectrum, and color change can be observed by naked eyes (S, wang, et al chemistry-A European Journal,16 (2010): 5600-5606;Qingmei Chen,et al.Talanta,221 (2021) 121476;Changqing Zhu,et al.Analytical Methods,8 (2016): 6560-6565). Although the colorimetric detection is realized to a certain extent by the method, the sensitivity does not meet the actual detection requirement, and the resolution of naked eyes is low, so that the colorimetric detection is only the shade change of one color.
Disclosure of Invention
The invention aims to provide a method based on Au-Fe 3 O 4 The preparation method and the application of the salmonella recognition immune probe of the nano material can be used for rapidly detecting salmonella in food by a magnetic capture and colorimetric method.
In order to achieve the above purpose, the technical solution adopted by the invention is as follows:
Au-Fe-based 3 O 4 The preparation method of the salmonella recognition immune probe of the nano material comprises the following steps:
(1) Preparation of Fe 3 O 4 Nanoparticles: adding 1-octadecene and oleic acid into a three-necked flask, stirring and mixing uniformly at 100-130 ℃, preserving heat for 15-30 min, and then injecting Fe (CO) 5 Heating to 280-300 ℃ and reacting for 1.5-2 h, bubbling with nitrogen in the whole process, and cooling to room temperature after the reaction is completed; then adding acetone for centrifugation, adding TMAH solution into the sediment product, and centrifugally separating to obtain Fe 3 O 4 Nanoparticle of Fe 3 O 4 Dispersing in deionized water to obtain Fe 3 O 4 The dispersion, designated MNPs;
(2) Preparation of Au-Fe 3 O 4 Heterodimeric nanoparticles: adding MNPs, deionized water and sodium citrate solution into a three-neck flask, stirring and mixing uniformly at 80-120 ℃, and then adding HAuCl 4 Stirring and mixing the solution continuously at 80-120 ℃ and reacting for 20-30 min; after the reaction was completed for 5min, an equal volume of HAuCl was again added 4 The solution and the sodium citrate solution with equal volume are stirred and mixed at the temperature of 80-120 ℃ and reacted for 3-6 min, and the Au-Fe is obtained after the reaction is finished 3 O 4 Heterodimeric nanoparticles, noted DBNPs; then cooling to room temperature, and placing into a refrigerator for preservation at 4 ℃ for standby;
(3) Preparation of Au-Fe 3 O 4 Heterodimer recognition probes: mixing the DBNPs prepared in the step (2) with HS-PEG-COOH, shaking at 37 ℃ for 50-60 min, then adding PBS solution with pH of 6.8 and concentration of 0.01mol/L, shaking gently at room temperature to activate carboxyl groups on the surface of the DBNPs, and incubating for 20-40 min; then, the solution was washed three times with PBS solution having a pH of 7.2 and a concentration of 0.01mol/L, and dispersed in PBS solution having a pH of 7.2 and a concentration of 0.01 mol/L; then adding salmonella monoclonal antibody and oscillating for 3-4 h, washing with PBS solution with pH of 7.2 and concentration of 0.01mmol/L to remove excessive salmonella monoclonal antibody after the reaction is completed,obtaining an immune probe which specifically recognizes salmonella, namely Au-Fe 3 O 4 Heterodimer recognition probes, designated as IDBNPs, were added to a PBS solution having a pH of 7.2 and a concentration of 0.01mol/L, and stored in a refrigerator at 4 ℃.
Further, in the step (1), 1-octadecene, oleic acid and Fe (CO) 5 The volume ratio of acetone, TMAH solution and deionized water is 10-20:1-2:0.2-0.4:10-20:10-25:10-20, the concentration of the TMAH solution is 10%, and the flow rate of nitrogen is 10-20 mL/min.
Further, in the step (2), MNPs, deionized water, sodium citrate solution and HAuCl are added 4 The volume ratio of the solution is 0.1-0.2:10-20:0.2:0.15-0.25, the mass concentration of the sodium citrate solution is 8-10 mg/mL, and the HAuCl is prepared by mixing the sodium citrate solution with the sodium citrate solution 4 The molar concentration of the solution was 10mmol/L.
Further, au-Fe obtained in the step (2) 3 O 4 The heterodimer nanoparticle is one-to-one dumbbell-structured heterodimer nanoparticle, wherein the diameter of the Au nanoparticle is 12-16 nm, and Fe 3 O 4 The diameter of the nano particles is 12-16 nm.
Another object of the present invention is to provide a Au-Fe based alloy 3 O 4 The application of the salmonella recognition immune probe of the nano material in detecting salmonella.
Further, the above applications include quantitative detection of salmonella in milk and fetal bovine serum by combining magnetic capture with uv-vis absorption spectroscopy or qualitative and semi-quantitative detection of salmonella in milk and fetal bovine serum by combining magnetic capture with colorimetry.
Further, the quantitative detection of salmonella in milk and fetal bovine serum by combining magnetic capture and ultraviolet-visible light absorption spectrum specifically comprises the following steps:
(a) Taking a milk or fetal bovine serum sample to be detected, adding an equal volume of IDBNPs into the sample, then gently shaking at 37 ℃, culturing at 37 ℃ for 30min, capturing salmonella bound with the IDBNPs by using a magnetic bracket, and separating a supernatant from a precipitate, wherein the magnetic attraction time is 10-20 min;
(b) Taking supernatant, and quantitatively detecting salmonella by using ultraviolet-visible light absorption spectrum.
Further, the qualitative and semi-quantitative detection of salmonella in milk and fetal bovine serum by combining magnetic capture and colorimetry specifically comprises the following steps:
(a) Taking a milk or fetal bovine serum sample to be detected, adding an equal volume of IDBNPs into the sample, then gently shaking at 37 ℃, culturing at 37 ℃ for 30min, capturing salmonella bound with the IDBNPs by using a magnetic bracket, and separating a supernatant from a precipitate, wherein the magnetic attraction time is 10-20 min;
(b) Taking supernatant, adding ABTS, observing the color change of the supernatant, and carrying out qualitative and semi-quantitative detection on salmonella in the sample.
Further, when the milk sample to be detected is taken in the step (a), acetic acid is added into the milk to be detected, stirring and standing are carried out until white precipitate is not generated, and supernatant is taken as the milk sample to be detected.
Further, the magnetic attraction time in the step (a) is 15min.
The beneficial effects of the invention are as follows:
(1) The Au-Fe based alloy prepared by the invention 3 O 4 The salmonella recognition immune probe of the nano material has the easy modification property of gold nano particles, LSPR property and the capturing and enriching function of the magnetic material, so that the recognition, capturing and detection integration of salmonella is realized;
(2) Au-Fe prepared by the invention 3 O 4 The salmonella identification immune probe not only can realize quantitative detection of salmonella, but also can realize qualitative and semi-quantitative detection of salmonella by a colorimetric method, and has the advantages of high detection speed, high sensitivity, low detection limit which can reach 10CFU/mL;
(3) Au-Fe prepared by the invention 3 O 4 The salmonella identification immune probe has strong anti-interference capability, simple and convenient operation and high detection speed, has high practical application value and can be used for milk by using a magnetic capture and colorimetric method to detect salmonellaAnd salmonella in fetal bovine serum.
Drawings
FIG. 1 shows Au-Fe in the present invention 3 O 4 A representation of the heterodimer, wherein (a) is Au seed -Fe 3 O 4 Transmission electron microscopy of heterodimers, (b) is Au seed -Fe 3 O 4 HAADF-STEM of heterodimer, (c) Au seed -Fe 3 O 4 A STEM element map of Au, fe, O of heterodimer and a superimposed map, (d) a TEM map of DBNPs, (e) an ultraviolet spectrogram of DBNPs;
FIG. 2 shows Au-Fe of the present invention 3 O 4 A characterization map of heterodimeric probes (IDBNPs), wherein (a) is the fluorescence spectrum (excitation wavelength 554 nm) of Cy 3-labeled IDBNPs and DBNPs; (b) fluorescence microscopy images of IDBNPs labeled with Cy 3; (c) fluorescence microscopy images of DBNPs labeled with Cy 3; (d) Is the ultraviolet visible spectrum of the IDBNPs and the color chart of the IDBNPs; (e) TEM images of IDBNPs (the inset shows the particle size distribution of IDBNPs);
FIG. 3 is a graph of the magnetic response rate of IDBNPs of this invention;
FIG. 4 is a graph showing the relationship between the detected signal values of the IDBNPs and different magnetic capture times;
FIG. 5 (a) is a graph of ultraviolet spectrum of the detection of salmonella at different concentrations by IDBNPs of the present invention, and (b) is a graph of the linear relationship between ultraviolet absorbance and salmonella concentration;
FIG. 6 is a graph showing the detection effect of IDBNPS based on ABTS and actual naked eyes;
FIG. 7 is a graph showing the effect of the IDBNPs of the present invention on detecting different bacteria, in FIG. 7, (a) is a histogram of signal values of the IDBNPs for detecting different bacteria (the inset is a corresponding ultraviolet-visible spectrum); (b) a colorimetric detection map of different species of bacteria; (c) For microscope images captured by IDBNPs, the scale is 10 μm, error line= ±sd (n=3);
FIG. 8 is a graph showing the effect of IDBNPs of the present invention on detection of Salmonella in different synthetic samples, (a) spectrum detection signal values of synthetic samples; (b) Colorimetric detection pictures of different synthetic samples, error line = ±sd (n=3).
Detailed Description
The invention provides a method based on Au-Fe 3 O 4 The invention relates to a preparation method of a salmonella recognition immune probe of a nano material and application thereof, and aims to make the purposes, the technical scheme and the effects of the invention clearer and more definite. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the invention.
Example 1
The embodiment provides a method based on Au-Fe 3 O 4 The preparation method of the salmonella recognition immune probe of the nano material specifically comprises the following steps:
(1) Preparation of Fe 3 O 4 Nanoparticles: into a three-necked flask, 20mL of 1-octadecene and 2mL of oleic acid were added, stirred and mixed uniformly at 120℃and kept at a temperature for 30min, and then 0.4mL of Fe (CO) was injected 5 Heating to 295 ℃ and reacting for 2 hours, bubbling nitrogen in the whole process, wherein the flow rate of the nitrogen is 15mL/min, and cooling to room temperature after the reaction is completed; then 20mL of acetone was added for centrifugation, and 25mL of TMAH solution (i.e., tetramethylammonium hydroxide solution, the concentration of which was 10%) was added to the precipitated product, followed by centrifugation to obtain Fe 3 O 4 Nanoparticle of Fe 3 O 4 Dispersing in 20mL deionized water to obtain Fe 3 O 4 The dispersion, designated MNPs;
(2) Preparation of Au seed -Fe 3 O 4 Heterodimeric nanoparticles: into a three-necked flask, 0.2mL of MNPs, 20mL of deionized water and 0.5mL of sodium citrate solution (the mass concentration of the sodium citrate solution is 10 mg/mL) were added, and the mixture was stirred and mixed uniformly at 100℃and then 0.25mL of HAuCl was added 4 Solution (HAuCl) 4 The molar concentration of the solution is 10 mmol/L), and the mixture is continuously stirred and mixed at 100 ℃ and reacted for 30min to obtain Au seed -Fe 3 O 4 Heterodimeric nanoparticles;
(3) At 5min intervals after the completion of the reaction in step (2), 0.25mL of HAuCl was added again 4 The solution and 0.5mL sodium citrate solution are stirred and mixed at 100 ℃ and reacted for 5min,after the reaction is finished, au-Fe is obtained 3 O 4 Heterodimeric nanoparticles, noted DBNPs; then cooling to room temperature, and placing into a refrigerator for preservation at 4 ℃ for standby;
(4) Preparation of Au-Fe 3 O 4 Heterodimer recognition probes: 15mL of DBNPs were mixed with 20mg of HS-PEG-COOH (mercapto-polyethylene glycol-carboxylic acid) and shaken at 37℃for 60min, then PBS solution with pH of 6.8 and concentration of 0.01mol/L was added, and the mixture was gently shaken at room temperature to activate carboxyl groups on the surface of DBNPs and incubated for 30min; then washed three times with PBS solution of pH 7.2 at a concentration of 0.01mol/L and dispersed in 15mL of PBS solution (pH 7.2 at a concentration of 0.01 mol/L); then 15 mug of salmonella monoclonal antibody is added and the reaction is oscillated for 4 hours, after the reaction is completed, the excess salmonella monoclonal antibody is washed and removed by PBS solution with pH of 7.2 and concentration of 0.01mol/L, thus obtaining the immune probe for specifically recognizing salmonella, which is marked as IDBNPs, and the immune probe is stored in a refrigerator at 4 ℃.
Au-Fe prepared by the preparation method 3 O 4 The heterodimer recognition probe is one-to-one dumbbell-structured heterodimer nano-particles, and the surface of the heterodimer recognition probe is coupled with a salmonella monoclonal antibody.
Example 2
The embodiment provides a method based on Au-Fe 3 O 4 The preparation method of the salmonella recognition immune probe of the nano material specifically comprises the following steps:
(1) Preparation of Fe 3 O 4 Nanoparticles: 15mL of 1-octadecene and 1mL of oleic acid were added to a three-necked flask, stirred and mixed well at 120℃and incubated for 30min, and then 0.3mL of Fe (CO) was injected 5 Heating to 295 ℃ and reacting for 2 hours, bubbling nitrogen in the whole process, wherein the flow rate of the nitrogen is 15mL/min, and cooling to room temperature after the reaction is completed; then 10mL of acetone was added for centrifugation, and 15mL of TMAH solution (i.e., tetramethylammonium hydroxide solution, the concentration of which was 10%) was added to the precipitated product, followed by centrifugation to obtain Fe 3 O 4 Nanoparticle of Fe 3 O 4 Dispersing in 20mL deionized water to obtain Fe 3 O 4 The dispersion, designated MNPs;
(2) Preparation of Au seed -Fe 3 O 4 Heterodimeric nanoparticles: adding MNPs (0.1 mL), deionized water (10 mL) and sodium citrate solution (0.3 mL) (the mass concentration of the sodium citrate solution is 10 mg/mL) into a three-necked flask, stirring and mixing uniformly at 80-120 ℃, and then adding HAuCl (0.15 mL) 4 Solution (HAuCl) 4 The molar concentration of the solution is 10 mmol/L), and the mixture is continuously stirred and mixed at 110 ℃ and reacted for 30min to obtain Au seed -Fe 3 O 4 Heterodimeric nanoparticles;
(3) At 5min intervals after the completion of the reaction in step (2), 0.15mL of HAuCl was again added 4 The solution and 0.15mL sodium citrate solution are stirred and mixed at 110 ℃ and reacted for 6min, and Au-Fe is obtained after the reaction is finished 3 O 4 Heterodimeric nanoparticles, noted DBNPs; then cooling to room temperature, and placing into a refrigerator for preservation at 4 ℃ for standby;
(4) Preparation of Au-Fe 3 O 4 Heterodimer recognition probes: 15mL of DBNPs were mixed with 20mg of HS-PEG-COOH (mercapto-polyethylene glycol-carboxylic acid) and shaken at 37℃for 60min, then PBS solution with pH of 6.8 and concentration of 0.01mol/L was added, and the mixture was gently shaken at room temperature to activate carboxyl groups on the surface of DBNPs and incubated for 30min; then washed three times with PBS solution of pH 7.2 at a concentration of 0.01mol/L and dispersed in 15mL of PBS solution (pH 7.2 at a concentration of 0.01 mol/L); then 15 mug of salmonella monoclonal antibody is added and the reaction is oscillated for 4 hours, after the reaction is completed, the excess salmonella monoclonal antibody is washed and removed by PBS solution with pH of 7.2 and concentration of 0.01mol/L, thus obtaining the immune probe for specifically recognizing salmonella, which is marked as IDBNPs, and the immune probe is stored in a refrigerator at 4 ℃.
Au-Fe prepared by the preparation method 3 O 4 The heterodimer recognition probe is one-to-one dumbbell-structured heterodimer nano-particles, and the surface of the heterodimer recognition probe is coupled with a salmonella monoclonal antibody.
Next, we performed correlation detection on DBNPs and IDBNPs prepared by the preparation method of example 1, characterized the same, and performed correlation detection on salmonella by using IDBNPs, and validated the effectiveness of IDBNPs in detecting salmonella.
1. Characterization of DBNPs
Taking the product Au obtained in the step (2) of the example 1 seed -Fe 3 O 4 Heterodimeric nanoparticle solution and product Au-Fe obtained in step (3) of example 1 3 O 4 Heterodimeric nanoparticle solutions, DBNPs, were prepared for examination by transmission electron microscopy or the like, as shown in fig. 1. FIG. 1 is a representation of DBNPs, wherein FIGS. 1 (a), (b), (c) are Au, respectively seed -Fe 3 O 4 As can be seen from FIGS. 1 (a), (b) and (c), the TEM image, HAADF-STEM image, and STEM element distribution and superposition image of Au, fe and O elements of the heterodimer nanoparticle seed -Fe 3 O 4 The heterodimer nanoparticle has a one-to-one dumbbell structure, namely gold nanoparticle and Fe 3 O 4 The nano particles form a one-to-one dumbbell structure, and the gold nano particles and Fe 3 O 4 The nano particles form good combination; FIG. 1 (d) is a TEM image of DBNPs, and it can be seen from FIG. 1 (d) that HAuCl is further added 4 Au-Fe obtained by solution and sodium citrate solution 3 O 4 Heterodimer nanoparticles still maintain a one-to-one dumbbell structure, and the gold nanoparticles and Fe 3 O 4 The nanoparticles each had a particle diameter of about 15nm, and the inset in FIG. 1 (d) shows a distribution of the particle diameters of DBNPs, showing that the average size of DBNPs is about 30nm; FIG. 1 (e) is an ultraviolet spectrum of DBNPs, showing that the maximum ultraviolet absorption wavelength of DBNPs is 520nm, and the DBNPs color is shown as reddish wine.
2. Characterization of IDBNPs
Taking the solution of IDBNPs obtained in the step (4) of the above example 1, namely Au-Fe 3 O 4 In the heterodimer recognition probe of example 1, in step (4), DBNPs were modified by adding HS-PEG-COOH, and the carboxyl groups on the surface of DBNPs were activated, and then salmonella monoclonal antibody was added, and the carboxyl groups on DBNPs were bound to the amino groups of the antibody by covalent coupling to obtain Au-Fe 3 O 4 Heterodimer recognition probes. To confirm this binding, the obtained IDBNPs, DBNPs were reacted with Cy 3-labeled goat anti-mouse IgG, respectively, and performedThe following detection is carried out, and the detection result is shown in FIG. 2. FIG. 2 is a representation of IDBNPs, wherein FIG. 2 (a) is a fluorescence spectrum of Cy 3-labeled IDBNPs and DBNPs, the excitation wavelength is 554nm, and it can be seen from FIG. 2 (a) that the maximum absorption peak (568 nm) of Cy3 appears in the fluorescence spectrum of IDBNPs, but not in the corresponding absorption peak in the fluorescence spectrum of DBNPs; FIGS. 2 (b) and (c) are fluorescence microscopy images of Cy 3-labeled IDBNPs and Cy 3-labeled DBNPs, in which a number of orange fluorescent spots representing antibodies were observed in the IDBNPs samples, no apparent fluorescence was found in the DBNPs, indicating that the Salmonella monoclonal antibodies were successfully bound to the DBNPs; FIG. 2 (d) is a graph of the ultraviolet visible spectrum of IDBNPs and the color of IDBNPs, the ultraviolet visible spectrum and the color being substantially consistent with DBNPs; FIG. 2 (e) is a TEM image of IDBNPs (inset shows the particle size distribution of IDBNPs), the diameter of IDBNPs is about 30+ -6 nm, the diameter is slightly increased, indicating that the surface of IDBNPs is coupled with salmonella monoclonal antibody.
3. Magnetic response properties of IDBNPs
Taking the IDBNPs solution obtained in the step (4) of example 1, namely Au-Fe 3 O 4 Heterodimer recognition probes, which use magnetic scaffolds for magnetic response detection, are shown in FIG. 3, FIG. 3 is a graph of the magnetic response rate of IDBNPs, illustrating Au-Fe 3 O 4 The heterodimer recognition probe has magnetic response performance, and as the magnetic attraction time increases, the IDBNPs are gradually enriched under the action of a magnetic field, but the time required for complete enrichment is longer, so that the better magnetic capture time needs to be determined.
We set 6 groups of 0.5mL concentrations 10 6 After the CFU/mL of the salmonella solution is respectively added with 0.5mL of IDBNPs and incubated by a constant temperature oscillator (37 ℃,120rpm,30 min), magnetic capture is carried out on the salmonella solution for different time by adopting a magnetic bracket, supernatant is separated, ultraviolet-visible light absorption spectrum detection is carried out on the supernatant, and the relation between the detection magnetic capture time and an ultraviolet spectrum detection signal value is obtained, as shown in figure 4, the maximum detection signal value is generated in the magnetic capture time of 15min, and the magnetic capture time can be selected to be 15min in the detection process.
4. Quantitative and qualitative detection of salmonella using IDBNPs
Respectively preparing 0.5mL of the solution with the concentration of 0, 10 and 10 2 、10 4 、10 6 After adding 0.5mL of IDBNPs respectively, incubating the mixture with a constant temperature oscillator (37 ℃ C., 120rpm,30 min), separating salmonella combined with the IDBNPs by using a magnetic bracket for 15min, separating the supernatant from the precipitate, and retaining the supernatant and the precipitate.
As shown in FIG. 5, the maximum ultraviolet absorption intensity of the supernatant was measured by ultraviolet-visible light absorption spectrum as shown in FIG. 5 (a), and a curve was drawn, and as shown in FIG. 5 (b), it was found that the absorbance was varied by 10-10% with respect to the salmonella concentration 3 The CFU/mL range shows good linear relation, the minimum detection limit is 10CFU/mL, the quantitative detection of salmonella can be realized by utilizing IDBNPs and combining magnetic capture and ultraviolet-visible light absorption spectrum, and in the practical use process, the trace detection is usually carried out by sampling, and the detection is 10-10 3 The linear relationship between CFU/mL salmonella concentration and ultraviolet absorbance is sufficient for routine detection.
The separated supernatant was taken and observed for color change, and 0.5mL of ABTS was added to the supernatant at a concentration of 5mg/mL to observe color change, as shown in FIG. 6. FIG. 6 is a photograph showing the change of the supernatant color after magnetic separation and the supernatant color after addition of ABTS with the concentration of salmonella, as can be seen from FIG. 6, the residual IDBNPs in the supernatant decreases after magnetic separation as the concentration of salmonella increases, and the supernatant color changes from red to light red as observed by naked eyes; then, after ABTS was added to the supernatant, the supernatant color changed from brown to reddish brown and finally to green as the salmonella concentration increased.
The color change is because the prepared IDBNPs have pseudo-catalase activity, when the ABTS is added into the supernatant, under the catalysis of free IDBNPs, the ABTS is oxidized into green chelate, namely OxABTS, and according to the color mixing principle, the green chelate can be blended with the original red of the supernatant to show rich color change. In addition, along with the increase of the concentration of salmonella, the concentration of IDBNPs in supernatant gradually decreases, so that the red color of the IDBNPs gradually becomes lighter, the reduction of the concentration of the IDBNPs weakens the catalytic performance of the IDBNPs on ABTS, the generated OxABTS green color also gradually weakens, different color changes can be shown according to a color mixing principle, the salmonella can be detected in a colorimetric way, the presence or absence of the salmonella can be judged through the color changes, and the concentration range of the salmonella can be roughly judged through the comparison of the colors, so that the qualitative and semi-quantitative detection of the salmonella is realized. And referring to FIG. 6, when the salmonella concentration is 10CFU/mL, the color change occurs, and the minimum detection limit of colorimetric detection can reach 10CFU/mL. The salmonella is detected by the colorimetric method, so that the method is rapid and sensitive.
5. Detection of selectivity and specificity of IDBNPs
To verify that IDBNPs are selective for salmonella only, this example also compares them to other common food-borne pathogens (e.g., escherichia coli, staphylococcus aureus, pseudomonas aeruginosa), as shown in fig. 7.
The specific operation process is that 0.5mL of the solution with the concentration of 10 is respectively prepared 6 CFU/mL Salmonella (SE), escherichia coli (e.coil), staphylococcus Aureus (SA), pseudomonas Aeruginosa (PAE) solutions were then added with 0.5mL of bnps, respectively, incubated with a constant temperature shaker (37 ℃,120rpm,30 min), and separated with a magnetic rack, i.e., the supernatant was separated from the precipitated complex, and the supernatant and the precipitated complex were retained, and a blank control was set, which was an equivalent amount of IDBNPs solution.
The magnetically separated precipitated complexes were resuspended in 2mL of PBS (0.01 m, ph=7.2), and acridine orange was added to a final concentration of 10 μg/mL, and then the solution was placed in a constant temperature shaker at 37 ℃ and 150rpm to oscillate for 30min away from light, after the reaction was completed, the fluorescent intensity of the precipitated complexes was detected by washing 3 times with a magnetic rack, and 0.5mL of ABTS having a concentration of 5mg/mL was added to the supernatant to observe a color change, and the detection result was shown in fig. 7. Fig. 7 is a graph showing the effect of detecting different bacteria by using IDBNPs, wherein fig. 7 (a) is a histogram of signal values of IDBNPs for detecting different bacteria (corresponding ultraviolet-visible spectrum is shown in the inset), which illustrates that when IDBNPs are incubated with other common food pathogens (e.g. escherichia coli, staphylococcus aureus, pseudomonas aeruginosa), detection of salmonella is only achieved, and the signal values of other pathogens are similar to those of a blank group; FIG. 7 (b) is a colorimetric detection of different species of bacteria, showing that only Salmonella changes in color and that the color is green, indicating that IDBNPs bind to Salmonella and that the content of free IDBNPs is reduced; FIG. 7 (c) is a microscopic image of the complex precipitate after magnetic separation, and it can be seen from FIG. 7 (c) that many green spots are observed in the sample of the combination of IDBNPs with Salmonella, as stained bacteria, no obvious spots are observed in the sample of the combination of IDBNPs with E.coli, staphylococcus aureus and Pseudomonas aeruginosa, and the above results indicate that the IDBNPs have good selectivity and specificity for Salmonella and can only capture Salmonella.
Example 3
In this example 3, the actual sample was tested by using the IDBNPs prepared in example 1, and the feasibility of testing the salmonella content of the IDBNPs in the actual sample was verified, wherein the selected samples were cow's milk and cow's serum, and the control group was PBS solution added with salmonella, and the effect graph of testing salmonella in different synthetic samples by using the IDBNPs is shown in fig. 8. The detection steps are as follows:
taking 0.5mL of milk and fetal bovine serum to be detected, and adding 0, 10 and 10 concentrations respectively 2 、10 4 、10 6 Mixing CFU/mL salmonella suspension (volume ratio is 9:1) to obtain a synthetic sample, adding 0.5mL of IDBNPs into the sample, then gently shaking at 37 ℃ and culturing at 37 ℃ for 30min, then capturing salmonella combined with the IDBNPs by using a magnetic bracket, and separating a supernatant from a precipitate, wherein the magnetic attraction time is 15min; when the milk sample to be detected is taken, acetic acid is firstly added into the milk to be detected, stirring and standing are carried out until white precipitate is not generated, the supernatant is taken as the milk sample to be detected, so as to prevent protein and the like from influencing the detection result, and in addition, the salmonella is added into the synthetic sample, so that the salmonella is ensured to be actually contained in the sample.
As shown in fig. 8 (a), fig. 8 (a) shows the spectrum detection signal values of the synthesized sample, and the spectrum detection signal values of the three are substantially the same, so that salmonella can be quantitatively detected by using the ultraviolet-visible light absorption spectrum.
In addition, the supernatant was taken and 0.5mL of ABTS at a concentration of 5mg/mL was added to observe the color change of the supernatant, and as shown in fig. 8 (b), fig. 8 (b) is a colorimetric detection picture of different synthetic samples, and the color change of the milk sample and the fetal bovine serum albumin sample is substantially the same as that in the ideal environment, i.e., PBS solution, indicating that qualitative and semi-quantitative detection of salmonella can be performed by a colorimetric method.
Through the detection, the IDBNPs also have good anti-interference capability, and can be applied to actual sample detection.
In the actual detection process of the sample to be detected, the following steps can be specifically adopted:
(1) Taking 0.5mL of milk or fetal bovine serum sample to be detected, adding 0.5mL of IDBNPs into the sample, then gently shaking at 37 ℃, culturing at 37 ℃ for 30min, capturing salmonella combined with the IDBNPs by using a magnetic bracket, and separating supernatant from precipitate after 15min of magnetic attraction; when taking a milk sample to be detected, adding acetic acid into the milk to be detected, stirring and standing until white precipitate is not generated, and taking supernatant as the milk sample to be detected;
(2) Taking supernatant, detecting absorbance of the supernatant by using ultraviolet-visible light absorption spectrum, and quantitatively detecting salmonella by referring to the linear relation between the change value of absorbance and salmonella concentration determined in the test process;
(3) And taking supernatant, adding 0.5mL of ABTS (anaerobic ammonium chloride) with the concentration of 5mg/mL, observing the color change of the supernatant, and colorizing according to the corresponding relation between the concentration and the color of the salmonella determined in the test process to roughly judge the concentration range of the salmonella, so that qualitative and semi-quantitative detection of the salmonella in the sample can be realized.
The parts which are not described in the invention can be realized by adopting or referring to the prior art.
It should be understood that the above description is not intended to limit the invention to the particular embodiments disclosed, but to limit the invention to the particular embodiments disclosed, and that the invention is not limited to the particular embodiments disclosed, but is intended to cover modifications, adaptations, additions and alternatives falling within the spirit and scope of the invention.

Claims (9)

1. Au-Fe-based 3 O 4 The preparation method of the salmonella recognition immune probe of the nano material is characterized by comprising the following steps:
(1) Preparation of Fe 3 O 4 Nanoparticles: adding 1-octadecene and oleic acid into a three-necked flask, stirring and mixing uniformly at 100-130 ℃, preserving heat for 15-30 min, and then injecting Fe (CO) 5 Heating to 280-300 ℃ and reacting for 1.5-2 h, bubbling with nitrogen in the whole process, and cooling to room temperature after the reaction is completed; then adding acetone for centrifugation, adding TMAH solution into the sediment product, and centrifugally separating to obtain Fe 3 O 4 Nanoparticle of Fe 3 O 4 Dispersing in deionized water to obtain Fe 3 O 4 The dispersion, designated MNPs;
(2) Preparation of Au-Fe 3 O 4 Heterodimeric nanoparticles: adding MNPs, deionized water and sodium citrate solution into a three-neck flask, stirring and mixing uniformly at 80-120 ℃, and then adding HAuCl 4 Stirring and mixing the solution continuously at 80-120 ℃ and reacting for 20-30 min; after the reaction was completed for 5min, an equal volume of HAuCl was again added 4 The solution and the sodium citrate solution with equal volume are stirred and mixed at the temperature of 80-120 ℃ and reacted for 3-6 min, and the Au-Fe is obtained after the reaction is finished 3 O 4 Heterodimeric nanoparticles, noted DBNPs; then cooling to room temperature, and placing into a refrigerator for preservation at 4 ℃ for standby;
in the step (2), the volume ratio of MNPs, deionized water, sodium citrate solution and HAuCl4 solution is 0.1-0.2: 10-20:0.2-0.5:0.15-0.25, wherein the mass concentration of the sodium citrate solution is 8-10 mg/mL, and the molar concentration of the HAuCl4 solution is 10mmol/L;
(3) Preparation of Au-Fe 3 O 4 Heterodimer recognition probes: mixing DBNPs prepared in the step (2) with HS-PEG-COOH, andshaking at 37 ℃ for 50-60 min, then adding PBS solution with pH of 6.8 and concentration of 0.01mol/L, gently shaking at room temperature to activate carboxyl groups on the surface of DBNPs, and incubating for 20-40 min; then, the solution was washed three times with PBS solution having a pH of 7.2 and a concentration of 0.01mol/L, and dispersed in PBS solution having a pH of 7.2 and a concentration of 0.01 mol/L; then adding salmonella monoclonal antibody and oscillating for 3-4 hours, after the reaction is completed, washing with PBS solution with pH of 7.2 and concentration of 0.01mol/L to remove redundant salmonella monoclonal antibody, thus obtaining the specific salmonella-recognition immune probe, namely Au-Fe 3 O 4 Heterodimeric probes, designated as IDBNPs, were stored in a refrigerator at 4 ℃.
2. An Au-Fe-based alloy according to claim 1 3 O 4 The preparation method of the salmonella recognition immune probe of the nano material is characterized in that 1-octadecene, oleic acid and Fe (CO) in the step (1) 5 The volume ratio of acetone, TMAH solution and deionized water is 10-20:1-2:0.2-0.4:10-20:10-25:10-20, the concentration of the TMAH solution is 10%, and the flow rate of nitrogen is 10-20 mL/min.
3. An Au-Fe-based alloy according to claim 1 3 O 4 The preparation method of the salmonella recognition immune probe of the nano material is characterized in that the Au-Fe obtained in the step (2) 3 O 4 The heterodimer nanoparticle is one-to-one dumbbell-structured heterodimer nanoparticle, wherein the diameter of the Au nanoparticle is 12-16 nm, and Fe 3 O 4 The diameter of the nano particles is 12-16 nm.
4. An Au-Fe-based alloy prepared by the method according to any one of claims 1 to 3 3 O 4 The application of the salmonella recognition immune probe of the nano material in detecting salmonella.
5. The Au-Fe-based alloy according to claim 4 3 O 4 Salmonella recognition immune probe of nano materialThe application of the needle in detecting salmonella is characterized in that the application comprises the quantitative detection of salmonella in milk and fetal bovine serum by combining magnetic capture and ultraviolet-visible light absorption spectrum or the qualitative and semi-quantitative detection of salmonella in milk and fetal bovine serum by combining magnetic capture and colorimetry.
6. The Au-Fe-based alloy according to claim 5 3 O 4 The application of the salmonella identification immune probe of the nano material in detecting salmonella is characterized in that the method comprises the following steps of:
(a) Taking a milk or fetal bovine serum sample to be detected, adding an equal volume of IDBNPs into the sample, then gently shaking at 37 ℃, culturing at 37 ℃ for 30min, capturing salmonella bound with the IDBNPs by using a magnetic bracket, and separating a supernatant from a precipitate, wherein the magnetic attraction time is 10-20 min;
(b) Taking supernatant, and quantitatively detecting salmonella by using ultraviolet-visible light absorption spectrum.
7. The Au-Fe-based alloy according to claim 5 3 O 4 The application of the salmonella identification immune probe of the nano material in detecting salmonella is characterized in that the method for qualitatively and semi-quantitatively detecting salmonella in milk and fetal bovine serum by combining a magnetic capture method and a colorimetric method comprises the following steps:
(a) Taking a milk or fetal bovine serum sample to be detected, adding an equal volume of IDBNPs into the sample, then gently shaking at 37 ℃, culturing at 37 ℃ for 30min, capturing salmonella bound with the IDBNPs by using a magnetic bracket, and separating a supernatant from a precipitate, wherein the magnetic attraction time is 10-20 min;
(b) Taking supernatant, adding ABTS, observing the color change of the supernatant, and carrying out qualitative and semi-quantitative detection on salmonella in the sample.
8. Au-Fe-based according to claim 6 or 7 3 O 4 Nanomaterial ofThe application of the salmonella identification immune probe in detecting salmonella is characterized in that when a milk sample to be detected is taken in the step (a), acetic acid is firstly added into the milk to be detected, stirring and standing are carried out until white precipitate is not generated, and supernatant fluid is taken as the milk sample to be detected.
9. Au-Fe-based according to claim 6 or 7 3 O 4 The application of the salmonella identification immune probe of the nano material in detecting salmonella is characterized in that the magnetic attraction time in the step (a) is 15min.
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