A highly sensitive and rapid colorimetric detection method for foodborne pathogenic bacteria

Abstract

The present invention provides a highly sensitive and rapid colorimetric detection method for foodborne pathogenic bacteria, comprising the following steps: preparing silver nanoparticles, gold nanoparticles and dendritic gold nanoparticles; modifying the silver nanoparticles, gold nanoparticles and dendritic gold nanoparticles with Salmonella antibodies, Staphylococcus aureus antibodies and Shigella antibodies respectively; preparing dopamine-coated modified aqueous magnetic fluid; sample detection, enriching the microorganisms in the detection sample by positive charging of the surface of magnetic nanoparticles; separating the microorganisms and matrix in the detection sample by applying an external magnetic field; using Salmonella, Staphylococcus aureus and Shigella as targets, and then detecting the pathogenic bacteria by dark field microscopy after reacting with the antibody-modified nanoparticles. The highly sensitive and rapid colorimetric detection method for foodborne pathogenic bacteria provided by the present invention does not require bacteria expansion for detection, has high detection sensitivity, and can achieve multi-target detection.

Description

Technical Field

[0001] The present invention relates to the technical field of pathogenic bacteria detection, and particularly relates to a highly sensitive and rapid colorimetric detection method for foodborne pathogenic bacteria. Background Art

[0002] Foodborne diseases refer to infections or poisonings caused by various pathogenic factors that enter the human body through food. It is one of the most widespread and common diseases in the world today. Recently, according to the statistical report of the World Health Organization (WHO), the actual incidence of foodborne diseases is 300-500 times more than the reported cases, and the number of people suffering from food contamination worldwide has reached hundreds of millions. Common foodborne diseases in developing countries include cholera, Campylobacter jejuni, Escherichia coli infection, salmonellosis, and shigellosis. The number of diarrhea cases in children under 5 years old worldwide is about 1.5 billion per year, of which more than 3 million die, and food factors account for a large proportion. It can be seen that foodborne microbial poisoning has always been the number one threat to food safety.

[0003] At present, the detection and identification of traditional foodborne pathogenic bacteria still remain at the levels of isolation and culture, morphological observation, biochemical identification, and serotyping. These methods are cumbersome to operate and have a long detection cycle, usually taking 5-14 days. Therefore, the detection results of pathogenic microorganisms often have a certain degree of lag. This is not conducive to the implementation of control measures by food safety supervision and inspection agencies, nor is it conducive to quickly taking corrective measures for potentially unsafe products, and is even less conducive to the correct treatment of poisoned patients clinically. After the implementation of the new standard of GB 29921-2013 "National Food Safety Standard Limits of Pathogenic Bacteria in Foods", a rapid detection method that is accurate, sensitive, time-saving, labor-saving, and low-cost for foodborne pathogenic bacteria has become an urgent need to ensure food safety.

[0004] All along, research institutions and scientific researchers at home and abroad have been committed to developing new rapid detection techniques and methods for foodborne pathogens. (1) Enzyme-linked immunosorbent assay (ELISA). ELISA is a detection technique that organically combines the specificity of antigen-antibody immune reactions and the highly efficient catalytic action of enzymes. It can be used to detect both antigens and antibodies. The ELISA method can be used to detect Listeria, Salmonella, Escherichia coli O157, Campylobacter, Pseudomonas, Shigella, Vibrio parahaemolyticus, Vibrio cholerae, Staphylococcus aureus, Bacillus cereus, etc. in food. In addition, the ELISA method can also be used to detect food-mediated viruses. At present, China has completed the isolation and identification of avian influenza epidemic strains, the development and application of avian influenza recombinant nucleoprotein diagnostic antigens, and established avian influenza immunoenzymatic diagnostic methods and techniques. (2) Miniature automatic fluorescence enzyme immunoassay (VIDAS). Using enzyme-linked fluorescence immunoassay technology, through antigen-antibody specific reactions, the target bacteria are isolated, and a special instrument automatically judges whether the sample is positive or negative according to the intensity of fluorescence. The VIDAS method for detecting Salmonella in food is more sensitive and specific than the conventional method. When detecting negative samples, it can quickly make a judgment of non-Salmonella, which is 2-3 days faster than the conventional method. (3) Impedance technology. The principle of impedance method is that during the growth and reproduction of bacteria, macromolecular electro-inert substances such as carbohydrates, proteins, and lipids in the culture medium are metabolized into small molecular substances with electroactivity such as lactate and acetate, which changes the impedance of the culture medium. By detecting the change in the impedance of the culture medium, the growth and reproduction characteristics of bacteria in the culture medium are judged, and the corresponding bacteria are detected. This method has the advantages of fast detection speed, high sensitivity, and good accuracy. At present, it has been used for the detection of total bacteria count, molds, yeasts, Escherichia coli, Salmonella, Staphylococcus aureus, etc., but the selectivity is not good enough. (4) Polymerase chain reaction (PCR) technology. Polymerase chain reaction technology is a technology that specifically amplifies the target DNA fragment under suitable conditions in vitro, using DNA as a template and a pair of artificially synthesized oligonucleotides as primers under the action of heat-resistant DNA polymerase. By designing the primer sequences, various pathogenic bacteria DNAs can be highly selectively and specifically recognized, thus realizing the detection of pathogenic microorganisms. (5) Immunomagnetic bead separation method. Immunomagnetic separation technology is to conjugate specific antibodies on the surface of magnetic particles, which specifically binds to the pathogenic bacteria to be detected in the sample. The magnetic particles carrying the pathogenic bacteria gather towards the magnetic pole direction under the action of an external magnetic field. The test sample mixture is discarded, so that the pathogenic bacteria are continuously separated and concentrated. It is a very effective method for separating target bacteria from food components. This method is actually more valuable in terms of separation and enrichment. (6) Gene chip technology. It is to spot various gene oligonucleotides on the surface of the chip. The DNA of the microbial sample is prepared into a fluorescent-labeled probe after PCR amplification, and then hybridized with the oligonucleotide spots on the chip. Finally, the fluorescence distribution pattern is quantitatively analyzed by a scanner to determine whether certain specific microorganisms exist in the test sample.In theory, gene chip technology can detect all potential pathogenic bacteria in one experiment, with high detection sensitivity, specificity, and rapidity and convenience. Therefore, it has good development prospects in the analysis and detection of pathogens.

[0005] However, there are still many problems to be solved in existing rapid detection technologies. For example: (1) The detection takes a long time. Although many new technologies have reduced the detection time to 24 - 36 hours, which is a great improvement, it still cannot meet the application in special scenarios. For example, when the life of a poisoned person is hanging by a thread and it is urgent to confirm the type of pathogenic bacteria for symptomatic treatment. (2) The sensitivity of many methods is insufficient. Among existing detection methods, there are still not many practical detection technologies with a detection sensitivity of more than 100 cfu / g. (3) Sample processing is still too complex. For example, in order to achieve better sensitivity, it is necessary to go through the process of culturing bacteria, which increases the detection time. (4) There are too many false positives in detection. For example, in probe technology, DNA contamination of samples easily causes negative samples to be detected as positive. (5) It is difficult to detect multiple target bacteria simultaneously.

[0006] In view of this, the purpose of the present invention is to provide a new method for detecting foodborne pathogenic bacteria to solve the above technical problems. Summary of the Invention

[0007] The technical problem to be solved by the present invention is to provide a highly sensitive and rapid colorimetric detection method for foodborne pathogenic bacteria, which does not require culturing bacteria, has high detection sensitivity, and can achieve multi-target detection.

[0008] To solve the above problems, the technical solution of the present invention is as follows:

[0009] A highly sensitive and rapid colorimetric detection method for foodborne pathogenic bacteria, comprising the following steps:

[0010] Step S1, preparing silver nanoparticles, gold nanoparticles, and dendritic gold nanoparticles;

[0011] Step S2, nanoparticle modification, using Salmonella antibodies, Staphylococcus aureus antibodies, and Shigella antibodies to modify silver nanoparticles, gold nanoparticles, and dendritic gold nanoparticles respectively;

[0012] Step S3, preparing and modifying magnetic nanoparticles to obtain a water-based magnetic fluid wrapped and modified with dopamine;

[0013] Step S4, sample detection, enriching microorganisms in the detection sample by positively charging the surface of magnetic nanoparticles; separating microorganisms and matrix in the detection sample by applying an external magnetic field; using Salmonella, Staphylococcus aureus, and Shigella as targets, and then detecting pathogenic bacteria by microscopic imaging technology after reacting with antibody-modified nanoparticles. Under the microscope, Salmonella presents blue bright spots, Staphylococcus aureus presents green bright spots, and Shigella presents red bright spots.

[0014] Further, in step S1, the preparation method of silver nanoparticles is as follows:

[0015] Prepare an aqueous solution with a volume of 100 mL containing 5 mmol / L sodium citrate and 0.025 mmol / L tannic acid, heat it for 10 - 20 min while stirring;

[0016] After heating to boiling, inject 1 mL of 25 mmol / L AgNO3 solution into the solution. The solution turns bright yellow. Centrifuge at 10000 rpm for 10 minutes, suck off the supernatant, and redisperse with ultrapure water to remove excess tannic acid to synthesize silver nanoparticle seeds with a diameter of 10 - 20 nm, and then cool the solution to 80 - 90 °C;

[0017] Add 100 μL of 25 mmol / L sodium citrate, 250 μL of 2.5 mmol / L tannic acid, and 250 μL of AgNO3 to the silver nanoparticle seed solution in sequence to prepare silver nanoparticles that appear blue under a dark - field microscope;

[0018] Purify the obtained silver nanoparticles by centrifuging at 10000 rpm for 10 - 15 min.

[0019] Further, in step S1, the preparation method of gold nanoparticles is as follows:

[0020] Add 1 mL of 24.24 mmol / L HAuCl4 solution to 99 mL of ultrapure water, place the solution in an oil - bath pot and stir while heating. When it boils, add 1 mL of 1% sodium citrate solution by mass. After the solution turns wine - red, continue heating for half an hour to prepare spherical gold nanoparticle seeds;

[0021] Cool the synthesized spherical gold nanoparticle seeds and add them to a 100 - mL capped glass bottle for storage in a 4 °C refrigerator;

[0022] Heat 200 μL of 24.24 mmol / L HAuCl4 solution, 2 mL of spherical gold nanoparticle seed solution, and 200 μL of 1% sodium citrate solution by mass to boiling for 25 - 35 min to prepare large - particle - size gold nanoparticles with a red solution and green appearance under a dark - field microscope.

[0023] Further, the preparation method of dendritic gold nanoparticles is as follows:

[0024] Add 1 mL of 24.24 mmol / L HAuCl4 solution to 99 mL of ultrapure water. Place the solution in an oil bath, heat it with stirring. When it boils, add 1 mL of 1% sodium citrate solution. After the solution turns wine red, continue heating for half an hour to prepare spherical gold nanoparticle seeds.

[0025] Cool the synthesized spherical gold nanoparticle seeds and add them to a 100 mL capped glass bottle, then store them in a refrigerator at 4 °C.

[0026] Add 200 μL of 24.24 mmol / L HAuCl4 solution, 2 mL of spherical gold nanoparticle seed solution, 200 μL of sodium citrate solution (1%), and 100 μL of hydroquinone solution (0.5%) to 20 mL of water in sequence. Stir rapidly for 2 minutes and let it stand at room temperature for 30 minutes to prepare dendritic gold nanoparticles with a blue solution and a red color under dark-field microscopy.

[0027] Furthermore, in step S2, the nanoparticle modification includes the following steps:

[0028] Dilute the corresponding nanoparticle solution with ultrapure water respectively, and then add an excessive amount of 1-mercaptoundecanoic acid to react covalently with the surface of the nanoparticles through Ag-S bonds and displacement reactions.

[0029] Stir the solution for 2 - 3 h, then add 1 μM of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 100 μM of N-hydroxysuccinimide, and stir for another 30 - 40 min to obtain activated nanoparticles.

[0030] Add Salmonella antibody, Staphylococcus aureus antibody, and Shigella antibody to the activated silver nanoparticle, gold nanoparticle, and dendritic gold nanoparticle solutions respectively. Stir at room temperature for 1 - 2 h, centrifuge to remove the supernatant, wash with ultrapure water, and finally disperse in phosphate buffer to obtain antibody-modified nanoparticles.

[0031] Furthermore, in step 3, the preparation and modification of magnetic nanoparticles include the following steps:

[0032] Dissolve FeCl3·6H2O and FeSO4·4H2O in deionized water. Under nitrogen protection, quickly add ammonia water at 78 - 82 °C. After the temperature stabilizes, gradually add 1% dopamine dropwise and stir for 80 - 100 min to prepare dopamine-coated and modified aqueous magnetic fluid.

[0033] Furthermore, the mass ratio of FeCl3·6H2O to FeSO4·4H2O is (1.85 - 2.15):1.

[0034] Further, in step S4, the specific steps of sample detection are as follows:

[0035] Take an appropriate amount of the sample and dissolve it in physiological saline, adjust the pH to neutral, add the magnetic nanoparticle solution of step S3, mix evenly and let it stand for a period of time; place a strong magnet at the bottom of the sample, pour off the upper-layer sample after the magnetic nanoparticles sink to the bottom, wash the precipitate with physiological saline to obtain the first reaction system (bacteria in the sample are enriched);

[0036] Respectively take antibody-modified silver nanoparticles, gold nanoparticles and dendritic gold nanoparticles and add them to the first reaction system for incubation for 10 - 30 min, and then wash with physiological saline to obtain the second reaction system (nanoparticles are anchored on the surface of bacteria);

[0037] Remove the strong magnet, add an appropriate amount of physiological saline to the second reaction system, shake well and incubate for 30 minutes, then place a strong magnet at the bottom again. After the magnetic nanoparticle-bacteria-gold / silver nanoparticles sink to the bottom, discard the upper-layer sample and wash it. Then add 1 mL of physiological saline to suspend it again, and pipette 200 μL and place it under a dark-field microscope for observation. Under the microscope, Salmonella shows blue bright spots, Staphylococcus aureus shows green bright spots, and Shigella shows red bright spots.

[0038] Compared with the prior art, the highly sensitive and rapid colorimetric detection method for foodborne pathogenic bacteria provided by the present invention has the following beneficial effects:

[0039] First, the highly sensitive and rapid colorimetric detection method for foodborne pathogenic bacteria provided by the present invention is based on the pathogenic bacteria enrichment technology of magnetic nanoparticles and the colorimetric technology of three-color noble metal nanoparticles. By positively charging the surface of magnetic nanoparticles, pathogenic bacteria and other live bacteria in food samples are identified, and the separation of bacteria and food matrix in the sample is achieved through an external magnetic field. Using common and high-incidence pathogenic microorganisms in food (Salmonella, Staphylococcus aureus and Shigella) as targets, after acting with antibody-modified nanoparticles, rapid detection and identification of pathogenic bacteria are achieved through dark-field microscopy imaging technology. Under the microscope, Salmonella shows blue bright spots, Staphylococcus aureus shows green bright spots, and Shigella shows red bright spots. Therefore, the highly sensitive and rapid colorimetric detection method for foodborne pathogenic bacteria provided by the present invention has a detection sensitivity of up to 100 bacteria / g, greatly shortens the detection time, and can achieve multi-target simultaneous detection.

[0040] Second, for the highly sensitive and rapid colorimetric detection method for foodborne pathogenic bacteria provided by the present invention, the modification of magnetic nanoparticles is achieved by molecular covalent coupling, connecting small molecules containing primary amines, secondary amines and tertiary amines to the surface of magnetic nanoparticles, and highly enriching microorganisms by positively charging the surface of magnetic nanoparticles. Description of the Drawings

[0041] To more clearly illustrate the technical solutions in the embodiments of the present invention, the following will briefly introduce the accompanying drawings required for the description of the embodiments. Obviously, the accompanying drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other accompanying drawings can be obtained based on these drawings.

[0042] Figure 1 It is the electron microscope image of the silver nanoparticles prepared in the present invention;

[0043] Figure 2 It is the ultraviolet-visible absorption spectrum of the silver nanoparticles and the modified silver nanoparticles prepared in the present invention;

[0044] Figure 3 It is the observation image of the silver nanoparticles under the dark-field microscope in the present invention;

[0045] Figure 4 It is the electron microscope image of the gold nanoparticles prepared in the present invention;

[0046] Figure 5 It is the ultraviolet-visible absorption spectrum of the gold nanoparticles and the modified gold nanoparticles prepared in the present invention;

[0047] Figure 6 It is the observation image of the gold nanoparticles under the dark-field microscope in the present invention;

[0048] Figure 7 It is the electron microscope image of the dendritic gold nanoparticles prepared in the present invention;

[0049] Figure 8 It is the ultraviolet-visible absorption spectrum of the dendritic gold nanoparticles and the modified dendritic gold nanoparticles prepared in the present invention;

[0050] Figure 9 It is the observation image of the dendritic gold nanoparticles under the dark-field microscope in the present invention;

[0051] Figure 10 It is the microscopic imaging of detecting Salmonella by the detection system of the present invention;

[0052] Figure 11 It is the microscopic imaging of detecting Staphylococcus aureus by the detection system of the present invention;

[0053] Figure 12 It is the microscopic imaging of detecting Shigella by the detection system of the present invention. Detailed implementation manners

[0054] To enable those skilled in the art to better understand the technical solutions in the embodiments of the present invention and make the above-mentioned objects, features, and advantages of the present invention more obvious and understandable, the following further describes the specific embodiments of the present invention.

[0055] In the ranges disclosed herein, the endpoints and any values are not limited to the exact ranges or values. These ranges or values should be understood to include values close to these ranges or values. For numerical ranges, the endpoint values of each range, between the endpoint values of each range, between the endpoint value of each range and a single point value, and between single point values can be combined with each other to obtain one or more new numerical ranges, and these numerical ranges should be regarded as specifically disclosed herein.

[0056] Example 1

[0057] The highly sensitive and rapid colorimetric detection method for foodborne pathogenic bacteria provided by the present invention includes the following steps:

[0058] Step S1, prepare silver nanoparticles, gold nanoparticles, and dendritic gold nanoparticles;

[0059] Specifically, the preparation method of silver nanoparticles is as follows:

[0060] Prepare an aqueous solution with a volume of 100 mL containing 5 mmol / L sodium citrate and 0.025 mmol / L tannic acid, heat it for 10 - 20 min while stirring;

[0061] After heating to boiling, inject 1 mL of 25 mmol / L AgNO3 solution into the solution. The solution turns bright yellow. Centrifuge at 10000 rpm for 10 minutes, suck off the supernatant, and redisperse with ultrapure water to remove excess tannic acid to synthesize silver nanoparticle seeds with a diameter of 10 - 20 nm. Then cool the solution to 80 - 90 °C;

[0062] Add 100 μL of 25 mmol / L sodium citrate, 250 μL of 2.5 mmol / L tannic acid, and 250 μL of AgNO3 to the silver nanoparticle seed solution in sequence to prepare silver nanoparticles that appear blue under a dark field microscope;

[0063] Purify the obtained silver nanoparticles by centrifuging at 10000 rpm for 10 - 15 min.

[0064] Please refer to Figures 1 to 3 , where Figure 1 is the electron microscope image of the silver nanoparticles prepared in the present invention; Figure 2 is the ultraviolet - visible absorption spectrum of the silver nanoparticles and the modified silver nanoparticles prepared in the present invention; Figure 3 is the observation image of the silver nanoparticles under a dark field microscope in the present invention.

[0065] The preparation method of gold nanoparticles is as follows:

[0066] Add 1 mL of 24.24 mmol / L HAuCl4 solution to 99 mL of ultrapure water. Place the solution in an oil bath and heat it with stirring. When it boils, add 1 mL of 1% sodium citrate solution. After the solution turns wine red, continue heating for half an hour to prepare spherical gold nanoparticle seeds;

[0067] Cool the synthesized spherical gold nanoparticle seeds and add them to a 100 mL capped glass bottle for storage in a refrigerator at 4 °C;

[0068] Heat 200 μL of 24.24 mmol / L HAuCl4 solution, 2 mL of spherical gold nanoparticle seed solution, and 200 μL of 1% sodium citrate solution to boiling for 25 - 35 min to prepare large-sized spherical gold nanoparticles with a red solution and green under dark-field microscopy.

[0069] Please refer to in combination Figures 4 to 6 , where Figure 4 is the electron microscopy image of the gold nanoparticles prepared in the present invention; Figure 5 is the ultraviolet-visible absorption spectrum of the gold nanoparticles and the modified gold nanoparticles prepared in the present invention; Figure 6 is the observation image of the gold nanoparticles in the present invention under dark-field microscopy.

[0070] The preparation method of dendritic gold nanoparticles is as follows:

[0071] Add 1 mL of 24.24 mmol / L HAuCl4 solution to 99 mL of ultrapure water. Place the solution in an oil bath and heat it with stirring. When it boils, add 1 mL of 1% sodium citrate solution. After the solution turns wine red, continue heating for half an hour to prepare spherical gold nanoparticle seeds;

[0072] Cool the synthesized spherical gold nanoparticle seeds and add them to a 100 mL capped glass bottle for storage in a refrigerator at 4 °C;

[0073] Add 200 μL of 24.24 mmol / L HAuCl4 solution, 2 ml of spherical gold nanoparticle seed solution, 200 μL of sodium citrate solution (1%), and 100 μL of hydroquinone solution (0.5%) to 20 mL of water in sequence, stir rapidly for 2 minutes, and let it stand at room temperature for 30 minutes to prepare dendritic gold nanoparticles with a blue solution and red under dark-field microscopy.

[0074] Please refer to in combination Figures 7 to 9 , where Figure 7It is the electron microscopy image of the dendritic gold nanoparticles prepared in the present invention; Figure 8 It is the ultraviolet-visible absorption spectrum of the dendritic gold nanoparticles and the modified dendritic gold nanoparticles prepared in the present invention; Figure 9 It is the observation image of the dendritic gold nanoparticles under a dark field microscope.

[0075] Step S2, nanoparticle modification: Salmonella antibody, Staphylococcus aureus antibody, and Shigella antibody are used to modify silver nanoparticles, gold nanoparticles, and dendritic gold nanoparticles respectively;

[0076] Specifically, the corresponding nanoparticle solutions are diluted with ultrapure water respectively, and then an excessive amount of 1-mercaptoundecanoic acid is added to covalently react with the surface of the nanoparticles through Ag-S bonds and displacement reactions;

[0077] The solution is stirred for 2 - 3 h, then 1 μM freshly prepared 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and 100 μM freshly prepared N-hydroxysuccinimide (NHS) are added, and it is stirred for another 30 - 40 min to obtain activated nanoparticles;

[0078] The Salmonella antibody, Staphylococcus aureus antibody, and Shigella antibody are respectively added to the activated silver nanoparticle, gold nanoparticle, and dendritic gold nanoparticle solutions, stirred at room temperature for 1 - 2 h, the supernatant is removed by centrifugation, washed with ultrapure water, and finally dispersed in a phosphate buffer solution to obtain antibody-modified nanoparticles.

[0079] The ultraviolet-visible absorption spectra of the modified silver nanoparticles, gold nanoparticles, and dendritic gold nanoparticles are respectively referred to Figure 2 、 Figure 5 and Figure 8 .

[0080] Step S3, prepare and modify magnetic nanoparticles to obtain dopamine-coated and modified water-based magnetic fluid;

[0081] The specific method is as follows:

[0082] Dissolve FeCl3·6H2O and FeSO4·4H2O in deionized water, quickly add ammonia water at 78 - 82 °C under nitrogen protection, dropwise add dopamine with a mass concentration of 1% after the temperature is stabilized, and stir for 80 - 100 min to prepare dopamine-coated and modified water-based magnetic fluid. For the prepared dopamine-coated and modified water-based magnetic fluid, small molecules containing primary amines, secondary amines, and tertiary amines are connected to the surface of the magnetic nanoparticles through molecular covalent coupling, and the high enrichment of microorganisms is achieved by positively charging the surface of the magnetic nanoparticles. Among them, the mass ratio of FeCl3·6H2O to FeSO4·4H2O is (1.85 - 2.15):1.

[0083] Step S4, sample detection: By positively charging the surface of magnetic nanoparticles, microorganisms in the detection sample are enriched; the separation of microorganisms and matrix in the detection sample is achieved by applying an external magnetic field; using Salmonella, Staphylococcus aureus, and Shigella as targets, after reacting with antibody-modified nanoparticles, pathogenic bacteria are detected by dark-field microscopy. Under the microscope, Salmonella shows blue bright spots, Staphylococcus aureus shows green bright spots, and Shigella shows red bright spots.

[0084] Specifically, it includes the following steps:

[0085] Take an appropriate amount of the sample and dissolve it in physiological saline, adjust the pH to neutral, add the magnetic nanoparticle solution in step S3, mix evenly and let it stand for a period of time; place a strong magnet at the bottom of the sample, pour off the upper-layer sample after the magnetic nanoparticles sink to the bottom, wash the precipitate with physiological saline to obtain the first reaction system;

[0086] Respectively take antibody-modified silver nanoparticles, gold nanoparticles, and dendritic gold nanoparticles and add them to the first reaction system for incubation for 10 - 30 min, then wash with physiological saline to obtain the second reaction system;

[0087] Remove the strong magnet, add an appropriate amount of physiological saline to the second reaction system, shake well and incubate for 30 min, then place a strong magnet at the bottom again. After the magnetic nanoparticle-bacteria-gold / silver nanoparticle sink to the bottom, discard the upper-layer sample and wash it. Then add 1 mL of physiological saline to suspend it, and pipette 200 μL and place it under a dark-field microscope for observation. Under the microscope, Salmonella shows blue bright spots, Staphylococcus aureus shows green bright spots, and Shigella shows red bright spots.

[0088] Please refer to Figures 10 to 12 , in which Figure 10 is the microscopic imaging diagram of the detection system of the present invention for detecting Salmonella; Figure 11 is the microscopic imaging diagram of the detection system of the present invention for detecting Staphylococcus aureus; Figure 12 is the microscopic imaging diagram of the detection system of the present invention for detecting Shigella. It can be seen from Figures 10 to 12 that the silver nanoparticles modified with Salmonella antibodies make Salmonella show blue bright spots, the gold nanoparticles modified with Staphylococcus aureus antibodies make Staphylococcus aureus show green bright spots, and the dendritic gold nanoparticles modified with Shigella antibodies make Shigella show red bright spots.

[0089] Example 2

[0090] Weigh 25 g of food sample (large pieces need to be crushed) into 225 mL of physiological saline and shake well; if the sample is strongly acidic or strongly alkaline, adjust the pH to neutral;

[0091] Add 0.01 g of dopamine-coated modified water-based magnetic fluid, shake well, and let stand for 10 - 120 min; place a strong magnet at the bottom of the sample, let stand for 3 - 10 minutes. After the magnetic nanoparticles settle to the bottom, pour out the upper food sample and add physiological saline for washing once to obtain the first reaction system.

[0092] Respectively pipette 10 μL of antibody-modified silver nanoparticle solution, gold nanoparticle solution, and dendritic gold nanoparticle solution into the first reaction system for incubation for 10 - 30 min, and then wash the magnetic nanoparticles twice with physiological saline to obtain the second reaction system.

[0093] Remove the strong magnet, add an appropriate amount of physiological saline to the second reaction system, shake well and incubate for 30 minutes. Then place a strong magnet at the bottom again. After the magnetic nanoparticle-bacteria-(gold or silver) nanoparticles settle to the bottom, discard the upper sample and wash. Add 1 mL of physiological saline again to suspend, and pipette 200 μL and observe under a dark-field microscope. Under the microscope, Salmonella shows blue bright spots, Staphylococcus aureus shows green bright spots, and Shigella shows red bright spots.

[0094] Using the detection method of the present invention, theoretically, the detection of a single bacterium can be achieved. According to the actual detection results, the detection sensitivity of the sample can reach 100 bacteria / g.

[0095] Compared with the prior art, the high-sensitivity and rapid colorimetric detection method for foodborne pathogenic bacteria provided by the present invention has the following beneficial effects:

[0096] First, the high-sensitivity and rapid colorimetric detection method for foodborne pathogenic bacteria provided by the present invention is based on the pathogenic bacteria enrichment technology of magnetic nanoparticles and the colorimetric technology of three-color noble metal nanoparticles. By positively charging the surface of magnetic nanoparticles, pathogenic bacteria and other live bacteria in food samples are identified. Through an external magnetic field, the separation of bacteria and food matrix in the sample is achieved. Using common and highly prevalent pathogenic microorganisms in food (Salmonella, Staphylococcus aureus, and Shigella) as targets, after reacting with antibody-modified nanoparticles, rapid detection and identification of pathogenic bacteria are achieved through dark-field microscopy imaging technology. Under the microscope, Salmonella shows blue bright spots, Staphylococcus aureus shows green bright spots, and Shigella shows red bright spots. Therefore, the high-sensitivity and rapid colorimetric detection method for foodborne pathogenic bacteria provided by the present invention has a detection sensitivity of up to 100 bacteria / g, the detection time is shortened to within 3 h, and multi-target simultaneous detection can be achieved.

[0097] Second, for the high-sensitivity and rapid colorimetric detection method for foodborne pathogenic bacteria provided by the present invention, the modification of magnetic nanoparticles is achieved by molecular covalent coupling. Small molecules containing primary amines, secondary amines, and tertiary amines are connected to the surface of magnetic nanoparticles, and high enrichment of microorganisms is achieved by positively charging the surface of magnetic nanoparticles.

[0098] The above has made a detailed description of the embodiments of the present invention, but the present invention is not limited to the described embodiments. For those skilled in the art, various changes, modifications, substitutions, and variations made to these embodiments without departing from the principle and spirit of the present invention still fall within the protection scope of the present invention.

Claims

1. A highly sensitive and rapid colorimetric detection method for foodborne pathogenic bacteria, characterized in that, It includes the following steps: Step S1, preparing silver nanoparticles, spherical gold nanoparticles and dendritic gold nanoparticles; the silver nanoparticles appear blue under a dark-field microscope, the spherical gold nanoparticle solution is red and appears green under a dark-field microscope, the dendritic gold nanoparticle solution is blue and appears red under a dark-field microscope; Step S2, nanoparticle modification, using Salmonella antibody, Staphylococcus aureus antibody and Shigella antibody to modify silver nanoparticles, gold nanoparticles and dendritic gold nanoparticles respectively; Step S3, preparing and modifying magnetic nanoparticles to obtain dopamine-coated and modified water-based magnetic fluid, and the specific method is as follows: Dissolve FeCl3·6H2O and FeSO4·4H2O in deionized water according to the mass ratio of (1.85 - 2.15):1, quickly add ammonia water at 78 - 82 °C under nitrogen protection, and gradually add dopamine with a mass concentration of 1% dropwise after the temperature stabilizes, and stir for 80 - 100 min to prepare dopamine-coated and modified water-based magnetic fluid; for the dopamine-coated and modified water-based magnetic fluid, small molecules containing primary amine, secondary amine and tertiary amine are connected to the surface of magnetic nanoparticles through molecular covalent coupling; Step S4, sample detection, enriching microorganisms in the detection sample by positive charging of the surface of magnetic nanoparticles, and the specific operation is as follows: take an appropriate amount of the sample and dissolve it in physiological saline, adjust the pH to neutral, add the dopamine-coated and modified water-based magnetic fluid in Step S3, mix evenly and then let it stand for a period of time; Separate microorganisms and matrix in the detection sample by applying an external magnetic field; using Salmonella, Staphylococcus aureus and Shigella as targets, and then detecting pathogenic bacteria by dark-field microscopy after reacting with antibody-modified nanoparticles, and Salmonella presents blue bright spots, Staphylococcus aureus presents green bright spots, and Shigella presents red bright spots under the microscope.

2. The highly sensitive and rapid colorimetric detection method for foodborne pathogenic bacteria according to claim 1, wherein In Step S1, the preparation method of silver nanoparticles is as follows: Prepare an aqueous solution with a volume of 100 mL containing 5 mmol / L sodium citrate and 0.025 mmol / L tannic acid, heat for 10 - 20 min while stirring; After heating to boiling, inject 1 mL of 25 mmol / L AgNO3 solution into this solution, the solution turns bright yellow, centrifuge at 10000 rpm for 10 minutes, suck off the supernatant, add ultrapure water to redisperse to remove excess tannic acid, synthesize silver nanoparticle seeds with a diameter of 10 - 20 nm, and then cool the solution to 80 - 90 °C; Add 100 μL of 25 mmol / L sodium citrate, 250 μL of 2.5 mmol / L tannic acid and 250 μL of AgNO3 to the silver nanoparticle seed solution in sequence to prepare silver nanoparticles that appear blue under a dark-field microscope; Purify the obtained silver nanoparticles by centrifuging at 10000 rpm for 10 - 15 min.

3. The highly sensitive and rapid colorimetric detection method for foodborne pathogenic bacteria according to claim 1, characterized in that, In Step S1, the preparation method of gold nanoparticles is as follows: Add 1 mL of 24.24 mmol / L HAuCl4 solution to 99 mL of ultrapure water. Place the solution in an oil bath and heat it with stirring. When it boils, add 1 mL of 1% sodium citrate solution. After the solution turns wine red, continue heating for half an hour to prepare spherical gold nanoparticle seeds. After cooling the synthesized spherical gold nanoparticle seeds, add them to a 100 mL capped glass bottle and store them in a refrigerator at 4 °C. Heat and boil 200 µL of 24.24 mmol / L HAuCl4 solution, 2 mL of spherical gold nanoparticle seed solution, and 200 µL of 1% sodium citrate solution for 25 - 35 min to prepare large-sized gold nanoparticles with a red solution and green under dark-field microscopy.

4. The highly sensitive and rapid colorimetric detection method for foodborne pathogenic bacteria according to claim 1, wherein The preparation method of dendritic gold nanoparticles is as follows: Add 1 mL of 24.24 mmol / L HAuCl4 solution to 99 mL of ultrapure water. Place the solution in an oil bath and heat it with stirring. When it boils, add 1 mL of 1% sodium citrate solution. After the solution turns wine red, continue heating for half an hour to prepare spherical gold nanoparticle seeds. After cooling the synthesized spherical gold nanoparticle seeds, add them to a 100 mL capped glass bottle and store them in a refrigerator at 4 °C. Add 200 µL of 24.24 mmol / L HAuCl4 solution, 2 ml of spherical gold nanoparticle seed solution, 200 µL of 1% sodium citrate solution, and 100 µL of 0.5% hydroquinone solution to 20 mL of water in sequence, stir rapidly for 2 minutes, and let it stand at room temperature for 30 minutes to prepare dendritic gold nanoparticles with a blue solution and red under dark-field microscopy.

5. The highly sensitive and rapid chromogenic detection method for foodborne pathogenic bacteria according to claim 1, characterized in that, In step S2, the nanoparticle modification includes the following steps: Dilute the corresponding nanoparticle solution with ultrapure water respectively, and then add an excessive amount of 1-mercaptoundecanoic acid to undergo a covalent reaction with the surface of the nanoparticles through Ag-S bonds and displacement reactions. Stir the solution for 2 - 3 h, then add 1 μM of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 100 μM of N-hydroxysuccinimide, and stir for another 30 - 40 min to obtain activated nanoparticles. Add Salmonella antibody, Staphylococcus aureus antibody, and Shigella antibody to the activated silver nanoparticle, gold nanoparticle, and dendritic gold nanoparticle solutions respectively, stir at room temperature for 1 - 2 h, centrifuge to remove the supernatant, wash with ultrapure water, and finally disperse in phosphate buffer to obtain antibody-modified nanoparticles.

6. The highly sensitive and rapid colorimetric detection method for foodborne pathogenic bacteria according to claim 1, wherein In step S4, the specific steps of sample detection are as follows: Take an appropriate amount of the sample and dissolve it in physiological saline, adjust the pH to neutral, add the dopamine-coated and modified water-based magnetic fluid in step S3, mix evenly and let it stand for a period of time; place a strong magnet at the bottom of the sample. After the magnetic nanoparticles sink to the bottom, pour out the upper-layer sample, and wash the precipitate with physiological saline to obtain the first reaction system. Respectively take antibody-modified silver nanoparticles, gold nanoparticles and dendritic gold nanoparticles and add them to the first reaction system for incubation for 10 - 30 min, and then wash with physiological saline to obtain the second reaction system; Remove the strong magnet, add an appropriate amount of physiological saline to the second reaction system, shake well and incubate for 30 minutes, then place the strong magnet at the bottom again. After the magnetic nanoparticles - bacteria - gold / silver nanoparticles sink to the bottom, discard the upper sample, wash, add 1 mL of physiological saline again to suspend, and then aspirate 200 µL and observe under a dark field microscope. Under the microscope, Salmonella shows blue bright spots, Staphylococcus aureus shows green bright spots, and Shigella shows red bright spots.

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