CN110687110A - A nano-gold colorimetric method for rapid detection of foodborne pathogens based on low pH - Google Patents

Abstract

The invention discloses a nano-gold colorimetric method for rapidly detecting food-borne pathogenic bacteria based on low pH, and belongs to the technical field of food safety. The nano gold particles obtained by a sodium citrate reduction method are subjected to charge interaction with bacteria in a low pH environment to change the aggregation state of the nano gold, which is expressed as the color change of a nano gold solution, and the detection result can be observed by naked eyes. The method can be used for detecting Staphylococcus aureus, Shigella, etc. within 5 min. The method has the advantages of high detection speed, convenient operation, simple reagent and no need of large-scale equipment, and is suitable for field detection or qualitative detection in mass products.

Description

A nano-gold colorimetric method for rapid detection of foodborne pathogens based on low pH

technical field

The invention relates to nanotechnology and biotechnology, and belongs to the technical field of food safety. Specifically, it particularly relates to a kind of nano-gold prepared by a traditional sodium citrate reduction method, a simple and fast method for detecting a variety of pathogenic bacteria in a low pH environment.

Background technique

Food-borne pathogens are extremely harmful to food safety, and the food-borne diseases caused by them are easy to spread and have serious harm to the human body. Therefore, the detection of pathogenic bacteria in food is of great significance and is an important social, economic and public health task. As the basis for many routine tests, traditional culture assays are internationally representative and standard, and are reliable methods for detecting foodborne pathogens, but these detection methods often require a lot of time, labor, and instrumental costs. Therefore, more rapid, convenient and suitable methods for detecting food-borne pathogens in some specific occasions such as on-site detection are more and more needed and valued by people.

Nanomaterials have developed rapidly in recent years and are widely used in biology, medicine and other fields. Nanomaterials have a larger specific surface area, among which gold nanoparticles have the characteristics of high absorption coefficient, strong scattering, and unique local surface plasmon resonance. The aggregation of gold nanoparticles can cause a red shift in the absorption spectrum, and because it is very close to the change of surface plasmon resonance, the gold nanoparticles solution changes from red to purple and blue depending on the degree of aggregation. And gold nanoparticles can be combined with some biomolecules such as proteins and nucleic acids. These unique properties of gold nanoparticles make them often used as colorimetric biosensors in detection, and have the advantages of fast, intuitive, accurate and efficient.

In view of this, the present invention is proposed.

SUMMARY OF THE INVENTION

In view of the problems existing in the prior art, the present invention provides a nano-gold colorimetric method for rapid detection of food-borne pathogenic bacteria based on low pH, which utilizes the unique optical properties of nano-gold and the negative charge on its surface, A principle is proposed based on the principle that the bacterial surface protein becomes positively charged at a pH lower than the isoelectric point of the bacterial surface protein, and the nano-gold is aggregated on the bacterial surface through electrostatic action, and then the colorimetric method is used to quickly and intuitively detect food in food. Methods for a variety of pathogenic bacteria. Therefore, the method includes: after preparing gold nanoparticles by the sodium citrate reduction method, adding the bacterial solution to be tested to the gold nanoparticles solution, adjusting the pH of the mixed solution with HCl of pH=1, and finally performing colorimetry with the control group to quickly and intuitively The final pH of the mixture is about 3.16. When the pH is too low, the nano-gold solution is unstable and cannot be detected; when the pH is greater than 3.5, it cannot be guaranteed that the pH is lower than the isoelectric point of all proteins, which affects the detection sensitivity. The method has the advantages of fast detection speed, convenient operation, simple reagents, and no need for instruments and equipment, and is suitable for on-site detection or qualitative detection in mass products.

The specific operation steps are as follows:

(1) Preparation of gold nanoparticles: 9.6 mL of ultrapure water was added with 20 µL of 0.5 M chloroauric acid and heated to boiling, then 400 µL of sodium citrate solution with a mass fraction of 1% was quickly added, and the heating and stirring were continued for about 10 min. The solution turned into a clear wine red and cooled to room temperature.

(2) Take the bacteria to be tested cultivated in the liquid medium, centrifuge and resuspend in sterilized ultrapure water as the sample solution to be tested.

(3) Prepare a hydrochloric acid solution with a pH of 1 as a low pH environmental regulator;

(4) Take 40 µL of the nano-gold solution prepared in (1), add 20 µL of ultrapure water and 20 µL of the pH 1 hydrochloric acid solution prepared in (3), as a control group.

(5) Detection of bacteria to be tested: Take 40 µL of the nano-gold solution prepared in (1), add 20 µL of the bacteria solution prepared in (2) and 20 µL of the hydrochloric acid solution with a pH of 1 prepared in (3), and compare For the control group prepared in (4), the color change of the solution was observed.

(6) Reading of the results: The control group prepared in (4) should keep the wine red color of the nano-gold solution. If the color of the control group also changes, the test result is invalid; (5) If the detection system changes from wine red to wine red Purple or blue, it is positive, indicating that the bacteria to be tested.

Further, the ultrapure water in the step (1) needs to be sterilized in advance.

Further, the bacteria to be tested in the step (2) are Staphylococcus aureus, Shigella, Pseudomonas aeruginosa, Vibrio parahaemolyticus, Salmonella typhimurium, Escherichia coli O157:H7 or Bacillus subtilis .

Further, the specific conditions in the step (2) refer to: after the bacteria to be tested are activated, cultured in a liquid medium to a logarithmic growth phase, and the bacteria are collected by centrifugation and resuspended in ultrapure water for detection.

The final pH value of the solution system in the detection method is 3.16, and at this pH, the nano-gold in the solution is in a stable dispersion state, that is, a wine-red solution.

The results of the detection method are read by the color change of gold nanoparticles. After adding the bacteria solution to be tested and adjusting the pH, the gold nanoparticles interact with the bacteria to be tested, so that the gold nanoparticles change from the original dispersed state to the aggregation state, which is expressed as From the original wine red to purple or blue.

The detection method is fast, and the results can be observed after adjusting the pH, and the time required is less than 5 minutes.

Compared with other traditional detection methods, the present invention has the following beneficial effects:

(1) The reagents used in the detection are stable and simple, and the operation is convenient and fast;

(2) The detection speed of the invention is fast. After adding the bacteria to be detected into the low pH nano-gold solution and mixing, the result can be read immediately, and the detection time is within 5 minutes.

(3) The present invention does not require complex instruments and equipment for detection, and the results can be read directly by the naked eye, and the detection results are highly intuitive.

Description of drawings

Figure 1 shows the UV-vis spectra of gold nanoparticles prepared by the sodium citrate reduction method at different pH in the present invention.

Figure 2 is the zeta potential diagram of gold nanoparticles in the control group of the detection system in the present invention.

Figure 3 is the UV-vis spectrum of gold nanoparticles when the low pH gold nanoparticles colorimetric method detects different concentrations of Staphylococcus aureus in the present invention.

Figure 4 shows the UV-vis spectra of gold nanoparticles when detecting different concentrations of Shigella by low pH gold nanoparticles colorimetric method in the present invention.

Fig. 5 is the FE-SEM images of the low pH nano-gold solution of the present invention when detecting Staphylococcus aureus under different magnifications.

FIG. 6 is the FE-SEM images of the low pH nano-gold solution of the present invention when Shigella is detected under different magnifications.

Detailed ways

The present invention will be further described below with reference to specific embodiments. It should be understood that the following examples are only used to illustrate the present invention rather than to limit the scope of the present invention, and those skilled in the art can make some non-essential improvements and adjustments according to the content of the above invention.

Example 1

The nano-gold colorimetric method of the present embodiment based on the rapid detection of food-borne pathogenic bacteria based on low pH, the steps are as follows:

(1) Preparation of gold nanoparticles: 9.6 mL of ultrapure water was added with 20 µL of final 0.5 M chloroauric acid and heated to boiling, then 400 µL of 1% sodium citrate solution was quickly added, and the heating and stirring continued for about 10 min , until the solution becomes transparent wine red, cooled to room temperature.

(2) Take Staphylococcus aureus and Shigella cultured in liquid medium, centrifuge and resuspend them in sterilized ultrapure water, measure their OD value, and dilute the bacterial solution to OD value of 0.5, 0.1 , 0.05 and 0.02.

(3) Prepare a hydrochloric acid solution with a pH of 1 as a low pH environmental regulator;

(4) Take 40 µL of the nano-gold solution prepared in (1), add 20 µL of ultrapure water and 20 µL of the pH 1 hydrochloric acid solution prepared in (3), as a control group.

(5) Detection of bacteria: Take 20 µL of the different concentrations of Staphylococcus aureus and Shigella bacteria prepared in (2) in a 1.5 mL centrifuge tube, respectively, and put them into each centrifuge tube containing the bacterial solution. Add 40 µL each of the gold nanoparticles solution prepared in (1), mix them evenly, and then add 20 µL of the hydrochloric acid solution with a pH of 1 prepared in (3) to each gold nanobacterium solution, and compare the control prepared in (4). group, observe the color change of the solution in each 1.5 mL centrifuge tube.

(6) Reading of the results: The color of the control group prepared in (4) remained the wine red of the nano-gold solution. When the OD value of Staphylococcus aureus was 0.5, the color of the nano-gold changed to blue, and then The OD value of the dilution is still wine red; when the OD value of Shigella is 0.5 and 0.1, the nano-gold solution turns blue, when the OD value is 0.05, the nano-gold solution turns purple, and when the OD value is 0.02, it is still Claret.

Therefore, in this example, the detection method proposed by the present invention can be used to detect Staphylococcus aureus and Shigella. Its detection sensitivity is OD 0.5 for Staphylococcus aureus, and the corresponding bacterial concentration is 1.6×10 ⁷ CFU/mL by the plate counting method; it is OD 0.05 for Shigella, and the corresponding bacterial concentration is obtained by the plate counting method. was 3.3×10 ⁵ CFU/mL.

Figure 1 shows the UV-vis spectra of gold nanoparticles prepared by sodium citrate reduction at different pH. The nano-gold solution with a concentration of 1 mM was mixed with equal volumes of ultrapure water (control) and HCl solution with a pH of 1-6, respectively. After standing for 10 min, the UV-vis spectrum was measured, and the color of the solution was photographed and recorded. . From the UV-vis absorption spectrum in Figure 1, it can be seen that the control group and pH 3-6 groups have the characteristic absorption peak (526 nm) of nano-gold particles, and the solution is wine red. However, the peaks in the pH 1 and pH 2 groups appeared red-shifted, indicating the aggregation of gold nanoparticles, and the color of the solution turned blue, and the gold nanoparticles were unstable. Therefore, the present invention selects the pH value with the final pH greater than 3 as the detection pH environment.

Fig. 2 is the zeta potential diagram of the gold nanoparticles of the control group in the detection system of the present invention; the final pH of the control group is about 3.16, and the charge of the gold nanoparticles of the control group is negatively measured through potential detection, and its zeta potential is -21.6±1.4 mV .

Figure 3 is the UV-vis spectrum of different concentrations of Staphylococcus aureus in low pH gold nanoparticles in the present invention. According to the detection method provided by the present invention, the UV-vis spectra of the control group and the groups with OD values of 0.1, 0.05 and 0.02 have the characteristic absorption peak (526 nm) of gold nanoparticles, and the solution is wine red. However, the UV-vis spectrum of the group with OD value of 0.5 showed a red shift, indicating the aggregation of gold nanoparticles on the bacterial surface at low pH, and the color of the solution changed to blue.

Figure 4 shows the UV-vis spectra of Shigella with different concentrations in low pH gold nanoparticles in the present invention. According to the detection method provided by the present invention, the UV-vis spectra of the control group and the group with an OD value of 0.02 have the characteristic absorption peak (526 nm) of the gold nanoparticles, and the solution appears wine red. However, the UV-vis spectra of the groups with OD values of 0.5, 0.1 and 0.05 showed a red shift, indicating the aggregation of gold nanoparticles on the bacterial surface at low pH, and the color of the solution changed to blue or purple.

Fig. 5 is the FE-SEM images of the detection of Staphylococcus aureus in the low pH nano-gold solution of the present invention under different magnifications. The mixture of the detection group with Staphylococcus aureus OD value of 0.5 was added dropwise to the silicon wafer and dried, and then observed under FE-SEM. It can be seen from the figure that because the pH is lower than the isoelectric point of the surface protein of Staphylococcus aureus, it is positively charged, and the negatively charged gold nanoparticles are adsorbed on its surface under the action of electrostatic force.

FIG. 6 is the FE-SEM images of Shigella detected in the low pH nano-gold solution of the present invention under different magnifications. The mixture of the detection group with Shigella OD value of 0.5 was added dropwise to the silicon wafer and dried, and then observed under FE-SEM. It can be seen from the figure that, unlike the phenomenon in which the gold nanoparticles are completely wrapped on the surface of the bacteria in the FE-SEM image of Staphylococcus aureus, the gold nanoparticles are only agglomerated and wrapped at both ends of Shigella, and the gold nanoparticles can be adsorbed under the same conditions. More Shigella, so the detection sensitivity is improved compared with Staphylococcus aureus.

At the same time, using the nano-gold colorimetric method for rapid detection of food-borne pathogenic bacteria based on low pH proposed by the present invention, Pseudomonas aeruginosa, Vibrio parahaemolyticus, Salmonella typhimurium, Escherichia coli O157:H7 and subtilis were detected. Bacillus. The detection sensitivity is OD 0.1 for Pseudomonas aeruginosa, OD 0.1 for Vibrio parahaemolyticus, OD 0.05 for Salmonella typhimurium, OD 0.5 for Escherichia coli O157:H7, OD 0.1 for Bacillus subtilis, and obtained by plate counting method. The corresponding bacterial solution concentrations were Pseudomonas aeruginosa, 4.5×10 ⁶ CFU/mL; Vibrio parahaemolyticus, 5.8×10 ⁶ CFU/mL; Salmonella typhimurium, 6.6×10 ⁶ CFU/mL; Escherichia coli O157 : H7, 4.4×10 ⁷ CFU/mL; Bacillus subtilis, 2.8×10 ⁵ CFU/mL.

The foregoing has shown and described the basic principles and main features of the present invention, as well as the advantages of the present invention. Those skilled in the art should understand that the present invention is not limited by the above-mentioned embodiments. The above-mentioned embodiments and descriptions only illustrate the principle of the present invention. Without departing from the spirit and scope of the present invention, the present invention will also have Various changes and modifications fall within the scope of the claimed invention. The claimed scope of the present invention is defined by the appended claims and their equivalents.

Claims (4)

1. A low pH-based nanogold colorimetric method for rapidly detecting food-borne pathogenic bacteria is characterized by comprising the following steps:

(1) preparing nano gold particles: adding 20 mu L of chloroauric acid with the concentration of 0.5M into 9.6 mL of ultrapure water, heating to boil, quickly adding 400 mu L of sodium citrate solution with the mass fraction of 1%, continuing heating and stirring for 10 min until the solution becomes transparent wine red, and cooling to room temperature;

(2) shake culturing the bacteria to be detected in a liquid culture medium at 37 ℃, and taking the bacteria as a sample to be detected under a specific condition;

(3) preparing a hydrochloric acid solution with pH =1, and providing a low pH environment for detection of bacteria to be detected;

(4) taking 40 muL of the nano-gold solution prepared in the step (1), and adding 20 muL of ultrapure water solution and 20 muL (3) of hydrochloric acid solution with pH =1 to serve as a control group;

(5) taking 40 muL of the nano-gold solution prepared in the step (1), adding 20 muL of bacterial liquid to be detected and 20 muL of hydrochloric acid solution with PH =1 prepared in the step (3), comparing the control group prepared in the step (4), and observing color change of the nano-gold solution;

(6) reading of the results: (4) the prepared control group should keep the wine red of the nano-gold solution, and if the color of the control group also changes, the detection result is invalid; (5) if the wine red color of the medium detection system is changed into purple or blue, the medium detection system is positive, and the existence of the bacteria to be detected is indicated.

2. The low-pH-based nanogold colorimetric method for rapidly detecting food-borne pathogenic bacteria according to claim 1, characterized in that: and (3) ultrapure water in the step (1) is required to be sterilized in advance.

3. The low-pH-based nanogold colorimetric method for rapidly detecting food-borne pathogenic bacteria according to claim 1, characterized in that: the bacteria to be detected in the step (2) are staphylococcus aureus, shigella, pseudomonas aeruginosa, vibrio parahaemolyticus, salmonella typhimurium, escherichia coli O157: H7 or bacillus subtilis.

4. The low-pH-based nanogold colorimetric method for rapidly detecting food-borne pathogenic bacteria according to claim 1, characterized in that: the specific conditions in the step (2) refer to: after the bacteria to be detected are activated, the bacteria are cultured in a liquid culture medium to the logarithmic growth phase, and the bacteria are centrifugally collected and resuspended in ultrapure water for detection.

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