CN110687110B - Nanogold colorimetric method for rapidly detecting food-borne pathogenic bacteria based on low pH - Google Patents
Nanogold colorimetric method for rapidly detecting food-borne pathogenic bacteria based on low pH Download PDFInfo
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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 and the like 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
Technical Field
The invention relates to a nanotechnology and a biotechnology, and belongs to the technical field of food safety. In particular to a simple and rapid method for detecting various pathogenic bacteria in a low pH environment by using nanogold prepared by a traditional sodium citrate reduction method.
Background
Food-borne pathogenic bacteria have great harm to food safety, and the caused food-borne diseases are easy to spread and have serious harm to human bodies. Therefore, the detection of pathogenic bacteria in food has important significance and is an important task in society, economy and public health. Traditional culture assays, which are the basis of many routine tests, are representative and standard internationally and reliable methods for detecting food-borne pathogens, but these assays often require significant time, labor and instrument costs. Therefore, methods for detecting food-borne pathogens that are faster, more convenient, and more suitable for specific situations, such as field testing, are increasingly in need and interest of people.
Nano materials have been developed rapidly in recent years, and are widely used in the fields of biology, medicine, and the like. The nano material has larger specific surface area, wherein the nano gold has the characteristics of high absorption coefficient, strong scattering property, unique local surface plasmon resonance and the like. The aggregation of the nano-gold particles causes a red shift in the absorption spectrum, while the nano-gold solution appears to change from red to purple, blue depending on the degree of aggregation, due to its close proximity to the change in surface plasmon resonance. And the gold nanoparticles can be combined with some biomolecules such as proteins, nucleic acids, etc. The unique properties of the nanogold enable the nanogold to be frequently used as a colorimetric biosensor to be applied to detection, and have the advantages of rapidness, intuition, accuracy and high efficiency.
Accordingly, the invention is provided.
Disclosure of Invention
Aiming at the problems in the prior art, the invention provides a nanogold colorimetric method for rapidly detecting food-borne pathogenic bacteria based on low pH, which provides a method for rapidly and intuitively detecting a plurality of pathogenic bacteria in food by utilizing the principle that protein on the surface of bacteria is changed into positive charge under the pH lower than the isoelectric point of the protein on the surface of the bacteria through electrostatic interaction based on the unique optical property of nanogold and negative charge carried by the surface of the nanogold. The method therefore comprises: after preparing the nanogold by using a sodium citrate reduction method, adding a bacterial solution to be detected into a nanogold solution, adjusting the pH of the mixed solution by using HCl with pH =1, and finally performing color comparison with a control group to quickly and visually read the result, wherein the final pH of the mixed solution is about 3.16. When the pH value is too low, the nano gold solution is unstable and cannot be detected; when the pH is more than 3.5, the pH cannot be guaranteed to be lower than the isoelectric points of all proteins, and the detection sensitivity is influenced. The method has the advantages of high detection speed, convenient operation, simple reagent and no need of instruments and equipment, and is suitable for field detection or qualitative detection in mass products.
The specific operation steps are as follows:
(1) preparing nano gold particles: adding 20 microliter of chloroauric acid with the concentration of 0.5M into 9.6 mL of ultrapure water, heating to boil, quickly adding 400 microliter of sodium citrate solution with the mass fraction of 1%, continuing heating and stirring for about 10 min until the solution becomes transparent wine red, and cooling to room temperature.
(2) The bacteria to be detected cultured in the liquid culture medium are taken, centrifuged and resuspended in sterilized ultrapure water as the sample solution to be detected.
(3) Preparing hydrochloric acid solution with pH of 1 as a low pH environment regulator;
(4) and (4) taking 40 muL of the nano-gold solution prepared in the step (1), adding 20 muL of ultrapure water and 20 muL of hydrochloric acid solution with the pH value of 1 prepared in the step (3), and taking the solution as a control group.
(5) And (3) detection of bacteria to be detected: and (3) taking 40 muL of the nano-gold solution prepared in the step (1), adding the bacterial liquid prepared in the step (2) and the hydrochloric acid solution with the PH of 1 prepared in the step (3), and comparing the control group prepared in the step (4) to observe the color change of the 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.
Further, the ultrapure water in the step (1) needs to be sterilized in advance.
Further, 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.
Further, 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 phase, and the bacteria are centrifugally collected and resuspended in ultrapure water for detection.
The final pH value of the solution system in the detection method is 3.16, and the nano gold in the solution is in a stable dispersion state under the pH value, namely, the nano gold is represented as a wine red solution.
The result of the detection method is read through the color change of the nanogold, the nanogold interacts with bacteria to be detected after the bacteria liquid to be detected is added and the pH value is adjusted, so that the nanogold is changed from the original dispersed state to the aggregated state and is shown to be changed from the original wine red to purple or blue.
The detection method is rapid in detection, the result can be observed after the pH value is adjusted, and the required time is less than 5 min.
Compared with other traditional detection methods, the method has the following beneficial effects:
(1) the reagent used for detection is stable and simple, and the operation is convenient and quick;
(2) the invention has high detection speed, and the bacteria to be detected can be immediately read after being added into the nano-gold solution with low pH value and uniformly mixed, and the detection time is within 5 min.
(3) The invention does not need complex instrument equipment for detection, can read the result directly by naked eyes, and has strong intuition of the detection result.
Drawings
FIG. 1 is UV-vis spectra of nano-gold prepared by sodium citrate reduction method in different pH values in the present invention.
FIG. 2 is a zeta potential diagram of the gold nanoparticles in the control group of the detection system of the present invention.
FIG. 3 is a UV-vis spectrum of nanogold when a low pH nanogold colorimetric method is used for detecting staphylococcus aureus with different concentrations in the invention.
FIG. 4 is a UV-vis spectrum of nano-gold in the detection of shigella at different concentrations by low pH nano-gold colorimetric method in the present invention.
FIG. 5 is an FE-SEM image of the low pH gold nanoparticle solution of the present invention for detecting Staphylococcus aureus under different magnifications.
FIG. 6 is an FE-SEM image of Shigella detection with low pH nanogold solution according to the invention under different magnifications.
Detailed Description
The present invention will be further described with reference to the following examples. It is to be understood that the following examples are illustrative only and are not intended to limit the scope of the invention, which is to be given numerous insubstantial modifications and adaptations by those skilled in the art based on the teachings set forth above.
Example 1
The low pH-based nanogold colorimetric method for rapidly detecting food-borne pathogenic bacteria comprises the following steps:
(1) preparing nano gold particles: adding 20 muL of chloroauric acid with final degree of 0.5M into 9.6 mL of ultrapure water, heating to boiling, quickly adding 400 muL of sodium citrate solution with mass fraction of 1%, continuously heating and stirring for about 10 min until the solution becomes transparent wine red, and cooling to room temperature.
(2) Respectively centrifuging and suspending Staphylococcus aureus and Shigella cultured in liquid culture medium in sterilized ultrapure water, measuring OD value, and diluting the bacteria solution to OD values of 0.5, 0.1, 0.05 and 0.02.
(3) Preparing hydrochloric acid solution with pH of 1 as a low pH environment regulator;
(4) and (4) taking 40 muL of the nano-gold solution prepared in the step (1), adding 20 muL of ultrapure water and 20 muL of hydrochloric acid solution with the pH value of 1 prepared in the step (3), and taking the solution as a control group.
(5) And (3) detection of bacteria: respectively taking staphylococcus aureus bacteria liquid and shigella bacteria liquid with different concentrations prepared in 20 muL (2) from 1.5 mL of centrifuge tubes, respectively adding 40 muL of the nano-gold solution prepared in the step (1) into each centrifuge tube filled with the bacteria liquid, uniformly mixing, then adding hydrochloric acid solution with pH of 1 prepared in 20 muL (3) into each nano-gold solution, comparing the control group prepared in the step (4), and observing the color change of the solution in each centrifuge tube of 1.5 mL.
(6) Reading of the results: (4) the color of the control group prepared in the step (1) is still wine red of the nanogold solution, when the OD value of staphylococcus aureus is 0.5, the nanogold changes into blue, and then the OD value of the dilution degree is still wine red; the nano gold solution turns blue when the OD value of the Shigella is 0.5 and 0.1, the nano gold solution turns purple when the OD value is 0.05, and the nano gold solution still turns wine red when the OD value is 0.02.
Therefore, in this example, the detection method proposed by the present invention can be used to detect staphylococcus aureus and shigella. The detection sensitivity of the kit to staphylococcus aureus is OD 0.5, and the concentration of corresponding bacterial liquid obtained by a plate counting method is 1.6 multiplied by 10 7 CFU/mL; the shigella is OD 0.05, and the corresponding bacterial liquid concentration is 3.3 × 10 by using a plate counting method 5 CFU/mL。
FIG. 1 is UV-vis spectra of nano-gold prepared by sodium citrate reduction method in different pH. And (3) respectively mixing the nano gold solution with the concentration of 1 mM with ultrapure water (control) and an HCl solution with the pH value of 1-6 in an equal volume, standing for 10 min, measuring the UV-vis spectrum of the mixture, and photographing and recording the color of the solution. As can be seen from the UV-vis absorption spectrum in FIG. 1, the control group and the groups having pH 3 to 6 had the characteristic absorption peak (526 nm) of the gold nanoparticles, and the solution appeared wine red. However, the peak values of the pH 1 and pH 2 show red shift, which indicates the aggregation of the nano-gold particles, and the solution color changes to blue, and the nano-gold is unstable. Therefore, the pH value with the final pH value larger than 3 is selected as the environment for detecting the pH value.
FIG. 2 is a zeta potential diagram of a control nanogold in a detection system according to the invention; the final pH of the control group is about 3.16, and the nano gold of the control group is negatively charged through potential detection, and the zeta potential of the nano gold is-21.6 +/-1.4 mV.
FIG. 3 shows the UV-vis spectra of Staphylococcus aureus in different concentrations in low pH nanogold according to the invention. According to the detection method provided by the 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 the gold nanoparticles, and the solution is wine red. However, the UV-vis spectrum with an OD of 0.5 shows a red shift, indicating that the gold nanoparticles aggregate on the bacterial surface at low pH and the solution turns blue in color.
FIG. 4 shows UV-vis spectra of Shigella at different concentrations in low pH nanogold in accordance with the present invention. According to the detection method provided by the invention, the UV-vis spectrums of the control group and the group with the OD value of 0.02 have the characteristic absorption peak (526 nm) of the gold nanoparticles, and the solution presents wine red. However, the UV-vis spectra with OD values of 0.5, 0.1 and 0.05 showed a red shift, indicating that the gold nanoparticles aggregated on the bacterial surface at low pH and the solution turned blue or purple in color.
FIG. 5 is an FE-SEM image of the low pH gold nanoparticle solution of the present invention under different magnifications when detecting Staphylococcus aureus. And dropwise adding the detection group mixed solution with the Staphylococcus aureus OD value of 0.5 onto a silicon wafer, drying, and observing under an FE-SEM. As can be seen from the figure, the pH value is lower than the isoelectric point of the protein on the surface of the staphylococcus aureus, so that the protein is positively charged, and the nano gold particles with negative charges are adsorbed on the surface of the staphylococcus aureus under the action of electrostatic force.
FIG. 6 is an FE-SEM image of Shigella detection under low pH nanogold solution of the invention under different magnifications. And (3) dropwise adding the detection group mixed solution with the Shigella OD value of 0.5 onto a silicon wafer, drying, and observing under an FE-SEM. As can be seen from the figure, the phenomenon that the gold nanoparticles are completely wrapped on the surfaces of the bacteria in the FE-SEM image of staphylococcus aureus is different, the gold nanoparticles are only gathered and wrapped at two ends of the Shigella in a large amount, and under the same condition, the gold nanoparticles can adsorb more Shigella, so that the detection sensitivity is improved compared with the detection sensitivity of the staphylococcus aureus.
Meanwhile, the nano-gold colorimetric method for quickly detecting the food-borne pathogenic bacteria based on low pH is used for detecting pseudomonas aeruginosa, vibrio parahaemolyticus, salmonella typhimurium, escherichia coli O157: H7 and bacillus subtilis. The detection sensitivity of the kit 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 and OD 0.1 for Bacillus subtilis, and the concentration of the corresponding bacterial liquid is Pseudomonas aeruginosa and 4.5 × 10 for Pseudomonas aeruginosa by plate counting method 6 CFU/mL; vibrio parahaemolyticus, 5.8X 10 6 CFU/mL; salmonella typhimurium, 6.6X 10 6 CFU/mL; escherichia coli O157H 7, 4.4X 10 7 CFU/mL; bacillus subtilis, 2.8X 10 5 CFU/mL。
The foregoing shows and describes the general principles and features of the present invention, together with the advantages thereof. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, which are described in the specification and illustrated only to illustrate the principle of the present invention, but that various changes and modifications may be made therein without departing from the spirit and scope of the present invention, which fall within the scope of the invention as claimed. The scope of the invention is defined by the appended claims and equivalents thereof.
Claims (2)
1. A low pH-based nanogold colorimetric method for rapidly detecting food-borne pathogenic bacteria is characterized by comprising the following steps:
step (1) preparation of 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;
step (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;
preparing a hydrochloric acid solution with pH =1, and providing a low pH environment for detection of bacteria to be detected;
step (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 of hydrochloric acid solution with pH =1 prepared in the step (3) to serve as a control group;
step (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;
reading the result of the step (6): keeping the drab color of the nano gold solution in the control group prepared in the step (4), and if the color of the control group is changed, the detection result is invalid; if the detection system in the step (5) is changed from wine red to purple or blue, the detection system is positive, and the bacteria to be detected exist;
the final pH value of the solution system in the detection method is 3.16;
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;
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.
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.
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