Method for simultaneously detecting various food-borne pathogenic bacteria based on colorimetric array sensor constructed by monatomic nano-enzyme

Abstract

The invention discloses a method for simultaneously detecting various food-borne pathogenic bacteria by using a colorimetric array sensor constructed based on monatomic nano-enzyme, which realizes high-sensitivity, rapid and simultaneous detection of various food-borne pathogenic bacteria, improves the sensitivity of the sensor, shortens the detection time, simplifies the operation steps, reduces the detection cost, expands the detection range and has strong reliability. The method has important research significance in the field of food safety.

Description

Technical Field

[0001] The present invention relates to a method for detecting foodborne pathogens, and in particular to a method for simultaneously detecting multiple foodborne pathogens using a colorimetric array sensor constructed based on single-atom nanozymes, belonging to the field of biological detection technology. Background Art

[0002] Food pathogens are pathogenic bacteria that can cause food poisoning or spread through food. Pathogenic bacteria directly or indirectly contaminate food and water sources. Oral infection in humans can lead to the occurrence of intestinal infectious diseases, food poisoning, and the spread of infectious diseases in livestock and poultry. Foodborne pathogens are an important source of food safety issues.

[0003] Common foodborne pathogen detection methods include:

[0004] 1. Enzyme-linked immunosorbent assay (ELISA). ELISA has strong specificity and high reaction sensitivity. Currently, there are commercialized Listeria monocytogenes detection kits, but they require pre-enrichment, which is time-consuming and the preparation of monoclonal antibodies is expensive.

[0005] 2. Immunochromatographic test strips: Although the immunochromatographic test strips are simple to operate, require low technical skills from the test personnel, and take a short time, their sensitivity is poor.

[0006] 3. Polymerase chain reaction technology (PCR). PCR detection has the characteristics of high sensitivity, which greatly shortens the time of pre-enrichment, but its economic cost is relatively high and requires high technical operation level.

[0007] 4. Loop-mediated isothermal amplification (LAMP). The LAMP method has high sensitivity, but its amplification principle is complex and requires the design of multiple primers, resulting in a high cost for biomarkers.

[0008] 5. Biosensors. The detection method of biosensors takes a short time and can be reused, but the sensitivity needs to be improved. Most of these technologies can only detect one type of foodborne pathogens, the experimental steps are complicated, rely on professional technicians, and the detection cost is expensive. Summary of the invention

[0009] The purpose of the present invention is to overcome the deficiencies of the above-mentioned prior art. The present invention provides a method for simultaneously detecting multiple foodborne pathogens based on a colorimetric array sensor constructed based on single-atom nanozymes, and utilizes the differences between different foodborne pathogens to achieve simultaneous detection of multiple foodborne pathogens.

[0010] To achieve the above object, the present invention adopts the following technical solutions:

[0011] The method for simultaneously detecting multiple foodborne pathogens using a colorimetric array sensor constructed based on single-atom nanozymes has the following specific steps:

[0012] (1) Using Fe-NC single-atom nanozymes to construct colorimetric array sensors;

[0013] (2) Use colorimetric array sensors to detect different types and concentrations of foodborne pathogens, obtain colorimetric data, perform data analysis, and draw a standard curve;

[0014] (3) The colorimetric array sensor is applied to actual sample detection, and the quantitative detection of multiple foodborne pathogens is achieved through standard curves.

[0015] As one of the preferred technical solutions, the multiple foodborne pathogens include: Staphylococcus aureus (S. aureas), Salmonella (SE), Vibrio vulnificus (VV), Vibrio harveyi (VH), Listeria monocytogenes (LM), and Vibrio parahaemolyticus (VP).

[0016] As one of the preferred technical solutions, in step (1), the Fe-NC single-atom nanozyme is prepared by the following method: first, ferric chloride and polyvinyl pyrrolidone are added to a saturated chloride solution, stirred for 2 hours until uniformly dispersed, and then vacuum freeze-dried to obtain a light yellow powder, and finally the powder is annealed, washed, vacuum dried, and calcined to obtain.

[0017] As one of the further preferred technical solutions, the usage ratio of saturated chloride salt solution, ferric chloride and polyvinyl pyrrolidone is 10mL:18mg:1g, wherein the saturated chloride salt solution is a saturated potassium chloride solution or a saturated sodium chloride solution.

[0018] As one of the further preferred technical solutions, the process conditions of the annealing treatment are: heating to 900° C. at 5° C. / min, annealing for 2 hours at the temperature under a nitrogen atmosphere, and naturally cooling to room temperature.

[0019] As one of the further preferred technical solutions, the washing process conditions are: washing with 0.5 mol / L sulfuric acid solution and deionized water alternately for 3 times to remove the template polyvinyl pyrrolidone.

[0020] As one of the further preferred technical solutions, the process conditions for vacuum drying are: vacuum drying at 60° C. for 12 hours.

[0021] As one of the further preferred technical solutions, the calcination process conditions are: calcination at 900° C. for 2 hours.

[0022] As one of the preferred technical solutions, in step (1), the method for constructing the colorimetric array sensor is as follows:

[0023] (1-1) First, a variety of foodborne pathogens are cultured separately to obtain corresponding bacterial solutions, and then gradient dilutions are performed to obtain samples of different concentrations;

[0024] (1-2) Then, the sample to be tested, Fe-NC single-atom nanozymes, and hydrogen peroxide solution of different concentrations are mixed and incubated, and TMB (tetramethylbenzidine) solution, OPD (o-phenylenediamine) solution, ABTS (2-2'-diamine-bis(3-ethylbenzothiazoline-6-sulfonic acid) diamine salt) solution, and buffer solution are added to react, and the construction of the colorimetric array sensor is completed.

[0025] As one of the further preferred technical solutions, in step (1-1), the concentration of the sample to be tested is 10 9 CFU / ml, 10 8 CFU / ml, 10 7 CFU / ml, 10 6 CFU / ml, 10 5 CFU / ml.

[0026] As one of the further preferred technical schemes, in step (1-2), the volume ratio of the sample to be tested, Fe-NC single-atom nanozyme, hydrogen peroxide solution, TMB solution, OPD solution, ABTS solution, and buffer solution is 1:1:1:1:1:1:1:5; wherein the concentration of Fe-NC single-atom nanozyme is 0.02 mg / ml, the mass concentration of hydrogen peroxide solution is 10%, the concentration of TMB solution is 0.625 mM, the concentration of OPD solution is 0.625 mM, the concentration of ABTS solution is 0.625 mM, and the buffer solution is NaAc-HAc buffer with a pH of 4.

[0027] As one of the further preferred technical solutions, in step (1-2), the incubation conditions are: incubation at 37°C for 30 minutes; and the reaction conditions are: reaction at 37°C for 15 minutes.

[0028] As one of the preferred technical solutions, in step (2), principal component analysis (PCA), hierarchical cluster analysis (HCA) or radar chart analysis is performed on foodborne pathogens of different types and concentrations, and the data of the first influencing factor (PC1) is analyzed and processed to obtain a linear relationship equation between concentration and PC1, and a standard curve is drawn.

[0029] As one of the preferred technical solutions, in step (3), the actual sample includes but is not limited to: lake water, sea water, tap water, and coconut juice.

[0030] As one of the preferred technical solutions, in step (3), the actual sample is tested using a spike recovery method.

[0031] Beneficial effects of the present invention:

[0032] The present invention constructs a colorimetric array sensor based on Fe-NC single-atom nanozymes, which can achieve high-sensitivity and rapid simultaneous detection of multiple foodborne pathogens, improve the sensitivity of the sensor, shorten the detection time, simplify the operation steps, reduce the detection cost, expand the detection range, and has strong reliability, which has important research significance in the field of food safety.

[0033] The working principle of the present invention is as follows: the Fe-NC single-atom nanozyme prepared by the biomimetic method of the present invention has high peroxidase-like activity, and can be used as a signal amplification catalyst in the colorimetric reaction to oxidize colorless tetramethylbenzidine (TMB) into a blue oxidation state TMBox, oxidize colorless o-phenylenediamine (OPD) into a yellow oxidation state OPDox, and oxidize light green 2-2'-diamine-bis(3-ethylbenzothiazoline-6-sulfonic acid) diamine salt (ABTS) into a green oxidation state ABTSox. The differences of foodborne pathogens are used to construct a colorimetric sensor array, and a variety of foodborne pathogens and their mixtures are successfully identified through data analysis methods such as principal component analysis (PCA).

[0034] In summary, traditional biosensor technology can only detect a single substance to be tested, while the present invention achieves the simultaneous detection of multiple foodborne pathogens and their mixtures, optimizes the operating steps, shortens the detection time, reduces the experimental cost, improves the sensitivity of the biosensor, expands the detection range, has strong stability, and the experimental results obtained are more accurate and reliable. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 is a comparison between Fe-NC single-atom nanozymes and other single-atom nanozymes; A is the absorption spectra of different single-atom nanozymes (the curves are as follows: (a) Fe-NC; (b) Fe-NC / S; (c) Fe-P / NC); B is that Fe-NC single-atom nanozymes can catalyze TMB, OPD and ABTS simultaneously;

[0036] Figure 2 is the PCA graph of foodborne pathogens at different concentrations, where A is 10 8 CFU, B is 10 7 CFU, C is 10 6 CFU, D is 10 5 CFU;

[0037] Figure 3 is the standard curve, where A is Listeria LM and B is Vibrio vulnificus VV;

[0038] Figure 4are mixtures of foodborne pathogens at different ratios, where A is a mixed bar graph of S. aureas: VV at different ratios (1:1, 3:7, 7:3, 1:9 and 9:1), B is a mixed bar graph of S. aureas: VP at different ratios (1:1, 3:7, 7:3, 1:9 and 9:1), C is a PCA graph of S. aureas: VV at different ratios (1:1, 3:7, 7:3, 1:9 and 9:1), and D is a PCA graph of S. aureas: VP at different ratios (1:1, 3:7, 7:3, 1:9 and 9:1);

[0039] Figure 5 This is the PCA diagram of six foodborne pathogens detected by actual samples, where A is sea water, B is lake water, C is tap water, and D is coconut juice. DETAILED DESCRIPTION

[0040] The present invention is further described below in conjunction with the accompanying drawings and embodiments. It should be noted that the following description is only for explaining the present invention and does not limit its contents.

[0041] The reagents required in the experiment were sterilized by high temperature and high pressure, and all experimental operations were carried out in a clean bench. The clean bench was sterilized by ultraviolet irradiation for 15 minutes before use to ensure that the entire experiment was carried out in a sterile environment.

[0042] 1. Preparation of Fe-NC single-atom nanozymes

[0043] 2. Preparation of Fe-NC: 18 mg of ferric chloride and 1 g of polyvinyl pyrrolidone were added to 10 mL of saturated potassium chloride or sodium chloride solution, stirred for 2 hours until uniformly dispersed, and then vacuum freeze-dried to obtain a light yellow powder. Finally, the powder was annealed at 900 ° C (heated to 900 ° C at 5 ° C / min) under a nitrogen flow for 2 hours and naturally cooled to room temperature. Then, the sample was washed alternately with 0.5 M dilute sulfuric acid and deionized water three times to remove the template. Then it was dried in a vacuum oven at 60 ° C for 12 hours. Finally, it was calcined at 900 ° C for 2 hours to obtain Fe-NC.

[0044] 2. Optimization of experimental conditions

[0045] In order to ensure the best catalytic effect of Fe-NC single-atom nanozyme, the reaction conditions were optimized by the control variable method: before the experiment, the Fe-NC single-atom nanozyme suspension was ultrasonically treated for 30 minutes to ensure its uniform dispersion in the solution. The experimental conditions included the pH value of NaAc-HAc buffer (1.0-8.0), the concentration of Fe-NC single-atom nanozyme (0.0-0.06 mg / ml), the concentration of TMB (0.0-0.875 mM), the concentration of OPD (0.0-0.875 mM), the concentration of ABTS (0.0-0.875 mM) and the reaction temperature (25-55 °C).

[0046] The controlled variable method was used to conduct a single-factor experiment to explore the optimal experimental conditions (the Fe-NC single-atom nanozyme suspension was ultrasonically treated for 30 minutes to ensure its uniform dispersion in the solution).

[0047] The concentration of Fe-NC single-atom nanozyme was selected as 0.05mg / ml, the concentration of TMB was 0.5mM, the concentration of H2O2 was 10%, and the reaction temperature was 30℃. The pH of the buffer solution was changed (1-8) to explore the optimal pH value of the reaction. 25μL Fe-NC single-atom nanozyme, 25μL TMB, 25μL H2O2 and 125μL buffer were added to the enzyme-labeled well and reacted at 30℃ for 15min. The absorbance value at 650nm was detected using a multifunctional microplate reader. The data analysis showed that the absorbance value reached when the pH of the buffer was 4 was the largest, so the catalytic effect was best under this condition.

[0048] The pH value of the buffer solution was 4, the TMB concentration was 0.5mM, the H2O2 mass concentration was 10%, and the reaction temperature was 30°C. The concentration of Fe-NC single-atom nanozyme was changed (0.0-0.06mg / ml) to explore the optimal reaction concentration of Fe-NC single-atom nanozyme. 25μL Fe-NC single-atom nanozyme, 25μL TMB, 25μL H2O2 and 125μL buffer were added to the ELISA well and reacted at 30°C for 15min. The absorbance value at 650nm was detected using a multifunctional ELISA reader. The data analysis showed that when the concentration of Fe-NC single-atom nanozyme reached 0.02mg / ml, the catalytic rate was significantly slowed down, so the catalytic effect was best under this condition.

[0049] The pH of the buffer was 4, the concentration of Fe-NC single-atom nanozyme was 0.02 mg / ml, the concentration of H2O2 was 10%, the reaction temperature was 30°C, and the TMB concentration (0.0-0.875 mM) was changed to explore the optimal substrate concentration of the reaction. Analysis of the experimental data showed that the best catalytic effect was achieved when the TMB concentration was 0.625 mM.

[0050] The pH of the buffer solution was 4, the concentration of Fe-NC single-atom nanozyme was 0.04 mg / ml, the concentration of H2O2 was 10%, and the concentration of TMB was 0.0625 mM. The reaction temperature was changed to explore the effect of temperature on the catalytic reaction. The absorbance at 650 mM was detected by an ELISA instrument and the best catalytic effect was achieved at a reaction temperature of 35°C.

[0051] 3. The pH of the buffer solution is 4, the concentration of Fe-NC single-atom nanozyme is 0.02 mg / ml, the reaction temperature is 35°C, and the TMB concentration is 0.0625 mM. The H2O2 concentration is changed to explore the optimal reaction conditions. The absorbance at 650 mM was detected by an ELISA instrument and the best catalytic effect was achieved when the H2O2 mass concentration was 10%. Therefore, the optimal experimental conditions were 0.02 mg / ml Fe-NC single-atom nanozyme concentration, 0.625 mM TMB concentration, pH 4, 10% H2O2 mass concentration, and 35°C reaction temperature.

[0052] 4. Cultivation of foodborne pathogens

[0053] In a sterile environment, Staphylococcus aureus (S. aureas), Salmonella (SE), Vibrio vulnificus (VV), Harveyella (VH), Listeria (LM) and Vibrio parahaemolyticus (VP) were inoculated with 1 ml of liquid culture medium respectively and the liquid culture medium was placed in a constant temperature shaker at 220 r / min and 37°C for 10 hours to obtain the original bacterial solution. In a sterile environment, 1 ml of the original bacterial solution was centrifuged at high speed (10000 r / min) for 10 minutes, and after discarding the supernatant, 1 ml of 0.85% sodium chloride aqueous solution was added to purify the strain. The purified six foodborne pathogens were gradiently diluted to obtain samples of different concentrations (the pathogen concentration was 10 9 CFU / ml, 10 8 CFU / ml, 10 7 CFU / ml, 10 6 CFU / ml, 10 5 CFU / ml). Take 1 ml of different types of foodborne pathogens and spread them on solid culture medium, culture and count them overnight at 35°C (the average value of 3 groups of data is taken) (Listeria and Vibrio vulnificus were purchased from Beina Biotechnology, Vibrio harveyi was purchased from Shanghai Fuxiang Biotechnology Co., Ltd., and solid culture medium was purchased from Solebao Biotechnology Co., Ltd.).

[0054] 5. Construction of Colorimetric Sensor Array

[0055] Fe-NC single-atom nanozymes have high peroxidase-like activity, such as Figure 1 As shown in A, compared with other nanozymes of the same type under the same experimental conditions at the same concentration (Fe-NC / S[1] and Fe-P / NC [2] All were prepared by the applicants themselves). The absorbance value of Fe-NC single-atom nanozyme at 650nm is significantly higher than that of other nanozymes (curve a). In addition, Fe-NC single-atom nanozyme can simultaneously catalyze TMB, OPD and ABTS ( Figure 1 Therefore, Fe-NC single-atom nanozymes are good materials for constructing colorimetric sensing arrays.

[0056] Under the optimal conditions, 25 μL of 6 different concentrations of foodborne pathogens and ultrapure water (3 blank control groups), 25 μL of Fe-NC single-atom nanozymes and 10% H2O2 solution were added to the ELISA wells and incubated at 37°C for 30 min. Then, 25 μL of TMB solution (0.625 mM), 25 μL of OPD solution (0.625 mM), 25 μL of ABTS solution (0.0625 mM) and 125 μL of buffer solution were added and reacted at 35°C for 15 min. UV-vis related experimental data were recorded according to the above experimental scheme.

[0057] In order to further verify the recognition performance of the colorimetric sensor array, experiments were conducted on mixing foodborne pathogens in different proportions. Staphylococcus aureus (S. aureas) and Vibrio vulnificus (VV) were uniformly mixed in a ratio of 1:1, 3:7, 7:3, 1:10, and 10:1 (volume ratio) and then the experiment was carried out according to the above experimental scheme. The experimental results showed good discrimination. Staphylococcus aureus (S. aureas) and Vibrio parahaemolyticus (VP) were uniformly mixed in a ratio of 1:1, 3:7, 7:3, 1:10, and 10:1 (volume ratio) and the above experiment was repeated. The results are as follows Figure 4 As shown in the figure, the constructed colorimetric sensor array has good discrimination for different proportions of foodborne pathogens, which is clearly distinguished in the bar graph. PCA was also used for analysis. Figure 4 All confidence intervals in C and D do not overlap, demonstrating that the constructed colorimetric sensor array can effectively identify mixtures of two foodborne pathogens in different ratios.

[0058] 6. Drawing of standard curve ( Figure 2 , Figure 3 )

[0059] Under optimal conditions, the colorimetric sensor array was used to detect different types and concentrations of foodborne pathogens to obtain colorimetric data. 9 CFU / ml, 10 8 CFU / ml, 10 7 CFU / ml, 10 6 CFU / ml, 10 5CFU / ml) of foodborne pathogens were subjected to principal component analysis (PCA) and the data of the first influencing factor (PC1) were analyzed and processed to obtain the linear relationship equation between concentration and PC1: the linear equation between the first influencing factor and concentration of Listeria monocytogenes (LM) was y=1.3487x-3.0525; the linear equation between the first influencing factor and concentration of Vibrio vulnificus (VV) was y=1.3487x-3.0525.

[0060] 7. Actual sample testing

[0061] The actual samples were tested using the spike recovery method. Six concentrations of 10 6 CFU / ml of foodborne pathogens (Staphylococcus aureus (S.aureas), Salmonella (SE), Vibrio vulnificus (VV), Harvey (VH), Listeria (LM), Vibrio parahaemolyticus (VP)). 25 μL of the four samples and the four samples without foodborne pathogens, 25 μL of Fe-NC single-atom nanozymes and 10% H2O2 solution were added to the enzyme-labeled wells and incubated at 37°C for 30 minutes. Then, 25 μL of TMB solution (0.625 mM), 25 μL of OPD solution (0.625 mM), 25 μL of ABTS solution (0.0625 mM) and 125 μL of buffer solution were added and reacted at 35°C for 15 minutes. UV-vis related experimental data were recorded according to the above experimental scheme.

[0062] The constructed colorimetric sensor array has been shown to be highly reliable and can be used in seawater ( Figure 5 Chinese A), tap water ( Figure 5 Middle C), Lake ( Figure 5 B) and coconut milk ( Figure 5 D) Detection of foodborne pathogens in samples.

[0063] Although the above describes the specific implementation mode of the present invention in conjunction with the accompanying drawings, it is not intended to limit the scope of protection of the present invention. Based on the technical solution of the present invention, various modifications or variations that can be made by those skilled in the art without creative work are still within the scope of protection of the present invention.

[0064] References

[0065] [1] RuiLi,Xiaoting He,Rida Javed,et al.Switching on-off-oncolorimetric sensor based on Fe-N / S-C single-atom nanozyme for ultrasensitiveand multimodal detection of Hg 2+ .Science of The Total Environment,2022,834:155428.

[0066] [2]Ying Li,Rida Javed,Rui Li,et al.Acolorimetric smartphone-basedsensor for on-site AA detection in tropical fruits using Fe-P / N-C single-atomnanoenzyme.Food Chemistry,2022,22,135017。

Claims

1. A method for simultaneously detecting multiple foodborne pathogens based on a colorimetric array sensor constructed using single-atom nanozymes, characterized in that: The specific steps are as follows: (1) Using Fe-NC single-atom nanozymes to construct colorimetric array sensors; (2) Use colorimetric array sensors to detect different types and concentrations of foodborne pathogens, obtain colorimetric data, perform data analysis, and draw a standard curve; (3) The colorimetric array sensor is applied to actual sample detection, and the quantitative detection of multiple foodborne pathogens is achieved through standard curves.

2. The method according to claim 1, characterized in that The various foodborne pathogens include: Staphylococcus aureus, Salmonella, Vibrio vulnificus, Harveyella, Listeria, and Vibrio parahaemolyticus.

3. The method according to claim 1, characterized in that: In step (1), the Fe-NC single-atom nanozyme is prepared by the following method: first, ferric chloride and polyvinyl pyrrolidone are added to a saturated chloride solution, stirred for 2 hours until uniformly dispersed, then vacuum freeze-dried to obtain a light yellow powder, and finally the powder is annealed, washed, vacuum dried, and calcined to obtain.

4. The method according to claim 1, characterized in that In step (1), the method for constructing the colorimetric array sensor is as follows: (1-1) First, a variety of foodborne pathogens are cultured separately to obtain corresponding bacterial solutions, and then gradient dilutions are performed to obtain samples of different concentrations; (1-2) Then, the sample to be tested, Fe-NC single-atom nanozyme, and hydrogen peroxide solution of different concentrations are mixed and incubated, and TMB solution, OPD solution, ABTS solution, and buffer solution are added to react, thereby completing the construction of the colorimetric array sensor.

5. The method according to claim 4, characterized in that In step (1-1), the concentration of the sample to be tested is 10 9 CFU / ml, 10 8 CFU / ml, 10 7 CFU / ml, 10 6 CFU / ml, 10 5 CFU / ml.

6. The method according to claim 4, characterized in that In step (1-2), the volume ratio of the sample to be tested, Fe-NC single-atom nanozyme, hydrogen peroxide solution, TMB solution, OPD solution, ABTS solution, and buffer solution is 1:1:1:1:1:1:1:5; wherein the concentration of Fe-NC single-atom nanozyme is 0.02 mg / ml, the mass concentration of hydrogen peroxide solution is 10%, the concentration of TMB solution is 0.625 mM, the concentration of OPD solution is 0.625 mM, the concentration of ABTS solution is 0.0625 mM, and the buffer solution is a NaAc-HAc buffer with a pH of 4.

7. The method according to claim 4, characterized in that In step (1-2), the incubation conditions are: incubation at 37°C for 30 minutes; and the reaction conditions are: reaction at 37°C for 15 minutes.

8. The method according to claim 1, characterized in that: In step (2), principal component analysis, cluster analysis or radar chart analysis is performed on foodborne pathogens of different types and concentrations, and the data of the first influencing factor is analyzed and processed to obtain a linear relationship equation between concentration and PC1, and a standard curve is drawn.

9. The method according to claim 1, characterized in that: In step (3), the actual samples include but are not limited to: lake water, sea water, tap water, and coconut water.

10. The method according to claim 1, characterized in that In step (3), the actual sample is tested using a spike recovery method.

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