Colorimetric detection method for escherichia coli based on p-benzoquinone mediated manganese dioxide nanosheet decomposition

Abstract

The invention discloses an Escherichia coli colorimetric detection method based on p-benzoquinone mediated manganese dioxide nanosheet decomposition, which comprises the following steps: the manganese dioxide nanosheet has oxidase-like activity, and directly catalyzes a colorless substrate 3, 3 ', 5, 5'-tetramethylbenzidine (TMB) to generate a blue oxidation product oxTMB (the characteristic absorption peak is 652nm) under an acidic condition without the participation of hydrogen peroxide (H2O2) and HRP (horse radish peroxidase). The escherichia coli reduces p-benzoquinone into hydroquinone (HQ), so that the manganese dioxide nanosheets are decomposed into manganese ions (Mn < 2 + >), oxidase-like activity of the manganese dioxide nanosheets is lost, and generation of oxTMB is prevented. During detection, the absorbance of the solution is reduced along with the increase of the concentration of escherichia coli at 652 nm, and the color fades from blue to colorless. The method does not need high-end instruments, and is simple in detection process, low in cost, high in sensitivity and selectivity and suitable for rapidly and conveniently detecting escherichia coli.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a colorimetric detection method for Escherichia coli based on the decomposition of manganese dioxide nanosheets mediated by p-benzoquinone. Background Art

[0002] Escherichia coli (E. coli) is a common microorganism that causes food and drinking water contamination. Ingestion can cause cholecystitis, cholangitis, urinary tract infection, diarrhea, renal failure, neonatal meningitis, Crohn's disease, and even death. Therefore, a sensitive and rapid method for detecting E. coli is crucial to reduce economic losses and maintain public safety.

[0003] E. coli can be detected by traditional culture techniques, which are simple and easy to operate, but bacterial culture takes 2-3 days to provide results, and the application of this technology in field detection is limited due to the need for instruments such as bacterial incubators and trained staff. Currently, methods based on polymerase chain reaction, DNA microarray, DNA sequencing, enzyme-linked immunosorbent assay, immunochromatography lateral flow chromatography, quartz crystal microbalance system, surface plasmon resonance, and flow cytometry have been used to detect E. coli contamination in food and water. However, these methods require sample pretreatment, expensive instruments and reagents, and professional personnel to operate the instruments, and are prone to produce erroneous results due to cross-reactions.

[0004] Optical biosensors based on absorbance, fluorescence, luminescence or scattering measurements have attracted increasing attention due to their outstanding performance in detecting pathogens. In particular, colorimetric biosensors have shown great potential in routine and on-site detection of pathogens due to their advantages such as high sensitivity, visual detection and short response time. Summary of the invention

[0005] The present invention discloses a colorimetric detection method for Escherichia coli based on the decomposition of manganese dioxide nanosheets mediated by p-benzoquinone. The method has the advantages of high sensitivity, rapid response, simple operation and visual detection, and does not require aptamer labeling and complex operations, precise experimental instruments and specialized operators, which greatly reduces the detection cost and labor cost.

[0006] To achieve the above objectives, the present invention is implemented through the following technical solutions:

[0007] A colorimetric detection method for Escherichia coli based on p-benzoquinone-mediated decomposition of manganese dioxide nanosheets, characterized in that it comprises the following steps:

[0008] (1) Manganese dioxide nanosheets (MnO 2 Preparation of NS);

[0009] (2) Escherichia coli (E. coli) reduces benzoquinone (BQ) to produce hydroquinone (HQ);

[0010] (3) Manganese dioxide nanosheets (MnO 2 NS) solution, vortex to mix evenly, and react fully at room temperature; the temperature range of room temperature is 18℃ to 28℃, and all room temperatures in this article are considered to be 18℃ to 28℃. Then add sodium acetate (NaAc·HAc) buffer and 3,3',5,5'-tetramethylbenzidine (TMB) solution, vortex to mix evenly, and then react in the dark; finally, use a UV-visible spectrophotometer to collect the absorption spectrum curve of the reaction solution, and establish a calibration curve with the concentration of Escherichia coli (E.coli) based on the absorption peak intensity at 652nm;

[0011] (4) Add manganese dioxide nanosheets (MnO 2 NS) solution, vortex to mix it evenly, and react it fully at room temperature; then add sodium acetate (NaAc·HAc) buffer and 3,3',5,5'-tetramethylbenzidine (TMB) solution, vortex to mix it evenly, that is, mix it by a vortex oscillator, and vortex is used to simplify it in this application; and react in the dark; finally, use a UV-visible spectrophotometer to collect the absorption spectrum curve of the reaction solution, compare the absorption peak intensity of the sample to be tested at 652nm with the established calibration curve, and determine the concentration of Escherichia coli (E.coli) in the sample to be tested.

[0012] Preferably, in step (1), the manganese dioxide nanosheets (MnO 2 The preparation method of NS) is as follows:

[0013] Quickly pour the manganese dichloride (MnCl2) solution into the hydrogen peroxide (H 2 O 2 ) and tetramethylammonium hydroxide in a mixed solution. The entire mixing process needs to be completed quickly to prevent local oversaturation. At this time, the solution turns dark brown. After continuous stirring at room temperature, the unreacted impurities are removed by centrifugation. Then, the precipitated manganese dioxide is carefully washed with ultrapure water and methanol, and placed in an oven to dry, and finally block manganese dioxide is obtained. The block manganese dioxide is dissolved in water, and nanosheets are prepared by ultrasound. The undispersed particles are removed by centrifugation, and the manganese dioxide nanosheets (MnO 2 The supernatant of NS was stored in a refrigerator at 4°C until use.

[0014] Preferably, in step (3), the concentration of p-benzoquinone (BQ) is 6 mM.

[0015] Preferably, in step (3), the Escherichia coli (E. coli) reduces p-benzoquinone (BQ) to hydroquinone (HQ): p-benzoquinone (BQ) and Escherichia coli (E. coli) are mixed and placed in a 37°C bacterial incubator for reaction for 1h-1.5h.

[0016] Preferably, in step (3), the concentration of the Escherichia coli (E. coli) suspension is 101 to 106 CFU / mL.

[0017] Preferably, in step (3), the manganese dioxide nanosheets (MnO 2 NS), sodium acetate (NaAc·HAc) buffer, and 3,3',5,5'-tetramethylbenzidine (TMB) were 50 μL, 300 μL, and 3 μL, respectively. 2 The concentrations of NS and 3,3',5,5'-tetramethylbenzidine (TMB) were 1 mg / mL and 50 mM, respectively.

[0018] The concentration of NaAc·HAc buffer was 0.2 mol / L and pH 3.6.

[0019] Preferably, the volumes of the Escherichia coli (E. coli) bacterial solution and para-benzoquinone (BQ) are both 100 μL.

[0020] Preferably, the H 2 O 2 The volume of the mixed solution of 20 mL and tetramethylammonium hydroxide is 20 mL, and the volume of the MnCl2 solution is 10 mL.

[0021] Preferably, the stirring time is 12h

[0022] Preferably, the solution is centrifuged for 20 min at a rotation speed of 2000 rpm.

[0023] Preferably, the temperature of the drying oven is 60°C.

[0024] Preferably, the time for ultrasonically preparing the nanosheets is 12 hours.

[0025] The beneficial effects of the present invention are as follows:

[0026] 1. The present invention utilizes p-benzoquinone to mediate the decomposition of manganese dioxide nanosheets and constructs a colorimetric analysis method to detect Escherichia coli. First, p-benzoquinone is reduced to hydroquinone by the cell respiration of Escherichia coli. Hydroquinone has reducing properties and can decompose the added manganese dioxide nanosheets into manganese ions, causing it to lose its oxidase-like activity and be unable to continue to catalyze 3,3',5,5'-tetramethylbenzidine (TMB) to produce a blue product oxTMB. As the concentration of Escherichia coli increases, the color of the solution gradually fades from blue to colorless, which is visible to the naked eye. Visual detection of Escherichia coli can be achieved by using the change in solution color. In addition, by recording the UV-visible absorption spectrum of the reaction solution, the concentration of Escherichia coli is monitored based on the absorption peak intensity at 652nm.

[0027] 2. The present invention realizes rapid and sensitive detection of Escherichia coli based on the change of oxTMB absorbance. The analysis method is simple, and no complicated experimental instruments and professionals are required. The experimental steps are simple and easy to operate. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0029] Figure 1 A flow chart showing a method for colorimetric detection of Escherichia coli based on p -benzoquinone-mediated decomposition of manganese dioxide nanosheets;

[0030] Figure 2 The manganese dioxide nanosheets (MnO 2 NS) representation diagram; Figure 2 A is manganese dioxide nanosheets (MnO 2 NS) transmission electron microscopy (TEM) images; Figure 2 B is manganese dioxide nanosheets (MnO 2 NS) UV-visible spectrum; Figure 2 C is manganese dioxide nanosheets (MnO 2 NS) particle size distribution diagram;

[0031] Figure 3 A feasibility analysis diagram of a colorimetric detection method for Escherichia coli based on benzoquinone-mediated decomposition of manganese dioxide nanosheets is shown; Figure 3 a in the figure is manganese dioxide nanosheet (MnO 2 NS), the reaction of p-benzoquinone (BQ) and 3,3',5,5'-tetramethylbenzidine (TMB), Figure 3 b in the figure is manganese dioxide nanosheets (MnO2 NS), p-benzoquinone (BQ), Escherichia coli (E. coli), and 3,3',5,5'-tetramethylbenzidine (TMB);

[0032] Figure 4 The relationship between E. coli concentration and the colorimetric reaction results is shown; Figure 4 A is the UV absorption spectrum of E. coli at different concentrations and its corresponding visual detection photos; Figure 4 B shows the linear relationship between the change in the absorption peak intensity at 652 nm and the logarithmic concentration of E. coli, with the concentration range of E. coli being 101 CFU / mL to 106 CFU / mL;

[0033] Figure 5 A selective colorimetric detection method for Escherichia coli based on the p-benzoquinone-mediated decomposition of manganese dioxide nanosheets was demonstrated. DETAILED DESCRIPTION

[0034] The present application is further described in detail below in conjunction with the examples. It is particularly noted that if no specific conditions are specified in the examples below, the reaction is carried out under conventional conditions or conditions recommended by the manufacturer. Unless otherwise specified, the raw materials used in the examples below can be sourced from common commercial sources.

[0035] Reagents and instruments used in this experiment:

[0036] LB broth was purchased from Qingdao Haibo Biotechnology Co., Ltd. (Qingdao, China);

[0037] Agar powder was purchased from Beijing Solebow Technology Co., Ltd. (Beijing, China);

[0038] 3,3′,5,5′-Tetramethylbenzidine (TMB) and p-benzoquinone (BQ) were purchased from Shanghai MacLean Biochemical Technology Co., Ltd. (Shanghai, China);

[0039] Sodium acetate trihydrate, glacial acetic acid, manganese chloride, hydrogen peroxide, tetramethylammonium hydroxide, methanol, and dimethyl sulfoxide were purchased from Sinopharm Chemical Reagent Co., Ltd.

[0040] The culture medium used in the present invention is:

[0041] LB broth (5 g / L yeast extract, 10 g / L trypsin, and 5 g / L NaCl, pH 7.0);

[0042] LB plates (5 g / L yeast extract, 10 g / L trypsin, 15 g / L agar and 5 g / L NaCl, pH 7.0).

[0043] Manganese dioxide nanosheets (MnO 2NS) morphology (JEOL JEM 2100F, Japan). The UV-visible spectrum of the reaction solution was measured on a UV-visible spectrophotometer (Shimadzu UV-1900i, Japan). The particle size of the sample was measured by a Zetasizer Nano ZS nanoparticle size analyzer (Malvern, UK) at 25°C, and measured three times after dilution with deionized water. The absorbance value of the bacterial solution at a wavelength of 600nm was measured by a Varioskan LUX multifunctional microplate reader (Thermo Fisher Scientific, USA). Sample centrifugation was completed on a Centrifuge 5418R refrigerated desktop microcentrifuge (Eppendorf, Germany). Bacterial culture was carried out in an MJ150F-I bacterial incubator (Shanghai Hengyi Scientific Instrument Co., Ltd.).

[0044] For the sake of brevity and clarity, the commonly used terms in the following text, such as benzoquinone (BQ), manganese dioxide nanosheets (MnO 2 NS), Escherichia coli (E.coli), 3,3',5,5'-tetramethylbenzidine (TMB), etc. will be referred to by their full names without using their corresponding abbreviations.

[0045] Example 1

[0046] A colorimetric detection method for Escherichia coli based on the decomposition of manganese dioxide nanosheets mediated by p-benzoquinone, wherein the synthesis steps of the manganese dioxide nanosheets are as follows:

[0047] 10 mL of manganese dichloride solution was quickly added to 20 mL of a mixed solution containing hydrogen peroxide and tetramethylammonium hydroxide. The entire mixing process needed to be carried out quickly to avoid local oversaturation. At this time, the solution immediately turned dark brown and continued to be vigorously stirred for 12 hours at room temperature. Then, it was centrifuged at 2000 rpm for 20 minutes. Finally, it was washed with water and methanol for 3 times respectively, and dried in a drying oven at 60°C to remove excess water and solvent, and finally block manganese dioxide was obtained;

[0048] In order to prepare flake manganese dioxide, water was added to dissolve the block manganese dioxide and ultrasonicated for 12 hours, then centrifuged at 2000 rpm for 20 minutes, and the supernatant was taken for use.

[0049] Figure 2 is a characterization diagram showing manganese dioxide nanosheets, where Figure 2 A is a TEM image of manganese dioxide nanosheets, showing that the manganese dioxide nanosheets are in the form of single-layer sheets and can be evenly dispersed in the solution; Figure 2 B is the UV-visible spectrum of manganese dioxide nanosheets, which shows that manganese dioxide nanosheets have an absorption peak at 360 nm; Figure 2C is a particle size distribution diagram of manganese dioxide nanosheets, indicating that the average size of manganese dioxide nanosheets is 200 nm.

[0050] Example 2

[0051] A colorimetric detection method for Escherichia coli based on the decomposition of manganese dioxide nanosheets mediated by p-benzoquinone, the feasibility of which is analyzed as follows:

[0052] (1) Dilute the bacteria with PBS buffer solution (0.01 M, pH = 7.4) to prepare a concentration of 10 6 CFU / mL of E. coli suspension;

[0053] (2) Take the E. coli suspension (10 6 CFU / mL, 100 μL) was evenly mixed with p-benzoquinone solution (6 mM, 100 μL) and incubated in a 37 °C bacterial incubator for 1 h;

[0054] (3) After the incubation, add manganese dioxide nanosheets (1 mg / mL, 50 μL) and react for 20 min, then add 300 μL of sodium acetate buffer solution and 3,3',5,5'-tetramethylbenzidine solution (50 mM, 3 μL), vortex and react for 5 min in the dark. After the reaction, use a UV-visible spectrophotometer to record the absorption spectrum from 200 to 800 nm.

[0055] Figure 3 The results showed that when there was no E. coli, the manganese dioxide nanosheets had oxidase activity and could oxidize 3,3',5,5'-tetramethylbenzidine to generate a blue product oxTMB, with a high absorbance at 652nm. However, when E. coli was present, p-benzoquinone was reduced to hydroquinone, which was reductive and could decompose the manganese dioxide nanosheets into Mn 2+ , which makes the manganese dioxide nanosheets lose their oxidase activity and fail to oxidize 3,3',5,5'-tetramethylbenzidine to generate oxTMB, so the absorbance value at 652nm decreases. The above shows that Escherichia coli (E. coli) can mediate the generation of hydroquinone from benzoquinone, thereby decomposing manganese dioxide nanosheets, and the established colorimetric analysis method is feasible.

[0056] Example 3

[0057] The establishment of calibration curves between different E. coli concentrations and oxTMB to detect different concentrations of E. coli includes the following steps:

[0058] First, dilute the E. coli solution into 10 6 , 10 5 , 10 4 , 10 3 , 10 2 , 101 CFU / mL, then take 100μL of Escherichia coli culture solution of different concentrations and mix it evenly with p-benzoquinone (6mM, 100μL), put it in a 37℃ bacterial incubator for 1h, then add manganese dioxide nanosheets (1mg / mL, 50μL) to react for 20min, then add 300μL of sodium acetate buffer solution and 3,3',5,5'-tetramethylbenzidine solution (50mM, 3μL), vortex and react for 5min in the dark. After the reaction, the absorption spectrum of 200-800nm ​​was recorded using a UV-visible spectrophotometer.

[0059] The PBS buffer solution (10 mM, pH 7.4) contains Na 2 HPO 4 (0.72g), KH 2 PO 4 (0.12g), NaCl (4g) and KCl (0.1g);

[0060] Sodium acetate buffer solution (0.2 M, pH 3.6) contains NaAc·3H 2 O (1.0206 g) and HAc (5.3208 mL).

[0061] Figure 4 A shows that as the concentration of E. coli increases (from a to g), the amount of hydroquinone generated gradually increases, the manganese dioxide nanosheets in the system are gradually decomposed, and the oxTMB product in the system decreases, so the absorbance at 652nm gradually decreases; Figure 4 B is the linear relationship between the change of the absorption peak intensity at 652nm and the logarithmic concentration of E. coli. The concentration range of E. coli is 10 1 CFU / mL~10 6 CFU / mL, the detection limit is 5 CFU / mL;

[0062] Example 4

[0063] A colorimetric detection method for Escherichia coli based on the decomposition of manganese dioxide nanosheets mediated by p-benzoquinone, wherein the selectivity verification steps are as follows:

[0064] The bacterial suspensions of Escherichia coli, Staphylococcus aureus and Pseudomonas aeruginosa were diluted to 10 6CFU / mL, 100 μL of bacterial solution of Escherichia coli, Staphylococcus aureus and Pseudomonas aeruginosa were taken and mixed evenly with p-benzoquinone (6mM, 100 μL), placed in a 37°C bacterial incubator for 1 hour, and then added manganese dioxide nanosheets (1 mg / mL, 50 μL) for 20 minutes, and then added 300 μL of sodium acetate buffer solution and 3,3',5,5'-tetramethylbenzidine solution (50mM, 3 μL), vortexed and reacted for 5 minutes in the dark. After the reaction, the absorption spectrum of 200-800nm ​​was recorded using a UV-visible spectrophotometer.

[0065] In order to further study the applicability of this method in actual samples, the detection method established in Example 1 was used to determine the content of Escherichia coli in sterilized milk samples by the standard addition method. The experimental results are shown in Table 1. The recovery rate was between 101.7% and 117.9%, and the relative standard deviation (RSD) was less than 7.92%, indicating that this method has high accuracy and is suitable for the detection of Escherichia coli content in actual milk samples.

[0066] Table 1

[0067]

[0068] Figure 5 The results showed that neither Staphylococcus aureus nor Pseudomonas aeruginosa could reduce the production of oxTMB, and the absorption peak intensity at 652nm was significantly higher than that of the Escherichia coli group, indicating that the constructed method can selectively detect Escherichia coli.

[0069] The above descriptions are merely embodiments of the present invention and are not intended to limit the patent scope of the present invention. Any equivalent structure or equivalent process transformation made using the contents of the present invention specification and drawings, or directly or indirectly applied in other related technical fields, are also included in the patent protection scope of the present invention.

Claims

1. A colorimetric detection method for Escherichia coli based on the decomposition of manganese dioxide nanosheets mediated by p-benzoquinone, characterized in that: The following steps are involved: (1) Preparation of manganese dioxide nanosheets (MnO2 NS); (2) Escherichia coli (E. coli) reduces benzoquinone (BQ) to produce hydroquinone (HQ); (3) Add manganese dioxide nanosheet (MnO2 NS) solution to the mixed solution of Escherichia coli (E. coli) and benzoquinone (BQ) with different concentrations, vortex to mix them evenly, and fully react at room temperature between 18°C ​​and 28°C; then add sodium acetate (NaAc·HAc) buffer and 3,3',5,5'-tetramethylbenzidine (TMB) solution, vortex to mix them evenly, and then react in the dark; finally, use a UV-visible spectrophotometer to collect the absorption spectrum curve of the reaction solution, and establish a calibration curve with the concentration of Escherichia coli (E. coli) based on the absorption peak intensity at 652nm; (4) Add manganese dioxide nanosheet (MnO2NS) solution to the mixture of the sample to be tested and benzoquinone (BQ), vortex to mix them evenly, and react them fully at room temperature; then add sodium acetate (NaAc·HAc) buffer and 3,3',5,5'-tetramethylbenzidine (TMB) solution, vortex to mix them evenly, and react in the dark; finally, use a UV-visible spectrophotometer to collect the absorption spectrum curve of the reaction solution, compare the absorption peak intensity of the sample to be tested at 652nm with the established calibration curve, and determine the concentration of Escherichia coli (E. coli) in the sample to be tested.

2. The method for colorimetric detection of Escherichia coli based on p-benzoquinone-mediated decomposition of manganese dioxide nanosheets according to claim 1, characterized in that: In step (1), the synthesis steps of the manganese dioxide nanosheets (MnO2NS) are as follows: quickly pouring a manganese dichloride (MnCl2) solution into a mixed solution containing hydrogen peroxide (H2O2) and tetramethylammonium hydroxide, the entire mixing process needs to be completed quickly to prevent local supersaturation, at which time the solution turns dark brown, and after continuous stirring at room temperature, centrifugation is performed to remove unreacted impurities, and then the precipitated manganese dioxide is carefully washed with ultrapure water and methanol, and placed in an oven for drying to finally obtain block manganese dioxide; dissolving the block manganese dioxide in water, preparing nanosheets using ultrasound, centrifuging to remove undispersed particles, and storing the supernatant containing manganese dioxide nanosheets (MnO2NS) in a refrigerator at 4°C for standby use.

3. The method for colorimetric detection of Escherichia coli based on p-benzoquinone-mediated decomposition of manganese dioxide nanosheets according to claim 1, characterized in that: In step (3), the concentration of p-benzoquinone (BQ) is 6 mM. P-benzoquinone (BQ) is mixed with Escherichia coli (E. coli) and then placed in a 37° C. bacterial incubator for reaction for 1 h to 1.5 h.

4. The method for colorimetric detection of Escherichia coli based on p-benzoquinone-mediated decomposition of manganese dioxide nanosheets according to claim 1, characterized in that: In step (3), the concentration of Escherichia coli (E. coli) is 10 1 ~10 6 CFU / mL.

5. The method for colorimetric detection of Escherichia coli based on p-benzoquinone-mediated decomposition of manganese dioxide nanosheets according to claim 1, characterized in that: In step (3) and step (4), the volumes of the manganese dioxide nanosheets (MnO2 NS), sodium acetate (NaAc·HAc) buffer, and 3,3',5,5'-tetramethylbenzidine (TMB) are 50 μL, 300 μL, and 3 μL, respectively, and the concentrations of the manganese dioxide nanosheets (MnO2 NS) and 3,3',5,5'-tetramethylbenzidine (TMB) are 1 mg / mL and 50 mM, respectively.

6. The method for colorimetric detection of Escherichia coli based on p-benzoquinone-mediated decomposition of manganese dioxide nanosheets according to claim 2, characterized in that: The volume of the manganese dichloride (MnCl2) solution is 10 mL, and the volume of the mixed solution of hydrogen peroxide (H2O2) and tetramethylammonium hydroxide is 20 mL.

7. The method for colorimetric detection of Escherichia coli based on p-benzoquinone-mediated decomposition of manganese dioxide nanosheets according to claim 2, characterized in that: The stirring time is 12h.

8. The method for colorimetric detection of Escherichia coli based on p-benzoquinone-mediated decomposition of manganese dioxide nanosheets according to claim 2, characterized in that: The centrifugation time is 20 min and the rotation speed is 2000 rpm.

9. The method for colorimetric detection of Escherichia coli based on p-benzoquinone-mediated decomposition of manganese dioxide nanosheets according to claim 2, characterized in that: The oven drying temperature is 60°C.

10. The method for colorimetric detection of Escherichia coli based on p-benzoquinone-mediated decomposition of manganese dioxide nanosheets according to claim 2, characterized in that: The time for preparing the nanosheets by ultrasound is 12 hours, the time for centrifugation is 30 minutes, and the rotation speed is 2000 rpm.

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