Application of fluorescence sensor based on manganese dioxide nanosheet response in escherichia coli detection

Abstract

The invention discloses application of a fluorescence sensor based on manganese dioxide nanosheet response in escherichia coli detection, and belongs to the technical field of biosensors. According to the method, firstly, p-benzoquinone (BQ) is reduced to generate hydroquinone (HQ) by using escherichia coli, then manganese dioxide nanosheets (MnO2NS) are added, the generated hydroquinone has reducibility, can decompose the manganese dioxide nanosheets into Mn < 2 + >, lose oxidase-like activity and cannot oxidize fluorescent red dye (Amplex Red, AR), and by measuring the fluorescence spectrum of a reaction solution, the content of the manganese dioxide nanosheets in the reaction solution can be determined. The fluorescence intensity of the reaction system is reduced along with the increase of the concentration of the escherichia coli. According to the method, expensive precise instruments are not needed, special operators are trained, the detection method is simplified, the detection cost of the escherichia coli is reduced, the detection is rapid and simple, the sensitivity is high, and the selectivity is good.

Description

Technical Field

[0001] The invention belongs to the technical field of biosensors, and in particular relates to the application of a fluorescence sensor based on the response of manganese dioxide nanosheets in the detection of Escherichia coli. Background Art

[0002] Escherichia coli is a Gram-negative, facultative anaerobic bacterium that is one of the most common pathogens of foodborne diseases and is the main cause of most foodborne diseases in the United States and around the world. Millions of people are infected with bacteria each year, and most of these bacterial infections are transmitted through food. Therefore, real-time, in-situ measurement and analysis of E. coli is an important means to reduce unnecessary losses caused by bacterial infections.

[0003] The traditional bacterial counting method is the standard method for detecting live bacteria, but the operation process is cumbersome, time-consuming, and has a certain false negative rate. In recent years, biological detection methods based on modern molecular technology have developed rapidly and have been widely used in the detection of Escherichia coli, such as polymerase chain reaction (PCR). PCR technology has strong specificity, high sensitivity, rapidity and accuracy, but PCR reactions are easily contaminated. Trace amounts of RNA or DNA contamination in the sample will produce highly misleading results, and it is also easy to produce primer dimers (PD) that compete with the target sequence, which will affect the amplification efficiency of PCR.

[0004] Although ELISA has good sensitivity, high specificity, simplicity and rapidity, it also has some inevitable defects, such as cross-reaction, high selectivity for reagents, and difficulty in analyzing multiple components at the same time. Other detection methods such as flow cytometry can detect pathogenic bacteria in a non-culturable state (VBNC) and can penetrate the cell membrane of pathogenic bacteria and bind to nucleic acids for live bacteria detection, but this method often has high requirements for cell activity and concentration, lacks the availability of specific dyes, and is easily affected by dead cells and causes nonspecific staining. Summary of the invention

[0005] In view of the defects in the above-mentioned prior art, the purpose of the present invention is to design and provide a fluorescent sensor based on the response of manganese dioxide nanosheets, a preparation method and its application in detecting Escherichia coli. The fluorescent biosensor of the present invention has the advantages of high sensitivity, rapid response, simple operation and good selectivity, does not need to be coupled with a specific aptamer, does not require complicated operations and specialized operators, and greatly reduces the detection cost and labor cost.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions:

[0007] In one aspect, the present invention provides the use of manganese dioxide nanosheets as fluorescent sensors in the qualitative and quantitative detection of Escherichia coli.

[0008] The application, the method for preparing the manganese dioxide nanosheets comprises:

[0009] Weigh hydrogen peroxide and tetramethylammonium hydroxide solution, mix them, quickly add MnCl2 solution, vigorously stir at room temperature, centrifuge for the first time, wash, dry to obtain blocky manganese dioxide, add it into water for ultrasonic dissolution, centrifuge it for the second time, take the supernatant, and obtain manganese dioxide nanosheets as fluorescent sensors.

[0010] In the application, the volume ratio of the hydrogen peroxide, tetramethylammonium hydroxide solution and MnCl2 solution is (1-2):(1-2):(1-2);

[0011] The concentration of the hydrogen peroxide is 3wt%;

[0012] The concentration of the tetramethylammonium hydroxide solution is 1.2M;

[0013] The concentration of the MnCl2 solution is 0.3M.

[0014] In the application, the time of rapid addition is within 15 seconds;

[0015] The vigorous stirring time is 10 to 12 hours;

[0016] The conditions of the first centrifugation are: time 20-30 min, speed 2000 rpm;

[0017] The ultrasound time is 12 to 14 hours;

[0018] The conditions for the second centrifugation are: time 30-40 min, rotation speed 2000 rpm.

[0019] In the application, the washing is washing with water and methanol three times respectively.

[0020] In a second aspect, the present invention provides a method for detecting the concentration of Escherichia coli in a sample using a fluorescence sensor based on the response of manganese dioxide nanosheets, comprising the following steps:

[0021] (1) preparing Escherichia coli of various concentrations, adding p-benzoquinone, manganese dioxide nanosheets and PBS buffer, reacting at room temperature, adding a fluorescent substrate to obtain a mixed solution, reacting in the dark, and measuring to obtain a calibration curve between the Escherichia coli concentration and the fluorescence intensity;

[0022] (2) adding p-benzoquinone to the sample solution to be tested, incubating, adding manganese dioxide nanosheets and PBS buffer, reacting at room temperature, adding a fluorescent substrate to obtain a mixed solution, performing a light-protected reaction, and measuring the fluorescence intensity of the mixed solution after completion;

[0023] (3) Substituting the fluorescence intensity obtained in step (2) into the calibration curve in step (1) to determine the concentration of Escherichia coli in the sample solution to be tested.

[0024] In the method, the volume ratio of the benzoquinone, manganese dioxide nanosheets, PBS buffer and fluorescent substrate in steps (1) and (2) is (100-200):(30-60):(110-220):(10-20);

[0025] The volume ratio of Escherichia coli to p-benzoquinone in step (1) is 1:1;

[0026] The volume ratio of the sample solution to be tested to p-benzoquinone in step (2) is 1:1;

[0027] The concentration of Escherichia coli in step (1) is 1.0×10 1 CFU / mL-1.0×10 8 CFU / mL.

[0028] In the method, the concentration of p-benzoquinone is 4 to 8 mM;

[0029] Preferably, the concentration of benzoquinone is 6 mM.

[0030] The method, the incubation and culture conditions are: temperature 37°C, time 1 to 1.5 hours;

[0031] The reaction time at room temperature is 30 to 40 minutes;

[0032] The light-avoiding reaction time is 30 to 40 minutes;

[0033] The measurement method is carried out at an excitation wavelength of 560 nm.

[0034] In the method, the fluorescent substrate is fluorescent red dye (Amplex Red).

[0035] Compared with the prior art, the present invention has the following beneficial effects:

[0036] 1. In the method for detecting Escherichia coli based on a fluorescent sensor responsive to manganese dioxide nanosheets, the manganese dioxide nanosheets (MnO2 NS) have oxidase-like activity, and the fluorescent red dye (Amplex Red, AR) is a non-fluorescent peroxidase-like substrate that can be oxidized by the manganese dioxide nanosheets (MnO2 NS) to generate an oxidized product oxAR with strong fluorescence. The present invention first uses Escherichia coli to reduce benzoquinone (BQ) to generate hydroquinone (HQ), and then adds manganese dioxide nanosheets (MnO2 NS). HQ has a reducing property and can decompose the manganese dioxide nanosheets into Mn 2+, losing its oxidase-like activity and being unable to oxidize the fluorescent substrate AR (Amplex Red), which is manifested as a decrease in the fluorescence intensity of the reaction system as the concentration of E. coli increases.

[0037] 2. The present invention can realize rapid and sensitive detection of Escherichia coli according to the change of oxAR fluorescence intensity. The operation is simple, and no complicated aptamer labeling is required, which reduces the detection cost. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 Characterization of MnO2 NS, where A is the TEM image of MnO2 NS; B is the UV-visible spectrum of MnO2 NS; C is the particle size distribution of MnO2 NS; D is the FTIR spectrum of MnO2 NS;

[0039] Figure 2 This is the optimization result diagram of BQ concentration;

[0040] Figure 3 Flow chart of the method for detecting Escherichia coli based on the fluorescence sensor responsive to manganese dioxide nanosheets, wherein A is the reaction of the fluorescent substrate AR and MnO2 NS to generate oxAR, which is manifested as fluorescence enhancement; B is the reaction of p-benzoquinone and Escherichia coli to generate hydroquinone, and then hydroquinone decomposes MnO2 NS to generate Mn 2+ , AR can no longer be oxidized, and the fluorescence intensity of the reaction system decreases; the inset is the fluorescence spectrum obtained by detecting the reaction system of the fluorescent substrate AR and MnO2 NS (curve (a)) and the reaction system of benzoquinone, Escherichia coli, fluorescent substrate AR and MnO2 NS (curve (b));

[0041] Figure 4 A feasibility analysis diagram of the method for detecting Escherichia coli based on the fluorescence sensor responsive to manganese dioxide nanosheets;

[0042] Figure 5 Figure 1 is the fluorescence spectra of different concentrations of E. coli and the corresponding detection photos under 365nm ultraviolet light, where A is the relationship between different E. coli concentrations and oxAR fluorescence intensity; B is the calibration curve of oxAR fluorescence intensity and different concentrations of E. coli, where the illustrations a and b represent the concentration of E. coli at 10 8 , 5×10 6 , 10 4 , 10 3 , 10 2 , the color difference of the reaction system of 10 CFU / mL under ultraviolet light;

[0043] Figure 6 The results are the responses of the fluorescent sensor of the present invention to Escherichia coli and Staphylococcus aureus. DETAILED DESCRIPTION

[0044] The present application will be 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 obtained from common commercial sources.

[0045] Reagents and instruments used in this experiment:

[0046] LB broth was purchased from Qingdao Haibo Biotechnology Co., Ltd. (Qingdao, China). Agar powder was purchased from Beijing Solebao Technology Co., Ltd. (Beijing, China). p-Benzoquinone was purchased from Shanghai MacLean Biochemical Technology Co., Ltd. (Shanghai, China). Tetramethylammonium hydroxide, hydrogen peroxide, methanol, potassium chloride, sodium chloride, anhydrous disodium hydrogen phosphate, and potassium dihydrogen phosphate were purchased from Sinopharm Chemical Reagent Company (Shanghai, China).

[0047] The culture medium used in the present invention: LB broth (5g / L yeast extract, 10g / L trypsin and 5g / L NaCl, pH7.0); LB plate (5g / L yeast extract, 10g / L trypsin, 20g / L agar and 5g / L NaCl, pH7.0).

[0048] The morphology of MnO2 NS obtained by transmission electron microscopy (JEOL JEM 2100F, Japan). The UV-visible spectrum was measured on a UV-visible spectrophotometer (Shimadzu UV-1900i, Japan). The Fourier transform infrared (FTIR) spectrum was measured using a Thermo Scientific Nicolet IS50 spectrometer from the United States. The particle size analysis of the samples was measured at 25°C by a Zetasizer Nano ZS nanoparticle size analyzer (Malvern, UK) and measured three times after dilution with deionized water. The fluorescence spectrum was measured using an Edinburgh Fs5 integrated fluorescence spectrometer (Edinburgh, UK). The absorbance values ​​of the bacterial solution at a wavelength of 600nm were measured by a Varioskan LUX multifunctional microplate reader (Thermo Fisher Scientific, USA). The model of the ultrasonic instrument was KQ3200DE from Kunshan Ultrasonic Instrument Co., Ltd. All samples were centrifuged in a Centrifuge 5418R refrigerated benchtop microcentrifuge (Eppendorf, Germany).

[0049] Embodiment 1:

[0050] 1. The synthesis steps of the fluorescence sensor based on the response of manganese dioxide nanosheets are as follows:

[0051] 10 mL of 3 wt% hydrogen peroxide was mixed with 10 mL of 1.2 M tetramethylammonium hydroxide solution and then quickly added into 10 mL of 0.3 M MnCl2 solution within 15 seconds. The solution was observed to immediately turn dark brown. The suspension was then vigorously stirred at room temperature for 12 h. The resulting solution was centrifuged in a centrifuge for 20 min (2000 rpm), then washed three times with water and methanol respectively, and dried in a drying oven at 60 °C to finally obtain blocky MnO2 NS.

[0052] Then, 10 mg of blocky manganese dioxide was weighed and dissolved in 10 mL of ultrapure water and ultrasonicated for 12 h to completely disperse the MnO2 NS. The dispersion was centrifuged in a centrifuge for 30 min (2000 rpm), and the supernatant was retained for the next experiment.

[0053] Figure 1 shows the characterization results of MnO2 NS, Figure 1 A is the TEM image of MnO2 NS, showing the morphology of MnO2 NS; Figure 1 B is the UV-visible spectrum of MnO2 NS, which shows that MnO2 NS has an absorption peak at 360nm; Figure 1 C is the particle size distribution diagram of MnO2 NS, which shows that the average size of MnO2 NS is 200 nm; Figure 1 D is the FTIR spectrum of MnO2 NS, which shows that MnO2 NS has -1 The band at 1630 cm is attributed to the stretching vibration of H2O and OH(NH) due to physical absorption. -1 The peak at 513 cm is attributed to the stretching vibration of C=O. -1 The characteristic peak at represents the symmetric stretching vibration of the Mn-O bond; the above data show that the above steps have successfully synthesized MnO2 NS.

[0054] Embodiment 2:

[0055] 1. Optimization of BQ concentration in the method of detecting Escherichia coli based on fluorescence sensor responsive to manganese dioxide nanosheets:

[0056] (1) Prepare a 10% PBS buffer solution (pH = 7.4). 8 CFU / mL of bacterial suspension;

[0057] (2) using PBS to prepare BQ solutions with concentrations of 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, and 8 mM;

[0058] (3) Pipette 100 μL of different concentrations of BQ and E. coli solution (10 8CFU / mL, 100 μL) were mixed in a 96-well plate and placed in a 37°C bacterial incubator for full incubation. Three sets of parallel samples were made. After 1 hour, a smartphone was used to take a photo to record the color change of BQ.

[0059] The results are as follows Figure 2 As shown, it was shown that the optimal concentration of Escherichia coli for reducing benzoquinone (BQ) was 6 mM.

[0060] 2. Feasibility verification of the method of detecting Escherichia coli based on the fluorescence sensor responsive to manganese dioxide nanosheets:

[0061] (1) Prepare a 10% PBS buffer solution (pH = 7.4). 8 CFU / mL of bacterial suspension;

[0062] (2) 100 μL of 10 8 CFU / mL bacterial solution and 100 μL PBS buffer (sample a), a mixed solution containing 100 μL PBS buffer and 100 μL 6 mM BQ (sample b), and a mixed solution containing 100 μL 10 8 The mixed solution of CFU / mL bacterial solution and 100 μL of 6 mM BQ (sample c) was placed in a 37°C bacterial incubator and incubated for 1 h;

[0063] (3) After the incubation, 30 μL of the MnO2 NS solution prepared in Example 1 and 110 μL of PBS were added to the three samples, and the mixture was reacted at room temperature for 30 min. Then, 10 μL of AR (fluorescent red dye, Amplex Red) solution was added, and the mixture was reacted in the dark for 30 min. The fluorescence spectrum of the solution was then measured using a fluorescence spectrometer with an excitation wavelength of 560 nm.

[0064] like Figure 3 A flow chart of the method for detecting Escherichia coli based on a fluorescent sensor responsive to manganese dioxide nanosheets. Figure 3 (a) is oxAR; (b) is AR; the inset is the fluorescence spectra of the BQ / MnO2 NS / AR reaction system in the absence of E. coli (curve a) and the presence of E. coli (curve b). It can be seen that in the presence of E. coli, MnO2 NS is decomposed into Mn 2+ , AR cannot be further oxidized and the fluorescence of the reaction system decreases.

[0065] The results are as follows Figure 4As shown, a is the fluorescence spectrum of the reaction between MnO2 NS and E. coli; b is the fluorescence spectrum of the reaction between MnO2 NS and BQ; c is the fluorescence spectrum of the reaction between MnO2 NS and BQ and E. coli. It can be seen that a shows that although E. coli has reducing properties, it is not enough to decompose MnO2 NS. MnO2 NS oxidizes AR to generate oxAR with strong fluorescence, which enhances the fluorescence of the system; b shows that in the absence of E. coli, BQ cannot be reduced to HQ, and MnO2 NS oxidizes the fluorescent substrate AR to generate oxAR, which enhances the fluorescence of the system; c shows that in the presence of E. coli, BQ is reduced to HQ, and HQ has a strong reducing property, which decomposes MnO2NS into Mn 2+ , AR cannot be oxidized and the fluorescence intensity of the reaction system decreases.

[0066] The above results show that the method of detecting Escherichia coli based on fluorescence sensor responsive to manganese dioxide nanosheets is feasible.

[0067] Embodiment 3:

[0068] Establishment of calibration curves between different E. coli concentrations and oxAR fluorescence intensity: Detection of different concentrations of E. coli includes the following steps:

[0069] First, dilute the E. coli solution to 10 8 , 5×10 6 , 10 4 , 10 3 , 10 2 , 10CFU / mL, 100μL of E. coli of different concentrations was evenly mixed with 100μL of BQ (6mM), and incubated in a 37℃ bacterial incubator for 1h, then 30μL of MnO2 NS and 110μL of PBS were added, and the reaction was carried out at room temperature for 30min, and then 10μL of AR was added, and the reaction was carried out in the dark for 30min, and finally the fluorescence spectrum of the sample was measured to obtain the calibration curve between the fluorescence intensity of E. coli of different concentrations and oxAR. Among them, the PBS buffer solution contains 0.72gNa2HPO4, 0.12gKH2PO4, 4gNaCl and 0.1gKCl, with a concentration of 10mM and pH 7.4.

[0070] The results are as follows Figure 5 As shown in A, with the increase of E. coli concentration (from a to g represent the concentration of 10 8 , 5×10 6 , 10 4 , 10 3 , 10 2 , 10CFU / mL of Escherichia coli liquid), MnO2 NS was decomposed into Mn 2+, the ability to oxidize AR becomes weaker and weaker, so the fluorescence intensity of the reaction system gradually decreases, and the color of the solution can be seen to change from blue to red under 365nm ultraviolet. Figure 5 B is the calibration curve between the fluorescence intensity of oxAR and the concentration of different E. coli solutions (Inset: the linear relationship between the fluorescence intensity of oxAR and the logarithmic concentration of E. coli, ranging from 10 1 -10 8 CFU / mL, and the detection limit is 1 CFU / mL).

[0071] Embodiment 4:

[0072] The selective verification of the method for detecting Escherichia coli based on the fluorescence sensor responsive to manganese dioxide nanosheets includes the following steps:

[0073] Dilute the bacterial suspension of Escherichia coli and Staphylococcus aureus to 10 7 CFU / mL, 100μL of Escherichia coli and Staphylococcus aureus culture liquid were respectively mixed with 100μL of BQ (6mM), and incubated in a 37℃ bacterial incubator for 1h, then 30μL of MnO2 NS and 110μL of PBS were added, and the reaction was carried out at room temperature for 30min, and then 10μL of AR was added, and the reaction was carried out in the dark for 30min, and finally the fluorescence spectrum of the sample was measured.

[0074] The results are as follows Figure 6 As shown, the fluorescence sensor based on the response of manganese dioxide nanosheets responds better to Escherichia coli than to Staphylococcus aureus.

[0075] Embodiment 5:

[0076] In order to further study the practicality of this method in actual samples, the content of E. coli in milk was detected by the standard addition method according to the above detection method. The milk was DeLunSu purchased from ordinary supermarkets. The experimental results are shown in Table 1 below. The recovery rate is between 99.3% and 109.7%, and the relative standard deviation (RSD) is less than 4.6%, indicating that the method of the present invention has good practicality and can be used to detect the content of E. coli in actual milk samples.

[0077] Table 1 Detection of Escherichia coli content in milk samples

[0078]

[0079] The above is only an exemplary embodiment of the present disclosure, but the protection scope of the present disclosure is not limited thereto. Any changes or substitutions that can be easily thought of by a person skilled in the art within the technical scope disclosed in the present disclosure should be included in the protection scope of the present disclosure. Therefore, the protection scope of the present disclosure should be based on the protection scope of the claims.

Claims

1. Application of manganese dioxide nanosheets as fluorescent sensors in qualitative and quantitative detection of Escherichia coli.

2. The use according to claim 1, characterized in that The method for preparing manganese dioxide nanosheets comprises: Weigh hydrogen peroxide and tetramethylammonium hydroxide solution, mix them, quickly add MnCl2 solution, vigorously stir at room temperature, centrifuge for the first time, wash, dry to obtain blocky manganese dioxide, add it into water for ultrasonic dissolution, centrifuge it for the second time, take the supernatant, and obtain manganese dioxide nanosheets as fluorescent sensors.

3. The use according to claim 2, characterized in that The volume ratio of the hydrogen peroxide, tetramethylammonium hydroxide solution and MnCl2 solution is (1-2):(1-2):(1-2); The concentration of the hydrogen peroxide is 3wt%; The concentration of the tetramethylammonium hydroxide solution is 1.2M; The concentration of the MnCl2 solution is 0.3M.

4. The use according to claim 2, characterized in that The time for the rapid addition is within 15 seconds; The vigorous stirring time is 10 to 12 hours; The conditions of the first centrifugation are: time 20-30 min, speed 2000 rpm; The ultrasound time is 12 to 14 hours; The conditions for the second centrifugation are: time 30-40 min, rotation speed 2000 rpm.

5. The use according to claim 2, characterized in that The washing is performed by washing with water and methanol for 3 times respectively.

6. A method for detecting the concentration of Escherichia coli in a sample using a fluorescence sensor based on the response of manganese dioxide nanosheets, characterized in that: The following steps are involved: (1) preparing Escherichia coli of various concentrations, adding p-benzoquinone, manganese dioxide nanosheets and PBS buffer, reacting at room temperature, adding a fluorescent substrate to obtain a mixed solution, reacting in the dark, and measuring to obtain a calibration curve between the Escherichia coli concentration and the fluorescence intensity; (2) adding p-benzoquinone to the sample solution to be tested, incubating, adding manganese dioxide nanosheets and PBS buffer, reacting at room temperature, adding a fluorescent substrate to obtain a mixed solution, performing a light-protected reaction, and measuring the fluorescence intensity of the mixed solution after completion; (3) Substituting the fluorescence intensity obtained in step (2) into the calibration curve in step (1) to determine the concentration of Escherichia coli in the sample solution to be tested.

7. The method according to claim 6, characterized in that The volume ratio of benzoquinone, manganese dioxide nanosheets, PBS buffer and fluorescent substrate in steps (1) and (2) is (100-200):(30-60):(110-220):(10-20); The volume ratio of Escherichia coli to p-benzoquinone in step (1) is 1:1; The volume ratio of the sample solution to be tested to p-benzoquinone in step (2) is 1:1; The concentration of Escherichia coli in step (1) is 1.0×10 1 CFU / mL~1.0×10 8 CFU / mL.

8. The method according to claim 6, characterized in that The concentration of p-benzoquinone is 4-8 mM; Preferably, the concentration of benzoquinone is 6 mM.

9. The method according to claim 6, characterized in that The incubation conditions are: temperature 37°C, time 1 to 1.5 hours; The reaction time at room temperature is 30 to 40 minutes; The light-avoiding reaction time is 30 to 40 minutes; The measurement method is carried out at an excitation wavelength of 560 nm.

10. The method according to claim 6, characterized in that The fluorescent substrate is a fluorescent red dye.

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