A Ni / Co (HHTP) MOF electrode material, a preparation method thereof, and its application in preparing an electrochemical aptamer sensor for detecting Escherichia coli O157:H7

Abstract

The present invention discloses a Ni / Co(HHTP)MOF electrode material, a preparation method thereof, and an application thereof in the preparation of an electrochemical aptamer sensor for detecting Escherichia coli O157:H7, belonging to the technical field of harmful microorganism detection. The electrochemical aptamer sensor is prepared by modifying a rod-shaped Ni / Co(HHTP)MOF material onto a glassy carbon electrode. The electrochemical aptamer sensor of the present invention can efficiently and specifically capture Escherichia coli O157:H7 and can be used to quickly and sensitively detect Escherichia coli O157:H7 in food. Compared with traditional bacterial detection methods, the method of the present invention has good selectivity, storage stability and reproducibility, a low detection limit, and a short detection time, providing a new idea for the detection of foodborne pathogens and having good application prospects.

Description

Technical Field

[0001] The present invention belongs to the technical field of harmful microorganism detection, and specifically relates to a Ni / Co (HHTP) MOF electrode material, a preparation method thereof, and an application of the Ni / Co (HHTP) MOF electrode material in preparing an electrochemical aptamer sensor for detecting Escherichia coli O157:H7. Background Art

[0002] Escherichia coli O157:H7 is a pathogenic enterohemorrhagic E. coli, representing a group of E. coli strains that cause bloody diarrhea and enteritis in humans. It is highly acid-resistant, withstanding temperatures of 37°C for five hours at a pH of 2.5-3. It is also cold-resistant, surviving for extended periods in the refrigerator. However, it is heat-sensitive, being inactivated in just one minute at 75°C. The dangers of E. coli O157:H7 are primarily due to the diseases it causes. It can be transmitted to humans through the consumption of contaminated foods, such as beef, raw milk, chicken, and chicken products. Symptoms of infection include abdominal cramps and diarrhea, which can sometimes progress to bloody diarrhea (hemorrhagic colitis). Fever and vomiting may also occur. The incubation period can range from three to eight days, with an average of three to four days. Most patients recover within one day, but a few, particularly young children and the elderly, may develop life-threatening conditions such as hemolytic uremic syndrome. Hemolytic uremic syndrome (HUS) is characterized by acute renal failure, hemolytic anemia, and thrombocytopenia, with a mortality rate of 3% to 5%. Furthermore, the infectious dose of E. coli O157:H7 is extremely low; it is estimated that 1% of individuals infected with Shiga toxin-producing E. coli develop HUS, 25% of HUS patients develop neurological complications (such as seizures, stroke, and coma), and approximately 5% of survivors develop sequelae of chronic kidney disease, which is usually mild. Therefore, E. coli O157:H7 poses a significant public health threat, and its spread needs to be controlled through strengthened food hygiene management and prevention of foodborne infections.

[0003] Traditional bacterial detection methods mainly use plate count technology, polymerase chain reaction (PCR), and enzyme-linked immunosorbent assay (ELISA). Although these methods have strong specificity and high sensitivity in detection, they are expensive, have strict technical requirements, and complex procedures, resulting in a time-consuming and labor-intensive detection process, complicated operations, and high costs. Therefore, it is very necessary to establish a method that can detect bacteria quickly, specifically, sensitively, and efficiently, with good stability and reproducibility, low detection limit, wide detection range, and high practical application value, in order to effectively control food contamination. Summary of the Invention

[0004] The present invention provides a Ni / Co(HHTP)MOF electrode material, which is prepared by the following method:

[0005] Ni(CH3COO)2·4H2O, Co(CH3COO)2·4H2O and HHTP were dissolved in a solvent and stirred for reaction. After the reaction was completed, the mixture was cooled to room temperature and the reaction solution was centrifuged to obtain a solid product. The product was then washed and dried to obtain a Ni / Co(HHTP)MOF electrode material.

[0006] In the above-mentioned method for preparing the Ni / Co(HHTP)MOF electrode material, each raw material is selected from the following parts:

[0007] Ni(CH3COO)2·4H2O 100-180 parts, Co(CH3COO)2·4H2O 50-90 parts, HHTP 120-200 parts, solvent 10-40 parts;

[0008] When the component is a solid component, the portion number represents milligrams; when the component is a liquid component, the portion number represents milliliters; in actual applications, the portion number can be enlarged or reduced in proportion to the portion number.

[0009] In a specific embodiment, each raw material is selected from the following parts:

[0010] Ni(CH3COO)2·4H2O 124 parts, Co(CH3COO)2·4H2O 62 parts, HHTP 162 parts, solvent 18 parts;

[0011] When the component is a solid component, the portion number represents milligrams; when the component is a liquid component, the portion number represents milliliters; in actual applications, the portion number can be enlarged or reduced in proportion to the portion number.

[0012] In the above-mentioned method for preparing the Ni / Co(HHTP)MOF electrode material, the solvent is a DMF aqueous solution, wherein the volume ratio of DMF to water is 1:3.

[0013] In the above-mentioned method for preparing Ni / Co(HHTP)MOF electrode material, the stirring reaction conditions are selected from: stirring reaction at 80-90°C for 1-60 minutes; preferably: stirring reaction at 85°C for 30 minutes.

[0014] The present invention provides the use of the above-mentioned Ni / Co(HHTP)MOF electrode material in preparing an electrochemical aptamer sensor for detecting Escherichia coli O157:H7.

[0015] The present invention provides an electrochemical aptamer sensor, which is prepared by the following method:

[0016] The Ni / Co(HHTP)MOF electrode material solution was evenly dropped onto the surface of a glassy carbon electrode and allowed to dry naturally at room temperature to obtain a Ni / Co(HHTP)MOF / GCE electrode. The aptamer solution was dropped onto the Ni / Co(HHTP)MOF / GCE electrode and incubated to obtain Apt / Ni / Co(HHTP)MOF / GCE. The BSA solution was then dropped onto the reaction area of ​​the Apt / Ni / Co(HHTP)MOF / GCE electrode and incubated to obtain BSA / Apt / Ni / Co(HHTP)MOF / GCE, i.e., an electrochemical aptamer sensor.

[0017] In the above-mentioned method for preparing the electrochemical aptamer sensor, the raw materials are selected from the following quantities:

[0018] 4-10 parts of Ni / Co(HHTP)MOF electrode material solution, 4-10 parts of aptamer solution, and 4-10 parts of BSA solution;

[0019] When the component is a solid component, the portion number represents milligrams; when the component is a liquid component, the portion number represents microliters; in actual application, the portion number can be enlarged or reduced proportionally.

[0020] In a specific embodiment, each raw material is selected from the following parts:

[0021] 7 parts of Ni / Co(HHTP)MOF electrode material solution, 5 parts of aptamer solution, and 5 parts of BSA solution;

[0022] When the component is a solid component, the portion number represents milligrams; when the component is a liquid component, the portion number represents microliters; in actual application, the portion number can be enlarged or reduced proportionally.

[0023] In the above-mentioned method for preparing the electrochemical aptasensor, the Ni / Co(HHTP)MOF electrode material solution refers to a PBS solution of Ni / Co(HHTP)MOF electrode material, and its concentration is 0.1-2.0 mg / mL, preferably 0.5 mg / mL.

[0024] In the above-mentioned method for preparing the electrochemical aptamer sensor, the aptamer solution refers to a PBS solution of the aptamer, and its concentration is 10 to 200 nM, preferably 100 nM.

[0025] In the above-mentioned preparation method of the electrochemical aptamer sensor, the incubation conditions are selected from: incubation at 30-40°C for 1-3 hours; preferably, the first incubation condition is: incubation at 37°C for 2 hours; the second incubation condition is: incubation at 37°C for 1 hour.

[0026] In the above-mentioned method for preparing the electrochemical aptamer sensor, the BSA solution refers to a BSA PBS solution, and its concentration is 0.1-1.0%, preferably 0.5%.

[0027] In the above-mentioned method for preparing the electrochemical aptamer sensor, the nucleic acid sequence of the aptamer is shown as SEQ ID NO: 1.

[0028] The present invention provides the use of the electrochemical aptamer sensor in detecting Escherichia coli O157:H7 in food.

[0029] The present invention provides a method for detecting Escherichia coli O157:H7 in food, comprising the following steps:

[0030] The working area of ​​the electrochemical aptamer sensor (the working area of ​​the modified glassy carbon electrode) was immersed in the sample to be tested and incubated at 37°C for 1 hour to achieve the binding of the aptamer and bacteria. The electrochemical aptamer sensor was rinsed three times with PBS buffer (pH 7.0) to eliminate unadsorbed bacteria. A conventional three-electrode system was used, in which the electrochemical aptamer sensor was used as the working electrode, the platinum wire was used as the counter electrode, and Ag / AgCl (KCl saturated) was used as the reference electrode. The three-electrode system was immersed in the electrolyte and electrochemical impedance spectroscopy was used for electrochemical detection to measure the interfacial reaction impedance value R. ct , R ct Substitute into the linear equation to calculate the concentration of E. coli O157:H7.

[0031] The above linear equation can be obtained as follows:

[0032] 5 μL of different concentration gradients (10~1×10 7 CFU / mL) of Escherichia coli O157:H7 bacteria were placed in the reaction area of ​​the electrochemical aptamer sensor (the working area of ​​the glassy carbon electrode) and incubated at 37°C for 1 hour to allow the aptamer to bind to the bacteria. The sensor was cleaned with PBS buffer (pH 7.0) and then formed into a three-electrode system with a platinum electrode and an Ag / AgCl electrode. The three-electrode system was immersed in 15 mL of an electrolyte containing 5 mmol / L potassium ferrocyanide and 0.1 mol / L KCl solution. Electrochemical impedance spectroscopy was used for electrochemical detection, and the bacterial concentration range was 10 to 1×10 7 The Nyquist plot of CFU / mL was obtained by fitting the data using ZSimpWin software to obtain the standard curve of Rct and the logarithm of bacterial concentration. The standard curve and linear equation were obtained by fitting using Origin software, and the linear relationship, linear equation and linear correlation coefficient R were obtained. 2 .

[0033] The beneficial effects of the present invention are:

[0034] This study prepared a rod-shaped Ni / Co(HHTP)MOF electrode material and used it as a sensitive platform for constructing electrochemical aptasensors for sensitive and selective detection of Escherichia coli O157:H7 in various environments. Due to its high electrochemical activity and uniform hybridization, Ni / Co(HHTP)MOF can be uniformly and stably modified onto GCE surfaces via strong electrostatic adsorption. Furthermore, Ni / Co(HHTP)MOF exhibits excellent biocompatibility, multivalent properties, and a large specific surface area, all of which facilitate the immobilization of large numbers of aptamer chains on the Ni / Co(HHTP)MOF surface. In the presence of bacteria, the aptamer and bacteria can hybridize to form a helical duplex, further leading to conformational changes in the aptamer. During the construction of the Ni / Co(HHTP)MOF-based aptasensor, electrode modification with Ni / Co(HHTP)MOF, aptamer immobilization, and bacterial detection all result in changes in the electrochemical signal. These changes are monitored using various electrochemical techniques, such as pulse voltammetry and electrochemical impedance spectroscopy.

[0035] Bimetallic MOFs and their derivatives exhibit a synergistic effect in enhancing electronic conductivity. The synergistic effect of bimetallic Ni / Co(HHTP)MOF can improve both electronic conductivity and chemical durability. This synergistic effect involves the rearrangement of metal atoms, forming an atomic and electronic structure more conducive to electronic conduction. Compared to monometallic MOFs, this synergistic effect results in higher electrical conductivity and electrochemical activity. This synergistic effect can enhance the electrochemical performance of the material, enabling it to excel in applications such as electrochemical energy storage.

[0036] The electrochemical aptamer sensor of the present invention can efficiently and specifically capture Escherichia coli O157:H7 and can be used to quickly and sensitively detect Escherichia coli O157:H7 in food. Compared with traditional bacterial detection methods, the method of the present invention has good selectivity, storage stability and reproducibility, a low detection limit and a short detection time, providing a new approach for the detection of foodborne pathogens and having good application prospects.

[0037] The electrochemical aptamer sensor constructed in the present invention combines the advantages of impedance detection technology, the specific recognition ability of the aptamer, and the excellent electronic conductivity of Ni / C(HHTP)MOF. It has the advantages of being fast, portable, simple, inexpensive, and label-free. It effectively shortens detection time and saves detection costs, makes up for the shortcomings of traditional detection methods, and has broad practical application prospects.

[0038] The electrochemical aptamer sensor of the present invention is used to detect Escherichia coli O157:H7. The method is simple to operate, does not require complex pretreatment of the sample to be tested, and has low detection cost. The electrochemical aptamer sensor of the present invention can effectively amplify the signal of Escherichia coli O157:H7 and can be used for low-concentration Escherichia coli O157:H7 detection. The sensitivity of the detection method of the present invention is 2.5 CFU / mL. The method of detecting Escherichia coli O157:H7 using the sensor of the present invention is short in time, with the entire detection process not exceeding 1 hour, and the detection speed is fast. The biosensor of the present invention can be used to detect Escherichia coli O157:H7 in food, realizing qualitative and quantitative analysis of Escherichia coli O157:H7.

[0039] In summary, the electrochemical aptamer sensor of the present invention can show good application prospects and value in the field of harmful microorganism detection technology. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 are the morphological characteristics of each material; among them, a is the SEM image of GCE, b is the SEM image of Ni / Co(HHTP)MOF / GCE electrode, c is the SEM image of Ni / Co(HHTP)MOF / GCE electrode after incubation with bacterial solution, d is the SEM image of BSA / Apt / Ni / Co(HHTP)MOF / GCE after incubation with bacterial solution, and e is the element distribution of Ni / Co(HHTP)MOF;

[0041] Figure 2 The differential pulse voltammograms of different modified electrodes in electrolyte;

[0042] Figure 3 The electrochemical impedance spectroscopy diagrams of different modified electrodes in electrolytes;

[0043] Figure 4 Nyquist plot of the electrochemical aptamer sensor after incubation with bacterial solutions of different concentrations for 1 h;

[0044] Figure 5 Standard curve for the electrochemical aptasensor to detect Escherichia coli O157:H7;

[0045] Figure 6 The histograms of the electrochemical aptasensor after incubation with different bacteria for 1 h;

[0046] Figure 7 The histograms before and after 15 days were saved for the electrochemical aptasensor;

[0047] Figure 8 For five identical electrochemical aptasensors and 10 3 Histogram of CFU / mL E. coli O157:H7 after 1 h of incubation. DETAILED DESCRIPTION

[0048] The materials used in the present invention are as follows:

[0049] The Escherichia coli O157:H7 (ATCC43895) used in the experiment was obtained from the Guangdong Provincial Center for Microbiological Culture Collection. The Escherichia coli aptamer used in the experiment was purchased from Shanghai Sangon Biotechnology Co., Ltd., and its nucleic acid sequence is as follows:

[0050] 5'-CCGGACCGCTTATGCCTTGCCATCTACAGAGCAGGTTGGACGG-3' (SEQ ID NO: 1).

[0051] Electrolyte containing 5 mM potassium ferricyanide and 0.1 M KCl: Add 3.7275 g potassium chloride (KCl), 1.6403 g potassium ferricyanide (K3[Fe(CN)6]), and 2.1121 g potassium ferrocyanide (K4[Fe(CN)6]·3H2O) to 1 L PBS, adjust the pH to 7.0, and shake to mix.

[0052] PBS buffer (1 L): Mix 6 g disodium hydrogen phosphate, 0.4 g sodium dihydrogen phosphate, and 9 g sodium chloride, add ddH2O (deionized water) to 1 L, adjust the pH to 7.0, shake to mix evenly, autoclave at 121°C for 2 h, and set aside.

[0053] Eosin-methylene blue solid medium (1 L): 5.445 g of eosin-methylene blue agar powder was added with ddH2O to 1 L, the pH was adjusted to 7.0, and the mixture was sterilized by autoclaving at 121°C for 2 h.

[0054] LB liquid medium (1 L): 10 g of peptone, 5 g of yeast powder, 10 g of NaCl, add ddH2O to 1 L, adjust the pH to 7.0, and sterilize by autoclaving at 121°C for 2 h.

[0055] Escherichia coli O157:H7 bacterial suspension culture:

[0056] Glycerol-frozen Escherichia coli O157:H7 was inoculated onto LB solid culture medium using the three-zone streak method and placed in a 37°C biochemical incubator for 18 to 24 hours to obtain a single colony. Pick an appropriate amount of single colonies of Escherichia coli O157:H7, inoculate them into a sterile cell culture tube containing LB liquid culture medium, and culture them in a 37°C constant temperature incubator at 150 rpm / min for 16 hours. The bacterial concentration was determined by an ultraviolet spectrophotometer using an optical density (OD) of 600 nm. The bacterial solution was then centrifuged at 4500 rpm / min for 15 minutes. After washing twice with ultrapure water, it was resuspended in PBS buffer and diluted to 10, 1×10 2 , 1×10 3 , 1×104 , 1×10 5 , 1×10 6 , 1×10 7 and 1×10 8 The bacterial solution was stored at 4°C until use.

[0057] In the present invention, the glassy carbon electrode is treated as follows before use:

[0058] The glassy carbon electrode was polished to a mirror finish using 1μm, 0.3μm, and 0.5μm alumina polishing powder, rinsed with ultrapure water, and then ultrasonicated in anhydrous ethanol and ultrapure water for 15 seconds each, repeated three times. A three-electrode system was formed by treating the glassy carbon electrode (working electrode), Ag / AgCl (KCl saturated) (reference electrode), and a platinum electrode (counter electrode). The system was immersed in 15mL of an electrolyte containing 5mM potassium ferrocyanide and 0.1M KCl, and cyclic voltammetry (CV) was performed using a Gamry Interface 11E electrochemical workstation. Parameter settings: potential scan range -0.2 to 0.6V, scan rate 0.1mV / s. When the scan curve stabilizes, the middle peak is stable, and the potential difference is ≤0.08V, the pretreatment of the glassy carbon electrode is complete, and a Bare GCE electrode is obtained.

[0059] In the electrochemical impedance spectroscopy (EIS) test of the following embodiments of the present invention, the electrochemical impedance spectroscopy (EIS) parameters are set as follows: the initial potential is obtained by open circuit potential scanning, the scanning frequency range is 0.01 Hz to 100 kHz, the amplitude is 5 mV, and the standing time is 2 s.

[0060] In the differential pulse voltammetry test of the following embodiment of the present invention, the differential pulse voltammetry (DPV) parameters are set as follows: initial potential is -0.2 V, final potential is 0.6 V, amplitude is 50 mV, pulse width is 0.1 s, and sampling width is 0.0167 s.

[0061] The other materials used in the present invention, unless otherwise stated, can be obtained through commercial channels. Unless otherwise specified, other terms used in the present invention generally have the meanings commonly understood by those of ordinary skill in the art. The present invention will be further described in detail below with reference to specific examples and data. The following examples are merely for illustration of the present invention and are not intended to limit the scope of the present invention in any way.

[0062] Example 1

[0063] The Ni / Co(HHTP)MOF electrode material was prepared as follows:

[0064] 124 mg of Ni(CH3COO)2·4H2O, 62 mg of Co(CH3COO)2·4H2O, and 162 mg of HHTP (2,3,6,7,10,11-hexahydroxytriphenylene hydrate) were dissolved in 18 mL of a solvent (DMF:H2O = 1:3) and stirred at 85°C for 5 minutes to thoroughly mix the reagents for subsequent oven synthesis. The solution was transferred to a glass bottle and reacted in an 85°C oven for 30 minutes before cooling to room temperature. After centrifugation and washing three times with ethanol, the product was dried in a vacuum oven at 60°C for 1 hour to obtain the Ni / Co(HHTP) MOF electrode material.

[0065] Example 2

[0066] Prepare the electrochemical aptasensor as follows:

[0067] 0.5 mg Ni / Co(HHTP)MOF electrode material was dissolved in 1 mL PBS and sonicated for 10 min. Then, 7 μL of the above electrode material solution (0.5 mg / mL) was evenly dropped on the surface of the glassy carbon electrode (GCE) and allowed to dry naturally at room temperature to obtain the Ni / Co(HHTP)MOF / GCE electrode. 5 μL of aptamer solution (100 nM) was dropped on the Ni / Co(HHTP)MOF / GCE electrode and incubated in a 37°C drying oven for 2 h to obtain Apt / Ni / Co(HHTP)MOF / GCE. Then, 5 μL of BSA solution (concentration 0.5%, solvent is PBS buffer) was dropped on the reaction area of ​​the Apt / Ni / Co(HHTP)MOF / GCE electrode, and the electrode was placed in a 37°C drying oven and incubated for 1 h to obtain BSA / Apt / Ni / Co(HHTP)MOF / GCE, i.e., the electrochemical aptamer sensor.

[0068] In the present invention, as a blocking agent, bovine serum albumin (BSA) molecules can occupy the residual sites to avoid nonspecific adsorption between bacteria and substances.

[0069] The electrochemical aptasensor BSA / Apt / Ni / Co(HHTP)MOF / GCE was mixed with Escherichia coli O157:H7 bacterial suspension (10 7 The prepared aptasensor was stored in a refrigerator at 4°C before use.

[0070] In order to explore the morphological characteristics of the above-mentioned electrode materials, scanning electron microscopy was used for characterization.

[0071] The test results are as follows Figure 1 As shown:

[0072] like Figure 1 As shown in a, the surface of the bare GCE is smooth. Figure 1 As shown in b, the surface of Ni / Co(HHTP)MOF / GCE becomes significantly rougher after MOF deposition and is composed of tightly connected particles. These particles are similar to 200nm thick rods, which makes the surface of the active material more exposed and shortens the path of charge transfer and electrolyte ion diffusion, thereby improving electronic conductivity. In addition, this structure obviously has a larger surface area and more E. coli sensing adsorption sites than smooth GCE. The above results show that Ni / Co(HHTP)MOF has been successfully grown on GCE. Figure 1 c shows that in the absence of aptamers and bovine serum albumin (BSA), the number of bacteria observed on the Ni / Co(HHTP)MOF / GCE surface after incubation with E. coli was low, indicating a low level of nonspecific adsorption of bacteria to the electrode surface. After further incubation with aptamers and BSA, a significant increase in the number of E. coli was observed on the electrode surface ( Figure 1 d). This increase is attributed to the specific binding affinity of the aptamer to the bacteria, resulting in higher capture efficiency. The bacteria were found to be wrapped by the material, indicating that E. coli had been successfully immobilized on the Ni / Co(HHTP)MOF / GCE surface. These results further confirmed that the prepared MOF / GCE sensor can effectively capture E. coli. In order to obtain the elemental composition of Ni / Co(HHTP)MOF, energy dispersive X-ray spectroscopy (EDS) was used to identify the elemental distribution of Ni / Co(HHTP)MOF. Figure 1 As shown in Figure e, C, O, Co, and Ni elements are uniformly distributed throughout the material.

[0073] Differential pulse voltammetry (DPV) and electrochemical impedance spectroscopy (EIS) were performed on each modified electrode using an electrochemical workstation. After the test, the residual liquid in the electrode reaction area was washed with PBS buffer. PBS buffer was added dropwise to the working area of ​​the electrode, and then the electrode was covered with a dustproof cap and stored in a refrigerator at 4°C until it was opened and used later.

[0074] The test results are as follows Figure 2 and Figure 3 As shown: different modified electrodes will lead to different changes in electrochemical signals, proving the feasibility of the present invention.

[0075] Principle of the Invention: Rod-shaped Ni / Co(HHTP)MOFs were prepared and used as a sensitive platform for constructing electrochemical aptamer sensors for sensitive and selective detection of Escherichia coli O157:H7 in various samples. Due to its high electrochemical activity and uniform hybridization properties, Ni / Co(HHTP)MOFs can be uniformly and stably modified onto GCE surfaces via strong electrostatic adsorption. Furthermore, Ni / Co(HHTP)MOFs exhibit excellent biocompatibility, multivalency, and a large specific surface area, all of which facilitate the immobilization of large quantities of aptamers on the Ni / Co(HHTP)MOF surface. In the presence of bacteria, a double-stranded helical structure is formed between the aptamer and the bacteria through hybridization reactions. During the construction of Ni / Co(HHTP)MOF-based electrochemical sensors, electrode modification of the Ni / Co(HHTP)MOF, aptamer immobilization, and bacterial detection result in changes in the electrochemical signal, which can be detected using electrochemical techniques such as dynamic pulse voltammetry (DPV) and electrochemical impedance spectroscopy (EIS).

[0076] Example 3

[0077] Detection of E. coli O157:H7:

[0078] 5 μL of different concentration gradients (10~1×10 7 A test Escherichia coli O157:H7 bacterial solution (CFU / mL) was placed in the reaction area (working area of ​​the glassy carbon electrode) of the electrochemical aptamer sensor prepared in Example 2 and incubated at 37°C for 1 hour to achieve aptamer-bacterial binding. The sensor was cleaned with PBS buffer (pH 7.0) and then formed into a three-electrode system with a platinum electrode and an Ag / AgCl electrode. The three-electrode system was immersed in 15 mL of an electrolyte containing 5 mmol / L potassium ferricyanide and 0.1 mol / L KCl solution. Electrochemical impedance spectroscopy (EIS) was used for electrochemical detection, and the bacterial concentration range was 10 to 1×10 7 The Nyquist plot of CFU / mL was fitted with the data using ZSimpWin software to obtain R ct The standard curve of the logarithm of bacterial concentration was fitted with Origin software to obtain the standard curve and linear equation, and the linear relationship, linear equation and linear correlation coefficient R were obtained. 2 , the experiment was repeated three times under the same conditions.

[0079] The test results are as follows Figure 4 and Figure 5 As shown:

[0080] Depend on Figure 4 The impedance spectrum shown shows that as the bacterial concentration increases, the semicircle of the Nyquist plot increases, indicating that the reaction resistance of the working electrode increases.

[0081] The impedance data was fitted using Origin software to obtain the linear equation R ct (Ω)=592.18197lgC E.coli (CFU / mL) + 98.84227, linear correlation coefficient (R 2 ) is 0.99262, indicating that it is linear in a wide range (10~1×10 7 CFU / mL), interface reaction resistance value R ct In the range of bacterial concentration, the relationship between the bacterial concentration and the logarithm of bacterial concentration increases linearly, such as Figure 5 As shown, the detection limit was 2.5 CFU / mL (S / N=3).

[0082] In the electrochemical sensor of the present invention, the two key issues in constructing an electrochemical aptamer sensor are the fixation of the aptamer, a biorecognition element, on the electrode surface and the enhancement of the signal during the recognition process. Both of these issues rely on the effectiveness of electrode modification. The aptamer can specifically immobilize E. coli, which is captured and adsorbed on the surface of the electrochemical sensor. The electron shielding effect of the cell membrane triggers changes in the interface, resulting in changes in the impedance spectrum and a weakening of the electrochemical signal.

[0083] Example 4

[0084] Selectivity, stability and reproducibility testing:

[0085] (1) Selectivity

[0086] Under the same conditions, the electrochemical aptamer sensor prepared in Example 2 was mixed with the same concentration (1×10 6 The aptamer was incubated with Escherichia coli O157:H7 (1000 CFU / mL), four potential interfering bacteria present in the laboratory (Staphylococcus aureus, Vibrio parahaemolyticus, Shigella, and Salmonella), and a mixture of these interfering bacteria and E. coli for one hour. Electrochemical impedance spectroscopy (EIS) was then performed on each of the three samples, using a PBS blank control as a control. The specificity of the electrochemical aptamer sensor was investigated by comparing the electron transfer resistance. The experiment was repeated three times.

[0087] The test results are as follows Figure 6 As shown:

[0088] When detecting the target strain O157:H7, the interface electronic reaction resistance value R ct The interfacial electronic reaction resistance was significantly greater than that of other interfering strains, mixed group and blank control group, indicating that the constructed sensor had good selectivity.

[0089] (2) Stability

[0090] Take the electrochemical sensor prepared in Example 2, add 1 μL of PBS buffer to the working area of ​​the sensor, and perform AC impedance testing. After the measurement, clean it with PBS, add 1 μL of PBS buffer to the working area, cover it with a small cap, and store it at 4°C for 15 days. Then, take out the sensor and perform AC impedance testing again to determine the stability of the electrochemical sensor. Repeat the experiment three times in parallel.

[0091] The test results are as follows Figure 7 As shown:

[0092] After being stored at 4°C for 15 days, the sensor can still maintain 90.58% of the initial signal value, and the interface reaction resistance is 1966Ω, indicating that the constructed sensor has good storage stability.

[0093] (3) Reproducibility

[0094] The reproducibility of the electrochemical aptamer sensor was evaluated by parallel experiments. Five electrochemical sensors prepared in Example 2 were used and mixed with a concentration of 1×10 3 After incubation with a 1000 CFU / mL E. coli O157:H7 bacterial solution for 1 hour, AC impedance spectroscopy was performed. The RSD of the fitted interfacial electron transfer resistance was calculated to determine the reproducibility of the electrochemical sensor. The experiment was repeated three times.

[0095] The test results are as follows Figure 8 As shown:

[0096] The interface reaction resistance R was calculated using Origin software. ct The relative standard deviation (RSD) was 3.6% after fitting, indicating that the electrochemical aptasensor has good reproducibility.

[0097] Application Examples

[0098] In order to further evaluate the feasibility of the application of the electrochemical aptamer sensor prepared by the present invention (Example 2) in the detection of food pathogens, different concentrations of Escherichia coli O157:H7 were added to fresh products, and the detection method described in Example 3 was used for detection. The interfacial reaction impedance value R ct , R ct Substitute into the linear equation described in Example 3 to calculate the concentration of E. coli O157:H7.

[0099] Compare the test results with the added concentration and calculate the recovery rate.

[0100] The calculation formula is:

[0101] Detection recovery rate = detection concentration / added concentration × 100%.

[0102] The test results are shown in Table 1:

[0103] Table 1 Recovery rates of spiked samples after adding different concentrations of E. coli O157:H7

[0104]

[0105] The results showed that the detection recovery rate was between 92.72% and 114.15%, indicating that the electrochemical aptamer sensor prepared by the present invention has good stability and strong practical applicability.

[0106] The preparation method of the present invention is based on the construction of an electrochemical sensor based on a conductive metal-organic framework and an aptamer. The aptamer can efficiently and specifically capture Escherichia coli O157:H7 and can be used to quickly and sensitively detect Escherichia coli O157:H7 in food. Compared with traditional bacterial detection, the method of the present invention has good selectivity, storage stability and reproducibility, a low detection limit and a short detection time, providing a new idea for the detection of foodborne pathogens and having good application prospects.

[0107] The electrochemical aptamer sensor constructed in the present invention for detecting Escherichia coli O157:H7 combines the advantages of impedance detection technology, the specific recognition ability of the aptamer, and the excellent electronic conductivity of Ni / Co(HHTP)MOF. It is fast, portable, simple, inexpensive, and does not require labeling. It effectively shortens detection time and saves detection costs, making up for the shortcomings of traditional detection methods and has broad practical application prospects.

[0108] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any other manner. Any person skilled in the art may utilize the above-disclosed technical content to modify or modify the present invention into equivalent embodiments. However, any simple modifications, equivalent variations, and modifications to the above embodiments that do not depart from the technical content of the present invention and are based on the technical essence of the present invention remain within the scope of protection of the present invention.

Claims

1. A method for detecting Escherichia coli O157:H7 in food, characterized in that: The steps include: The working area of ​​the electrochemical aptamer sensor was immersed in the sample to be tested and incubated at 37°C for 1 hour to achieve the binding of the aptamer and bacteria. The electrochemical aptamer sensor was rinsed three times with PBS buffer at pH 7.0 to eliminate unadsorbed bacteria. A conventional three-electrode system was used, in which the electrochemical aptamer sensor was used as the working electrode, the platinum wire was used as the counter electrode, and Ag / AgCl (KCl saturated) was used as the reference electrode. The three-electrode system was immersed in the electrolyte and electrochemical impedance spectroscopy was used for electrochemical detection to measure the interfacial reaction impedance value R. ct , R ct Substitute into the linear equation to calculate the concentration of E. coli O157:H7; The electrochemical aptamer sensor is prepared by the following method: A Ni / Co (HHTP) MOF electrode material solution was evenly dropped onto the surface of a glassy carbon electrode and allowed to dry naturally at room temperature to obtain a Ni / Co (HHTP) MOF / GCE electrode. An aptamer solution was dropped onto the Ni / Co (HHTP) MOF / GCE electrode and incubated to obtain Apt / Ni / Co (HHTP) MOF / GCE. A BSA solution was then dropped onto the reaction area of ​​the Apt / Ni / Co (HHTP) MOF / GCE electrode and incubated to obtain BSA / Apt / Ni / Co (HHTP) MOF / GCE, i.e., an electrochemical aptamer sensor. The Ni / Co (HHTP) MOF electrode material is prepared by the following method: Ni(CH3COO)2·4H2O, Co(CH3COO)2·4H2O and HHTP were dissolved in a solvent and stirred for reaction. After the reaction was completed, the mixture was cooled to room temperature and the reaction solution was centrifuged to obtain a solid product. The product was then washed and dried to obtain a Ni / Co (HHTP) MOF electrode material. The nucleic acid sequence of the aptamer is shown in SEQ ID NO:

1.

2. The detection method according to claim 1, wherein In the preparation method of the Ni / Co (HHTP) MOF electrode material, each raw material is selected from the following parts: 100-180 parts of Ni(CH3COO)2·4H2O, 50-90 parts of Co(CH3COO)2·4H2O, 120-200 parts of HHTP, and 10-40 parts of solvent.

3. The detection method according to claim 1, wherein In the preparation method of the Ni / Co (HHTP) MOF electrode material, the solvent is a DMF aqueous solution, wherein the volume ratio of DMF to water is 1:3; and / or the stirring reaction conditions are selected from: stirring the reaction at 80-90° C. for 1-60 minutes.

4. The detection method according to claim 1, wherein In the preparation method of the electrochemical aptamer sensor, the Ni / Co (HHTP) MOF electrode material solution is a PBS solution of Ni / Co (HHTP) MOF electrode material, and its concentration is 0.1-2.0 mg / mL.

5. The detection method according to claim 1, wherein In the preparation method of the electrochemical aptamer sensor, the raw materials are selected from the following quantities: 4-10 parts of Ni / Co (HHTP) MOF electrode material solution, 4-10 parts of aptamer solution, and 4-10 parts of BSA solution.

6. The detection method according to claim 1, characterized in that In the preparation method of the electrochemical aptamer sensor, the aptamer solution refers to a PBS solution of the aptamer, the concentration of which is 10-200 nM; and / or the BSA solution refers to a PBS solution of BSA, the concentration of which is 0.1-1.0%; and / or the incubation conditions are selected from: incubation at 30-40°C for 1-3 h.

7. The detection method according to claim 1, characterized in that The linear equation is obtained by the following method: Add 5 μL of different concentration gradients from 10 to 1×10 7 The Escherichia coli O157:H7 bacterial solution with a concentration of 100 CFU / mL was placed in the working area of ​​the electrochemical aptamer sensor and incubated at 37°C for 1 hour to achieve aptamer-bacterial binding. The sensor was cleaned with PBS buffer at pH 7.0 and then formed into a three-electrode system with a platinum electrode and an Ag / AgCl electrode. The three-electrode system was immersed in 15 mL of an electrolyte containing 5 mmol / L potassium ferrocyanide and 0.1 mol / L KCl solution. Electrochemical impedance spectroscopy was used for electrochemical detection, and the bacterial concentration range was 10 to 1×10 7 The Nyquist plot of CFU / mL was fitted with the data using ZSimpWin software to obtain R ct The standard curve of the logarithm of bacterial concentration was fitted with Origin software to obtain the standard curve and linear equation, and the linear relationship, linear equation and linear correlation coefficient R were obtained. 2 .

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