阅读说明：本技术 一种电化学生物传感器的制备方法及其在大肠杆菌检测中的应用 (Preparation method of electrochemical biosensor and application of electrochemical biosensor in escherichia coli detection ) 是由 黄启同 林小凤 杨民 曾卫佳 梅艳秋 何晨 于 2021-08-30 设计创作，主要内容包括：本发明公开了一种电化学生物传感器的制备方法及其在大肠杆菌检测中的应用。在玻碳电极表面滴入CDs-Fe-(3)O-(4)纳米复合材料,制备CDs-Fe-(3)O-(4)/GCE工作电极,激活电极后再浸泡在探针DNA中,得到DNA/CDs-Fe-(3)O-(4)/GCE探针电极,将探针电极浸入不同浓度的E.coli O157:H7溶液中,水浴反应得到大肠杆菌O157:H7-DNA/CDs-Fe-(3)O-(4)/GCE。本发明构建了一种基于CDs-Fe-(3)O-(4)复合纳米材料的灵敏电化学生物传感器,该电化学生物传感器对于E.coli O157:H7的测定具有良好的线性和检测限。此外,该生物传感器还成功应用于牛奶中E.coli O157:H7的测定,表明电化学生物传感器具有广阔的应用前景。(The invention discloses a preparation method of an electrochemical biosensor and application of the electrochemical biosensor in escherichia coli detection. Dripping CDs-Fe on the surface of glassy carbon electrode 3 O 4 Nanocomposite, preparation of CDs-Fe 3 O 4 Activating the working electrode of the/GCE electrode, and then soaking the working electrode in the probe DNA to obtain DNA/CDs-Fe 3 O 4 A GCE probe electrode, the probe electrode being immersed in different concentrations E.coli The Escherichia coli O157H 7-DNA/CDs-Fe is obtained by water bath reaction in O157H 7 solution 3 O 4 (ii)/GCE. The invention constructs a method based on CDs-Fe 3 O 4 Sensitive electrochemical biosensor of composite nanomaterial, the electrochemical biosensor is to E.coli The measurement of O157: H7 has good linearity and detection limit. In addition, the biosensor is successfully applied to milk E.coli Measurement of O157H 7, indicating electrochemical biographyThe sensor has wide application prospect.)

1. A preparation method of an electrochemical biosensor is characterized by comprising the following steps:

1) synthesis of CDs-Fe₃O₄Nanocomposite material

2) Preparation of DNA/CDs-Fe₃O₄/GCE probe electrode

Slowly dripping CDs-Fe on the surface of the clean glassy carbon electrode₃O₄Preparing CDs-Fe from nano composite material by room-temperature drying₃O₄The working electrode of/GCE followed by the addition of CDs-Fe₃O₄Soaking the GCE working electrode in a mixed solution of 0.1mM PBS, 8.00mM EDC and 8.00mM NHS for 3-3.5h to activate the electrode, washing with TE buffer solution, soaking the electrode in 0.1 μ M probe DNA for reaction for 24-25h, and washing with TE buffer solution to prepare DNA/CDs-Fe₃O₄a/GCE probe electrode, and finally storing the prepared electrode in an environment at 4 ℃;

3) preparation of biosensor

The prepared DNA/CDs-Fe₃O₄The GCE probe electrode being immersed in different concentrationsE. coliReacting in O157H 7 solution in water bath, and washing off the non-specifically adsorbed substances on the surface by TE buffer solution after the reaction is finishedE. coli O157H 7 to obtain Escherichia coli O157H 7-DNA/CDs-Fe₃O₄/GCE。

2. The method of claim 1, wherein the step 1) of synthesizing CDs-Fe₃O₄The method of the nanocomposite is as follows:

dissolving 3g of citric acid and 1g of glucose in water, reacting for 5-8 minutes under the condition that the microwave power is 750-850W, cooling and diluting with water, centrifuging for 15-20min, dialyzing for not less than 24 hours, freeze-drying to obtain CDs, and dissolving the CDs solid in 25mL of aqueous solution to obtain a CDs solution;

adding 1g of sodium acetate and 1g of ferric chloride into 15mL of CDs solution, ultrasonically treating the obtained mixed solution for 8-10 hours, placing the mixed solution into a high-pressure reaction kettle, reacting for 24-25 hours at the temperature of 200-220 ℃, cooling the solution, and centrifuging for 10-15min to obtain CDs-Fe₃O₄A nanocomposite material.

3. The method as claimed in claim 2, wherein the rotation speed of the centrifugation is 11000 and 13000 rpm.

4. The method of claim 1, wherein in step 2), the surface of the clean glassy carbon electrode is dripped with CDs-Fe₃O₄The volume of the nano composite material is 5.0-5.5 mu L, the volume of the mixed solution is 0.8-1.2mL, and the volume of the probe DN is 0.8-1.2 mL.

5. The method for preparing an electrochemical biosensor as claimed in claim 1, wherein the temperature of the water bath reaction in step 3) is 42 ℃ and the time is 35-40 min.

6. The method for preparing an electrochemical biosensor as claimed in claim 5, wherein the water bath reaction time in step 3) is 40 min.

7. Use of the electrochemical biosensor obtained by the preparation method according to any one of claims 1 to 6 in detection of Escherichia coli O157: H7.

Technical Field

The invention belongs to the field of electrochemical luminescence sensors, and particularly relates to a preparation method of an electrochemical biosensor and application of the electrochemical biosensor in detection of Escherichia coli O157: H7.

Background

In recent years, food poisoning caused by food-borne pathogenic bacteria frequently occurs worldwide and is the most prominent public health problem in the world. Coli (e.coli) O157: H7, one of the most harmful food-borne pathogenic bacteria, can cause hemorrhagic colitis, hemolytic uremic syndrome, and the like. More importantly, low doses of e.coli O157: H7 are pathogenic to humans and even severe, can lead to renal failure and death. Currently, over 200 million acute food-borne diseases are attributed to e.coli O157: H7 worldwide each year. Therefore, sensitive detection of e.coli O157: H7 in food is particularly important. Currently, the most commonly used methods for the assay of e.coli O157: H7 include bacterial culture counting, enzyme-linked immunosorbent assay, polymerase chain reaction, and the like. Nevertheless, the above methods still face several limitations in the detection of food-borne pathogens, including low sensitivity, complex pre-treatment and large environmental impact. Therefore, there is a need to develop a sensitive, rapid, simple and highly specific method for detecting e.coli O157: H7. Compared with the traditional method, the electrochemical biosensor has the advantages of high sensitivity, economy, accuracy and the like, so that the electrochemical biosensor is widely concerned in detection in E.coli O157: H7.

As is well known, nanomaterials have been widely used in the fields of biomedicine, analytical science, energy catalysis, etc. due to their excellent chemical, physical and biological properties. To date, some nanomaterials have been successfully applied to the construction of electrochemical sensors for e.coli O157: H7 detection, such as graphene/AuNPs, reduced graphene oxide/polyaniline/Au @ Pt/neutral red (rGO/PANI/Au @ Pt/Nr), rGO/Au @ Pt, rGO-polyvinyl alcohol/gold nanocomposite (AuNPs/rGO-PVA), and the like. Although the material makes a certain contribution to an electrochemical sensor for detecting E.coli O157: H7, the material still has the defects of complex preparation, insufficient economy and environmental protection, low sensor sensitivity and the like. Therefore, the preparation of the functional material which is easy to obtain, excellent in performance, economical and environment-friendly and used for constructing the novel electrochemical sensor has important significance for detecting E.coli O157: H7.

As a member of 'zero-dimensional' carbon nano materials, Carbon Dots (CDs) have the advantages of good biocompatibility, simple synthesis, good conductivity and the like, and can be used as one of ideal electrode materials. Fe₃O₄Has good magnetic function and can realize the magnetic recovery of the composite material.

Disclosure of Invention

The invention aims to provide a preparation method of an electrochemical sensor and application of the electrochemical sensor in detection of Escherichia coli O157: H7.

In order to achieve the purpose, the invention adopts the technical scheme that:

a preparation method of an electrochemical sensor comprises the following steps:

1) synthesis of CDs-Fe₃O₄Nanocomposite material

Dissolving 3g of citric acid and 1g of glucose in 5mL of water, reacting for 5-8 minutes under the condition that the microwave power is 750-;

adding 1g of sodium acetate and 1g of ferric chloride into 15mL of CDs solution, ultrasonically treating the obtained mixed solution for 8-10 hours, placing the mixed solution into a high-pressure reaction kettle, reacting for 24-25 hours at the temperature of 200-13000 ℃, cooling the solution, and centrifuging for 10-15 minutes at the rotating speed of 11000-13000rpm to obtain CDs-Fe₃O₄A nanocomposite;

2) preparation of DNA/CDs-Fe₃O₄/GCE probe electrode

Slowly dripping 5.0-5.5 mu L of CDs-Fe on the surface of a clean Glassy Carbon Electrode (GCE)₃O₄Preparing CDs-Fe from nano composite material by room-temperature drying₃O₄A GCE working electrode, prepared from CDs-Fe₃O₄The GCE working electrode is soaked in 0.8-1.2mL of mixed solution consisting of 0.1mM PBS, 8.00mM EDC and 8.00mM NHS for 3-3.5h to activate the electrode,then washing with TE buffer solution, soaking the electrode in 0.8-1.2ml 0.1 μ M probe DNA for reaction for 24-25h, washing with TE buffer solution to prepare DNA/CDs-Fe₃O₄a/GCE probe electrode, and finally, storing the prepared electrode in an environment at 4 ℃;

3) preparation of biosensor

The prepared DNA/CDs-Fe₃O₄Immersing the/GCE probe electrode into E.coli O157: H7 solutions with different concentrations, reacting in a water bath at 42 ℃ for 35-40min, and after the reaction is finished, washing off the E.coli O157: H7 which is not specifically adsorbed on the surface by using TE buffer solution to obtain Escherichia coli O157: H7-DNA/CDs-Fe₃O₄/GCE。

The invention constructs a method based on CDs-Fe₃O₄A sensitive electrochemical biosensor made of composite nano material is used for detecting E.coli O157: H7. CDs as one of carbon nano materials has good conductivity and large specific surface area, can improve the sensitivity of the sensor, contains rich carboxyl on the surface, can be used for fixing probe DNA, and can also be used as a reducing agent for synthesizing CDs-Fe₃O₄And (3) nano materials. Fe₃O₄The nanometer material can improve the performance of the electrochemical biosensor, and can realize the effect of the magnetic property on CDs-Fe₃O₄And (4) recovering. The electrochemical biosensor prepared by the invention has good specificity to E.coli O157: H7. Under the optimal condition, the electrochemical biosensor has good DA detection performance, and the linear range is 10-10⁸CFU/mL, detection limit of 6.88CFU/mL (3S/N). In addition, the electrochemical biosensor is successfully applied to detection of E.coli O157: H7 in milk, and the electrochemical biosensor has good application prospect.

Drawings

FIG. 1 is TEM and HRTEM images of CDs (A, B) and CDs-Fe3O4 nanomaterials (C, D).

FIG. 2 shows different electrodes at 1.0mM [ Fe (CN)₆]^3-/4Graphs of CVs in mixed solution with 0.1M KCl at a sweep rate of 0.1V/s.

FIG. 3 is a graph showing concentrations of 10CFU/mL (A) and 10⁸E.coli O157: H7 and DNA/CDs-Fe of CFU/mL (B)₃O₄Reaction time of/GCE.

FIG. 4 is 10⁵CFU/mL of different bacteria and DNA/CDs-Fe₃O₄Current response of the/GCE reaction (A) and 10⁵Current response in the co-presence of CFU/mL E.coli O157: H7 with different bacteria (B); wherein a is none, b is E.coli O157H 7, c is Staphylococcus aureus, d is Salmonella, e is Staphylococcus lactis, and f is Listeria.

FIG. 5 is DNA/CDs-Fe₃O₄Coli O157: H7(a to i:0, 10) with various concentrations of/GCE²,10³,10⁴,10⁵,10⁶,10⁷,10⁸CFU/mL) and a linear relationship of current to log concentration (B).

FIG. 6 is DNA/CDs-Fe₃O₄The reproducibility (A) and stability (B) of E.coli O157: H7 were determined by GCE.

Detailed Description

EXAMPLE 1 Synthesis of CDs-Fe₃O₄Nanocomposite material

3g of citric acid and 1g of glucose were dissolved in 5ml of water and reacted for 5 minutes under a microwave power of 800W. Then diluted with 30mL of water and centrifuged at 12000rpm for 15 min. And dialyzing for 24 hours, freezing and drying to obtain the synthesized CDs, and dissolving the CDs solid in 25mL of aqueous solution.

15mL of CDs solution is taken, 1g of sodium acetate and 1g of ferric chloride are added, the mixed solution is subjected to ultrasonic treatment for 8 hours, and the mixture is placed in a high-pressure reaction kettle and reacted for 24 hours at 210 ℃. Finally, after the solution is cooled, centrifuging for 10min at the rotating speed of 12000rpm to obtain CDs-Fe₃O₄A nanocomposite material.

EXAMPLE 2 preparation of electrode

1) Slowly dripping 5.0 mu L of CDs-Fe on the surface of a clean Glassy Carbon Electrode (GCE)₃O₄Preparing CDs-Fe from nano composite material by room-temperature drying₃O₄a/GCE working electrode. 2) Mixing CDs-Fe₃O₄the/GCE electrode was soaked in 1mL of a mixed solution of 0.1mM PBS, 8.00mM EDC and 8.00mM NHS for 3 hours to activate the electrode, and then washed with TE buffer. 3) Soaking the electrodeReaction in 1ml of 0.1. mu.M probe DNA for 24 hours, washing with TE buffer to prepare DNA/CDs-Fe₃O₄a/GCE probe electrode. Finally, the prepared electrode was stored at 4 ℃.

EXAMPLE 3 preparation of biosensor

The prepared DNA/CDs-Fe₃O₄the/GCE was immersed in different concentrations of E.coli O157: H7 solutions and reacted in a water bath at 42 ℃ for 40 min. After the reaction is finished, washing off the E.coli O157: H7 which is not specifically adsorbed on the surface by using TE buffer solution to obtain Escherichia coli O157: H7-DNA/CDs-Fe₃O₄/GCE。

Example 4

With [ Fe (CN) ]₆]^3-/4-The solution is an electrolyte, and the interlayer assembly of the modified electrode is characterized by adopting Cyclic Voltammetry (CV), Electrochemical Impedance Spectroscopy (EIS) and Differential Pulse Voltammetry (DPV).

1 characterization of the materials

CDs and CDs-Fe₃O₄The morphology of the nanomaterials was characterized by Transmission Electron Microscopy (TEM). FIG. 1A shows that the particle size distribution of the CDs prepared is relatively uniform, mainly at 3-4 nm. FIG. 1B is a high power transmission electron microscopy (HRTEM) image of CDs. The results show that CDs have a good lattice with a lattice spacing of 0.210nm, which is a typical (002) carbon crystal plane. As shown in FIG. 1C, CDs-Fe₃O₄The particle size range of the nano material is 15-40 nm. CDs-Fe₃O₄The HRTEM characterization of (FIG. 1D) shows that the nanomaterial has two lattice spacings, 0.345nm and 0.210nm, respectively, which correspond to Fe₃O₄The (220) crystal plane of (c) and the (002) crystal plane of carbon. The above morphology characterization shows that CDs-Fe₃O₄Nanomaterials have been successfully prepared.

2 characterization of electrochemical Properties

Due to [ Fe (CN)6]^3-/4-Has very good electrochemical activity, so that the electrochemical performance of the material is often characterized by the electrochemical performance of a working electrode or a modified electrode. In this example, the electrode performance was characterized mainly by Cyclic Voltammetry (CV), at 1.0mmol L-1[ Fe (CN)6]^3-/4-And 0.1mol of L-1KCl in the mixed aqueous solution, and measured by CV method using different electrodes, respectively, as shown inAs shown in fig. 2. On bare GCE, the redox peak current (I) has good reversibility, with the oxidation peak potential (Ipa) and reduction peak potential (Ipc) being-6.616A and 5.958A, respectively. When CDs are modified on the surface of GCE, Ipa and Ipc are respectively promoted to-12.29A and 12.06A, which shows that the prepared CDs have good conductivity. When the electrode is CDs-Fe₃O₄Ipa and Ipc are further enhanced for the case of/GCE, indicating Fe₃O₄The introduction of (2) can improve the conductivity of the electrode. However, when the probe DNA was reacted in the CDs Fe by a carboxyammoniation coupling reaction₃O₄On the/GCE surface, I decreases mainly because DNA molecules block [ Fe (CN)6]3-/4 in CDs-Fe₃O₄Diffusion of/GCE surface-. The above results also indicate that the probe DNA has been successfully immobilized on the surface of the working electrode.

3 reaction time

The interaction time of the probe electrode with the target substance is one of the key factors affecting the performance of the electrochemical biosensor. FIG. 3A shows that the optimal hybridization time was determined to be 40 minutes when the concentration of E.coli O157: H7 was 10 CFU/mL. However, when the concentration of E.coli O157: H7 was 10⁸The optimal hybridization time was determined to be 35 minutes at CFU/mL (FIG. 3B). Thus, in the electrochemical biosensor, E.coli O157: H7 and DNA/CDs-Fe₃O₄The optimal reaction time between/GCE was 40 minutes.

4 specific assay

The specificity of an electrochemical biosensor is an essential factor to ensure the accuracy of the biosensor. Therefore, a concentration (C) of 10 was investigated by DPV⁵CFU/mL of different bacteria for DNA/CDs-Fe₃O₄Interference of/GCE, such as: staphylococcus aureus, salmonella, staphylococcus lactis, and listeria. FIG. 4A shows DNA/CDs-Fe₃O₄the/GCE had little effect on other bacteria. However, the DNA/CDs-Fe3O4/GCE responded very strongly to E.coli O157: H7, with a 53.42% decrease in current signal. As shown in fig. 4B, other interfering substances had little effect on the measurement of e.coli O157: H7. The above results show that the DNA/CDs-Fe base₃O₄Electrochemical biosensor measurement of/GCE E E.coli O157: H7 has very excellent specificity.

5 sensitive assay

The invention explores the sensitivity and the detection range of the electrochemical biosensor through DPV. FIG. 5A shows the results when E.coli O157: H7 and DNA/CDs-Fe₃O₄Ipa decreased with increasing concentration of e.coli O157: H7 upon GCE interaction. The main possible reason is that the poor conductivity of e.coli O157: H7 affects the conductivity of the working electrode interface, resulting in a reduction of the electrochemical signal. When the concentration of E.coli O157H 7 is 10-10⁸In the range of CUF/mL, lgC and Ipa show a good linear relationship (FIG. 5B): ipa-1.5422 lgC-14.387. Meanwhile, the detection limit can reach 6.88CFU/mL, which shows that the kit has very good sensitivity.

6. Repeatability and stability experiments

To examine the reproducibility of the electrochemical biosensor, 6 DNAs/CDs-Fe were prepared₃O₄The working electrodes of the/GCE are used for detection 10 respectively⁵CUF/mL E.coli O157: H7. As shown in fig. 6A, the Relative Standard Deviation (RSD) of the test results was 2.6%, indicating that the electrochemical biosensor has good reproducibility. Meanwhile, the stability of the electrochemical biosensor is researched, and DNA/CDs-Fe is used₃O₄the/GCE was placed in a 4 ℃ dryer with a DPV detection of 10 concentration every 3 days⁵CUF/mL E.coli O157: H7. As can be seen from FIG. 6B, Ipa decreased only by 2.7% after 30d, indicating that the electrochemical biosensor based on DNA/CDs-Fe3O4/GCE has good stability.

7. Determination of E.coli O157H 7 in milk

Since milk is often contaminated with e.coli O157: H7, it is essential to measure e.coli O157: H7 in milk efficiently. Meanwhile, in order to verify the practical applicability of the electrochemical biosensor, the electrochemical biosensor was used to measure e.coli O157: H7 in milk. As shown in Table 1, various concentrations of Escherichia coli (10) were added to the samples³、10⁴、10⁵CUF/mL) with a recovery of between 95-102%, indicating that the method is feasible for the e.coli O157: H7 assay in big milk.

TABLE 1 determination of E.coli O157: H7 in milk by standard recovery method