CN112525965A

CN112525965A - Escherichia coli electrochemical detection method based on bacteria-mediated azido alkyne cycloaddition and atom transfer radical polymerization

Info

Publication number: CN112525965A
Application number: CN202011106591.2A
Authority: CN
Inventors: 许媛媛; 苗晋锋; 黎伟中; 李意; 鲍芳
Original assignee: Nanjing Agricultural University
Current assignee: Nanjing Agricultural University
Priority date: 2020-10-15
Filing date: 2020-10-15
Publication date: 2021-03-19
Anticipated expiration: 2040-10-15
Also published as: CN112525965B

Abstract

An Escherichia coli electrochemical detection method based on bacteria-mediated azido alkyne cycloaddition and atom transfer radical polymerization belongs to the technical field of life science analysis. The detection principle is as follows: will modify N₃Double-stranded DNA of the group is fixed on the surface of a gold electrode, and Cu is contained in the presence of Escherichia coli²⁺Is reduced to Cu by copper reductase NDH-2 in escherichia coli⁺And catalyze N₃The group and PBIB generate azide alkyne cycloaddition reaction, then a large amount of FMMA is connected and polymerized at the tail end of double-chain DNA through eATRP, finally the current intensity is measured through square wave voltammetry to be in positive correlation with the concentration of escherichia coli, a standard curve between an electrochemical signal and the concentration of the escherichia coli is drawn to obtain a linear equation, the electrochemical signal of a sample to be measured is measured, and the large amount of FMMA is calculated to realize large measurementSensitive detection of enterobacteria. The method provided by the invention has high selectivity on escherichia coli, is simple, rapid and sensitive, has good practical value and is convenient to popularize.

Description

Escherichia coli electrochemical detection method based on bacteria-mediated azido alkyne cycloaddition and atom transfer radical polymerization

Technical Field

An Escherichia coli electrochemical detection method based on bacteria-mediated azido alkyne cycloaddition and atom transfer radical polymerization belongs to the technical field of life science analysis.

Background

Coli is a commensal bacterium commonly found in the gastrointestinal tract of humans and animals, and mainly parasitizes in the large intestine, accounting for about 0.1% of intestinal bacteria. Pathogenic E.coli gastrointestinal infections are a significant cause of diarrhea outbreaks worldwide, particularly in developing countries. In recent years, food-borne diseases caused by escherichia coli have been prevalent, and escherichia coli has become an important test subject for food safety. At present, three methods for detecting escherichia coli in food are mainly used, namely a traditional microbial culture identification method, an immunological detection method and a PCR detection method. The traditional microbial culture is based on bacterial separation and culture, morphological identification and biochemical reaction to identify the bacterial species, is simple to operate and high in accuracy, but has the problems of long culture time, complex operation and the like, so that the application of the traditional microbial culture is severely limited; the immunological detection method has good specificity and sensitivity, but has the problems of long antibody preparation time, difficult antibody storage and the like; the PCR detection method has good specificity and sensitivity, and is widely applied to the detection of escherichia coli, but the method needs expensive instruments and professional operators, and is not suitable for field detection. Therefore, it is important to further research a new method with high sensitivity, easy operation and high selectivity.

Meanwhile, in order to improve detection performance, various signal amplification techniques such as hybrid chain reaction, rolling circle amplification reaction, nanomaterial, polymerization reaction, and the like are applied to the field of biosensing. Among them, Atom Transfer Radical Polymerization (ATRP) has advantages of controllable reaction process, wide reaction monomer range, etc., and has been proposed in the 90 s to be widely used in the fields of materials, synthesis, etc., and its application in biosensing is also favored by researchers.

Disclosure of Invention

Aiming at the defects of the prior art, the invention aims to provide an escherichia coli electrochemical detection method based on bacteria-mediated azido alkyne cycloaddition and atom transfer radical polymerization, which has the characteristics of high specificity, high sensitivity and the like.

In order to achieve the purpose, the technical scheme of the invention is as follows:

an Escherichia coli electrochemical detection method based on bacteria-mediated azido alkyne cycloaddition and atom transfer radical polymerization comprises the following steps:

(1) synthesis of dsDNA

Heating 2. mu.M ssDNAa and 2. mu.M ssDNA B at 95 ℃ for 10min, and then slowly cooling to room temperature;

(2) cultivation of Escherichia coli

Coli were grown overnight in LB medium, harvested in exponential growth phase by centrifugation, the supernatant was discarded, and the bacterial particles were then resuspended in PBS and bacterial concentration was monitored by measuring Optical Density (OD) at 600 nm. Before carrying out bacteria colorimetric detection experiment, OD of strain stock solution is added₆₀₀The value is adjusted to 0.1;

(3) pretreatment of gold electrodes

Polishing the gold electrode with 800-mesh, 1000-mesh, 2000-mesh and 5000-mesh sand paper for 3-5min respectively; then, respectively polishing the bare gold electrodes by using alumina suspension liquid with the particle size of 0.3 mu m and alumina suspension liquid with the particle size of 0.05 mu m, and then sequentially carrying out ultrasonic cleaning by using 99.99 percent ethanol and ultra-clean water; the treated gold electrode was then immersed in a freshly prepared solution of 30% H₂O₂And 98% of H₂SO₄1: 3V: V for 10 min; the electrode was then placed at 0.5M H₂SO₄The medium cyclic voltammetry is carried out at 0.1Vs in a potential range of-0.2-1.5V^-1Performing electrochemical treatment at the sweeping speed until a stable cyclic voltammogram is obtained; finally, washing the surface of the electrode by ultra-clean water, and drying by nitrogen;

(4) modification of gold electrodes

10 μ L of 1 μ M dsDNA formed in (2) above was dropped onto a clean gold electrode and incubated at 37 ℃ for 1.5 h; then washing the gold electrode twice by using TE buffer solution; then soaking the gold electrode in 2mM MCH solution dissolved in 70% alcohol for 0.5 h; then washing the electrode with TE buffer solution;

(5) click reaction

Will be modifiedThe gold electrode was immersed in a solution containing 50. mu.L of 4mM PBIB and 5. mu.L of 2mM CuSO₄And 1mL OD₆₀₀0.1 escherichia coli solution and incubating for 40min at 37 ℃ in the dark;

(6) eATRP reaction

Washing the gold electrode obtained after the step (5) for ten seconds by using a TE solution; followed by 50. mu.L of 10mM FMMA, 50. mu.L of 10mM Cu²⁺/Me₆TREN, 500. mu.L of 1M KBr, 900. mu.L of DMF and 3500. mu.L of 0.1mM KPF₆Applying a constant negative potential of-0.53V on the surface of the solution, and reacting for 40 min; and then scanning with linear voltammetry LSV to remove copper deposited on the electrode surface, which is set as: an initial potential of 0V; final potential 0.2V; scan rate 1.0Vs^-1(ii) a Finally, cleaning the electrode by using DMF and ultrapure water;

(7) electrochemical assay

Placing the gold electrode obtained in the above (6) in 1.0M LiClO₄Detection of de novo synthesized electroactive polymer on the electrode surface in solution and with square wave voltammetry SWV, set to: the initial potential is 0V, the final potential is 0.6V, the potential increment is 4.0mV, the pulse amplitude is 25mV, and the frequency is 15 Hz.

The method is designed and based on the bacteria-mediated CuAAC and eATRP principle to detect the Escherichia coli, avoids the use of nano materials and biological enzymes (which are easily influenced by external environments such as pH and temperature) in the current common signal strategy, multiplies the signals, greatly improves the sensitivity and has higher stability and reproducibility. A simple and sensitive method for detecting escherichia coli is established, and convenience is provided for future supervision.

Drawings

FIG. 1 is a schematic diagram of the construction principle of the detection system.

FIG. 2 is a graph showing the results of detection of E.coli at different concentrations: (A) square wave voltammogram (B) detected by escherichia coli with different concentrations is a linear relation graph of escherichia coli and corresponding current intensity.

Detailed Description

Example 1 sensitivity test

The experimental procedures are the same as those in the invention contents (1) to (7), and the experimental procedures are respectively examinedColi (10) of different concentrations in the target solution³CFU/mL、10⁴CFU/mL、10⁵CFU/mL、10⁶CFU/mL、10⁷CFU/mL、10⁸CFU/mL) generated current intensity to investigate the detection performance of the method of the present invention on E.coli. As shown in FIG. 2, the intensity of current increased with the increase in E.coli concentration because the greater the E.coli concentration, the Cu was present²⁺Is reduced to Cu⁺The greater the number of CuAAC and the more initiator PBIB is introduced, which in turn leads to the more FMMA electrochemical polymer monomers, the stronger and stronger the resulting amperage. The maximum peak intensity of current and the concentration of Escherichia coli are 10³CFU/mL to 10⁸The linear relation is good in the CFU/mL range, and the linear equation is that I ═ 0.3674+0.5941logC (R)²0.9974), the lowest detection limit is 90.89CFU/, L. The result shows that the method has higher detection sensitivity, lower detection limit and excellent performance.

Claims

1. An Escherichia coli electrochemical detection method based on bacteria-mediated azido alkyne cycloaddition and atom transfer radical polymerization belongs to the technical field of life science analysis. The detection principle is as follows: will modify N₃Double-stranded DNA dsDNA of the group is fixed on the surface of a gold electrode, and Cu is generated in the presence of Escherichia coli²⁺Is reduced to Cu by copper reductase NDH-2 in bacteria⁺And catalyze N₃The group and bromo-isobutyric propinyl ester PBIB generate azide cycloaddition reaction, then a large amount of FMMA is connected and polymerized at the tail end of double-chain DNA through eATRP, finally the current intensity is measured through a square wave voltammetry SWV and is in positive correlation with the concentration of escherichia coli, a standard curve between an electrochemical signal and the concentration of the escherichia coli is drawn to obtain a linear equation, and the electrochemical signal of a sample to be measured is measured, so that the sensitive detection of the escherichia coli can be realized through calculation.

2. The method for the electrochemical detection of escherichia coli based on bacteria-mediated azidoyne cycloaddition and atom transfer radical polymerization according to claim 1, wherein the step of dsDNA synthesis is as follows: mu.M ssDNA A and 2. mu.M ssDNA B were heated at 95 ℃ for 10min and then slowly cooled to room temperature.

3. The method for electrochemically detecting escherichia coli based on bacteria-mediated azido alkyne cycloaddition and atom transfer radical polymerization according to claim 1, wherein the escherichia coli is cultured by the following steps: coli were grown overnight in LB medium, harvested in exponential growth phase by centrifugation, the supernatant was discarded, and the bacterial particles were then resuspended in PBS and the bacterial concentration was monitored by measuring the optical density OD at 600 nm. Before carrying out bacteria colorimetric detection experiment, OD of strain stock solution is added₆₀₀The value was adjusted to 0.1.

4. The method for electrochemically detecting escherichia coli based on bacteria-mediated azido alkyne cycloaddition and atom transfer radical polymerization according to claim 1, wherein the gold electrode pretreatment step is as follows: polishing the gold electrode with 800-mesh, 1000-mesh, 2000-mesh and 5000-mesh sand paper for 3-5min respectively; then, respectively polishing the bare gold electrodes by using alumina suspension liquid with the particle size of 0.3 mu m and alumina suspension liquid with the particle size of 0.05 mu m, and then sequentially carrying out ultrasonic cleaning by using 99.99 percent ethanol and ultra-clean water; soaking the treated gold electrode in newly prepared goby solution for 10 min; the electrode was then placed at 0.5M H₂SO₄The medium cyclic voltammetry is carried out at 0.1Vs in a potential range of-0.2-1.5V^-1Performing electrochemical treatment at the sweeping speed until a stable cyclic voltammogram is obtained; finally, the surface of the electrode is washed by ultra-clean water and dried by nitrogen.

5. The method for electrochemically detecting escherichia coli based on bacteria-mediated azido alkyne cycloaddition and atom transfer radical polymerization according to claim 1, wherein the gold electrode modification step is as follows: 10 u L1 u M in claim 2 formed dsDNA is dropped on the clean gold electrode and at 37 degrees C were incubated for 1.5 h; then washing the gold electrode twice by using TE buffer solution; then soaking the gold electrode in 2mM MCH solution dissolved in 70% alcohol for 0.5 h; the electrodes were washed again with TE buffer.

6. The method for electrochemically detecting escherichia coli based on bacteria-mediated azido alkyne cycloaddition and atom transfer radical polymerization according to claim 1, wherein the click reaction is carried out by the following steps: soaking the modified gold electrode in a solution containing 50 μ L of 4mM PBIB and 5 μ L of 2mM CuSO₄And 1mL OD₆₀₀0.1 E.coli and incubated at 37 ℃ for 40min in the dark.

7. The method for electrochemically detecting escherichia coli based on bacteria-mediated azido alkyne cycloaddition and atom transfer radical polymerization as claimed in claim 1, wherein the steps for performing the etatr are as follows: washing the gold electrode obtained after the step of claim 6 with a TE solution for ten seconds; followed by 50. mu.L of 10mM FMMA, 50. mu.L of 10mM Cu²⁺/Me₆TREN, 500. mu.L of 1M KBr, 900. mu.L of DMF and 3500. mu.L of 0.1mM KPF₆Applying a constant negative potential of-0.53V on the surface of the solution, and reacting for 40 min; and then scanning with linear voltammetry LSV to remove copper deposited on the electrode surface, which is set as: an initial potential of 0V; final potential 0.2V; scan rate 1.0Vs^-1(ii) a Finally, the electrode was washed with DMF and ultrapure water.

8. The method for electrochemically detecting escherichia coli based on bacteria-mediated azido alkyne cycloaddition and atom transfer radical polymerization according to claim 1, wherein the electrochemical determination comprises the following steps: placing the gold electrode obtained by carrying out the process of claim 7 in 1.0M LiClO₄Detection of de novo synthesized electroactive polymer on the electrode surface in solution and with square wave voltammetry SWV, set to: the initial potential is 0V, the final potential is 0.6V, the potential increment is 4.0mV, the pulse amplitude is 25mV, and the frequency is 15 Hz.