CN114624305A - Renewable electrochemical sensor and construction method and application thereof - Google Patents
Renewable electrochemical sensor and construction method and application thereof Download PDFInfo
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Abstract
The invention provides a renewable electrochemical sensor and a construction method and application thereof, belonging to the technical field of biosensors; according to the invention, the renewable electrochemical sensor is constructed by taking the nanoporous gold as a signal conversion element, the adhesive protein modified by renewable segment nucleic acid short chain (L-DNA) as an intermediate connection element and the escherichia coli aptamer modified by renewable complementary segment nucleic acid short chain (C-DNA) as a biological recognition element, and can realize detection of different detection objects by replacing different types of biological recognition elements without preparing electrodes again, so that the renewable electrochemical sensor has good universality; the electrochemical sensor can be repeatedly used for many times, and a large amount of time is saved for simultaneously detecting pathogenic bacteria with different concentrations.
Description
Technical Field
The invention belongs to the technical field of biosensors, and particularly relates to a renewable electrochemical sensor and a construction method and application thereof.
Background
The electrochemical biosensor has the advantages of high sensitivity, good specificity, simple and convenient operation and the like, is widely concerned and researched in the fields of pathogenic bacteria, heavy metals, pesticide and veterinary drug residues and the like, and has great application potential in the fields of food safety detection, medicine, biology and the like. The core part of the electrochemical biosensor is generally composed of a biological recognition element, such as an aptamer, an antibody, etc., which is responsible for specific recognition and binding of a detection object, and a signal conversion element which amplifies and converts such a binding signal, thereby achieving specific and high-sensitivity detection of the detection object. In the construction of the biosensor, how to organically combine the biological recognition element with the signal conversion element not only maintains the recognition activity of the biological recognition material, but also ensures the effective electron transfer between the recognition material and the signal conversion element, and has a decisive role in the sensitivity and stability of the biosensor.
At present, the common strategy is to modify specific chemical groups on the biological recognition material to be combined with the signal conversion interface by means of adsorption or covalent combination. For example, when using an aptamer as a recognition element, a thiol group is usually modified at the end of the aptamer, and the aptamer is immobilized by forming a gold-sulfur covalent bond between the thiol group and a gold electrode. However, there are two disadvantages to this type of approach: the biological identification material is intensively distributed on the surface of the signal conversion element to cause a larger steric hindrance effect, so that the combination efficiency of the identification material is influenced; secondly, the combination of the biological recognition element and the detection object is usually not reversible, so that the recognition element and the signal conversion element must be prepared again for each detection, and the repeatability and reproducibility of the detection are greatly influenced. Therefore, the existing electrochemical sensor is difficult to repeatedly update and utilize, so that the repeatability and reproducibility of the electrochemical sensor are difficult to guarantee, the use cost is high, the operation is complicated, and the like.
Disclosure of Invention
Aiming at the defects in the prior art, the invention provides a renewable electrochemical sensor and a construction method and application thereof. According to the invention, the renewable electrochemical sensor is constructed by taking the nanoporous gold as a signal conversion element, the adhesive protein modified by renewable segment nucleic acid short chain (L-DNA) as an intermediate connection element and the escherichia coli aptamer modified by renewable complementary segment nucleic acid short chain (C-DNA) as a biological recognition element, and can realize detection of different detection objects by replacing different types of biological recognition elements without preparing electrodes again, so that the renewable electrochemical sensor has good universality; the electrochemical sensor can be repeatedly used for many times, and a large amount of time is saved for simultaneously detecting pathogenic bacteria with different concentrations.
According to the invention, firstly, a renewable electrochemical sensor is provided, which comprises a biological recognition element, an intermediate connection element and a signal conversion element, wherein the biological recognition element is an escherichia coli aptamer modified by a renewable complementary segment nucleic acid short chain (C-DNA), the intermediate connection element is an adhesion protein modified by a renewable segment nucleic acid short chain (L-DNA), and the signal conversion element is nano-porous gold.
The invention also provides a construction method of the renewable electrochemical sensor, which comprises the following steps:
(1) pretreating a glassy carbon electrode:
polishing the surface of the glassy carbon electrode by using a prepared alumina solution, then washing by using absolute ethyl alcohol, carrying out ultrasonic treatment in ultrapure water to remove impurities after washing, and finally carrying out ultrasonic treatment in H2SO4And (3) scanning the solution by cyclic voltammetry until a stable gold oxidation reduction peak is obtained, and then washing and naturally airing in a closed container.
Further, H2SO4The concentration of the solution was 0.5M.
(2) Preparation of free thiolated L-DNA:
thiolated short chain L-DNA, tris (2-carboxyethyl) phosphine (TCEP) and acetic acid were incubated in ultrapure water, and the mixture was then passed through MicroSpinTMG-25Columns to obtain free thiolated L-DNA, and placing in PBSE buffer solution for later use.
Further, the mass ratio of the thiolated short-chain L-DNA to the TCEP to the acetic acid is 1:1: 5; the initial concentration of TCEP was 10mM and the initial concentration of acetic acid was 500 mM.
Further, the nucleic acid sequence of the thiolated short chain L-DNA is: 5' - (SH-C)8)-TCCAGGC-3'。
Further, the incubation time is 60-90 min, and the purpose is to reduce the cross-linking of disulfide bonds.
(3) Electrode surface modification:
fixing the pretreated glassy carbon electrode, the cleaned platinum wire electrode and the reference electrode, then soaking the glassy carbon electrode, the cleaned platinum wire electrode and the reference electrode in a chloroauric acid solution, plating gold by adopting a linear scanning voltammetry method, and then putting the glassy carbon electrode, the cleaned platinum wire electrode and the reference electrode into a sulfuric acid solution for activation to obtain a nano porous gold modified glassy carbon electrode;
dropping the adhesive protein solution on the surface of the nanoporous gold modified glassy carbon electrode, naturally airing, then dropping a PBS solution containing 4- (N-maleimide methyl) cyclohexane-1-carboxylic acid sulfonic group succinimide ester sodium salt (sSMCC), then performing first incubation at room temperature in a dark place, dropping a PBSE buffer solution containing free thiolated L-DNA after incubation is finished, performing second incubation at room temperature in a dark place, finally dropping an escherichia coli aptamer with a terminal modified C-DNA, performing third incubation, and washing after incubation is finished to obtain the renewable electrochemical sensor.
Further, the volume ratio of the adhesion protein solution, the PBS solution containing sSMCC, the PBSE buffer solution containing free thiolated L-DNA and the aptamer of the end-modified C-DNA is 5:10:10: 4; wherein the concentration of the adhesion protein solution is 1mg/mL, the concentration of the PBS solution containing sSMCC is 0.05mol/L, the concentration of the PBSE buffer solution containing free thiolated L-DNA is 10 μ M, and the aptamer concentration of the end-modified C-DNA is 10 μ M.
Further, the nucleic acid sequence of the aptamer of the end-modified C-DNA is as follows: 5'-CCGGACGCTTATGCCTTGCCATCTACAGAGCAGGTGTGACGGGCCTGGAG-3' are provided.
Further, the time of the first incubation is 1-1.5 h; the time of the second incubation is 1.5-2 h; the third incubation condition is incubation for 2-4h at 4 ℃.
The invention also provides application of the renewable electrochemical sensor in detection of the concentration of food-borne pathogenic bacteria.
Further, the food-borne pathogenic bacteria are escherichia coli.
Compared with the prior art, the invention has the beneficial effects that:
in the invention, a renewable electrochemical sensor is prepared, wherein the renewable electrochemical sensor takes nano-porous gold as a signal conversion element, takes adhesive protein modified by renewable segment nucleic acid short chain (L-DNA) as an intermediate connection element, and takes Escherichia coli aptamer modified by renewable complementary segment nucleic acid short chain (C-DNA) as a biological recognition element. Wherein, the Escherichia coli aptamer modified by the renewable complementary segment nucleic acid short chain (C-DNA) is responsible for specific recognition and binding of a detection object, the nano-porous gold amplifies and converts the binding signal, and the splicing and fixing of the adhesion protein recognition element modified by the renewable segment nucleic acid short chain (L-DNA) and the signal conversion element are realized.
In the invention, the electrochemical biosensor is arranged in a solution to be detected to capture a target detection object after the signal conversion element, the intermediate connection element and the biological recognition element are assembled layer by layer, and the electrochemical signal of the sensor is changed due to the difference between the electrochemical characteristic of the target object and the electrochemical characteristic of the sensor, thereby realizing the qualitative or quantitative detection of the target detection object. After the detection is finished, the semi-permanent connection between the intermediate connection element and the biological recognition element is broken and separated through separation conditions such as high temperature, illumination and the like; after separation, the separation condition is removed, and the biological recognition element is added again and combined with the intermediate connecting element, so that the sensor is updated and utilized.
In the invention, the intermediate connecting element of the electrochemical biosensor comprises an adhesion section, a transition section and a renewable section, wherein the adhesion section can form stable permanent connection with the signal conversion element by means of covalent crosslinking, electrostatic adsorption and the like, the renewable section can form semi-permanent connection with the biological recognition element by means of hydrogen bonds and the like, and the transition section is an essential transition part when the adhesion section is converted into the renewable section. The semi-permanent connection may be broken under separation conditions of high temperature, light, etc. to allow the biometric element to be detached from the sensor surface. The biological recognition element comprises a renewable complementary segment and a target recognition segment, wherein the renewable complementary segment forms a semi-permanent connection through specific complementary coordination with the renewable segment of the intermediate connection element, and the target recognition segment is used for specifically recognizing and capturing target detection objects, such as bacteria, heavy metals, proteins and the like. The electrochemical biosensor is arranged in a solution to be detected to capture a target detection object after the assembly of the electrochemical biosensor layer by layer according to the signal conversion element, the intermediate connection element and the biological recognition element, and the electrochemical signal of the sensor is changed due to the difference between the electrochemical characteristic of the target object and the electrochemical characteristic of the sensor, so that the qualitative or quantitative detection of the target detection object is realized.
According to the electrochemical sensor, the sensor can be updated and reused, the electrode is prevented from being modified again, the repeatability and the reproducibility of the sensor are obviously improved, and the use cost of the sensor is reduced. The electrochemical sensor can realize the detection of different detection objects by replacing different types of biological recognition elements under the condition of not preparing electrodes again, and has good universality. The electrochemical sensor can be repeatedly used for many times, and a large amount of time is saved for simultaneously detecting pathogenic bacteria with different concentrations.
Drawings
FIG. 1 shows the structure of an L-DNA modified adhesion protein.
Fig. 2 is a flow chart of a method of constructing a renewable electrochemical sensor.
FIG. 3 is a diagram representing the construction process of an electrochemical biosensor based on an AC impedance method, wherein A is a bare GCE; b is Au/GCE; c is pro/Au/GCE; d is L-DNA/pro/Au/GCE; e is apt/L-DNA/pro/Au/GCE; coli/apt/L-DNA/pro/Au/GCE.
FIG. 4 is a representation of the process of modifying an adhesion protein based on surface enhanced Raman spectroscopy, wherein A is Au/GE; b is pro/Au/GE.
FIG. 5 shows electron transfer impedances of apt/L-DNA/pro/Au/GCE modified glassy carbon electrodes before (A) and after (B) melting of a regenerable sensor.
FIG. 6 is a graph of electrochemical response signal versus E.coli concentration, where A is the electrochemistry of the electrochemical biosensor for different concentrations of E.coliResponse (curves a-g, E.coli concentration 1.5 x 101，7.5*101，1.5*102，7.5*102，1.5*103，1.5*104，1.5*105) And B is a calibration curve of the relation between the electrochemical signal and the concentration of the escherichia coli.
FIG. 7 is a specificity diagram of the electrochemical biosensor, wherein A is Escherichia coli, B is Staphylococcus aureus, C is Salmonella, and D is blank PBS solution.
Detailed Description
The invention will be further described with reference to the following figures and specific examples, but the scope of the invention is not limited thereto.
The renewable electrochemical sensor provided by the embodiment of the invention takes nano-porous gold as a signal conversion element, takes the adhesion protein modified by a renewable segment nucleic acid short chain (L-DNA) as an intermediate connection element, and takes the escherichia coli aptamer modified by a renewable complementary segment nucleic acid short chain (C-DNA) as a biological recognition element.
The structure of the L-DNA modified adhesion protein is shown in figure 1. Catechol ligand 3, 4-dihydroxyphenylalanine (Dopa) is chelated with the nano-porous gold as an adhesion segment to be fixed on the surface of the glassy carbon electrode, free amino at the tail end of the zwitterionic peptide KE of the transition segment reacts with 4- (N-maleimidomethyl) cyclohexane-1-carboxylic acid sulfo-succinimidyl ester sodium salt (sSMCC) to provide maleimide groups, and L-DNA with the tail end modified with sulfydryl and the maleimide groups of the transition segment are subjected to cross-linking reaction to form a renewable segment.
The biometric element is composed of two parts. One part is a renewable complementary segment nucleic acid short chain C-DNA which is complementary with a renewable segment nucleic acid short chain L-DNA; the other part is a target recognition segment and is composed of an escherichia coli aptamer.
The detection principle is as follows:
as shown in fig. 2, the surface of the nanoporous gold modified glassy carbon electrode is connected with adhesion protein, and one end of the adhesion protein is complemented with the C-DNA modified escherichia coli aptamer to form a double chain; the aptamer is capable of specifically binding to escherichia coli when the sensor is contacted with escherichia coli; placing the sensor which is finished with detection in a water bath kettle at 58 ℃, melting L-DNA and C-DNA, and connecting the rest L-DNA/pro/Au/GCE modified electrode with an escherichia coli aptamer for detecting escherichia coli with different concentrations; the concentration of the escherichia coli solution is reflected by modifying various materials on the electrode and detecting the intensity of an electric signal generated on the glassy carbon electrode in the potassium ferricyanide solution.
In the present invention, the thiolated short chain L-DNA and the aptamer having a C-DNA modified at one end used are synthesized by Biotechnology engineering (Shanghai) Ltd; 4- (N-Maleimidomethyl) cyclohexane-1-carboxylic acid sulfosuccinimide ester sodium salt (sSMCC) was purchased from Wako glass Ltd, Zhenjiang, east China; tris (2-carboxyethyl) phosphine (TCEP) was purchased from national chemicals ltd; glassy carbon electrodes were purchased from shanghai chenhua instruments ltd; escherichia coli (No. CICC 10907) was purchased from China center for Industrial culture Collection of microorganisms.
Example 1: preparation of renewable electrochemical sensors
(1) Polishing a glassy carbon electrode by using alumina powder with three different particle sizes of 0.05 microns, 0.3 microns and 1 micron, respectively ultrasonically cleaning the glassy carbon electrode by using ultrapure water, 95% ethanol solution and ultrapure water for 5 minutes in sequence, and then performing cyclic voltammetry electrochemical scanning in 0.5M sulfuric acid solution (CHI660E electrochemical workstation, Shanghai Chenghua instruments, Ltd.), wherein the detection parameters are as follows: init E (V) ═ 0, High E (V) ═ 0.5, Low E (V) ═ 0, Scan Rate (V/S) ═ 0.1, Sweep Segments ═ 8, and Sensitivity (a/V) ═ 1E-5; until a stable peak shape appears, obtaining a pretreated glassy carbon electrode, as shown in fig. 3 (a);
(2) placing the pretreated glassy carbon electrode, a reference electrode and a counter electrode in a 1mg/mL chloroauric acid solution together for linear sweep voltammetry electrochemical scanning (CHI660E electrochemical workstation, Shanghai Chenghua instruments, Ltd.), wherein detection parameters are Init E (V) ═ 0.5 and Final E (V) ═ 0; scan Rate (V/S) is 0.1, Quiet time (S) is 200, Sensitivity (a/V) is 1 e-5; obtaining the nano porous gold modified glassy carbon electrode as shown in figure 3 (B);
(3) after washing and drying the glassy carbon electrode modified with the nano-porous gold, dropwise adding 5 mu L of 1mg/mL adhesive protein solution to the surface of the glassy carbon electrode, and naturally airing under a closed condition at room temperature to obtain a pro/Au/GCE modified electrode shown in figure 3 (C);
(4) washing the glassy carbon electrode modified with the adhesive protein in the step (3) by using ultrapure water, dropwise adding 10 mu L of 0.05M sSMCC solution to the surface of the glassy carbon electrode, and incubating for 1h at room temperature in a dark condition;
(5) mu.L of 10. mu.M thiolated short-chain L-DNA, 4. mu.L of 10mM tris (2-carboxyethyl) phosphine (TCEP) and 20. mu.L of 500mM acetic acid were each incubated in ultrapure water for 1h, and the mixture was passed through MicroSpinTM G-25Columns to obtain free thiolated L-DNA and placed in PBSE buffer solution for further use. Wherein the nucleic acid sequence of the thiolated short-chain L-DNA is: 5'- (SH-C8) -TCCAGGC-3';
(6) washing the glassy carbon electrode modified by the sSMCC in the step (4) by using ultrapure water, dripping 10 mu L of free thiolated L-DNA (deoxyribonucleic acid) with the concentration of 10 mu M on the surface of the glassy carbon electrode, and incubating for 1.5h at room temperature in a dark condition; obtaining the L-DNA/pro/Au/GCE modified electrode shown in figure 3 (D);
(7) and (4) washing the glassy carbon electrode modified by the L-DNA in the step (6) with ultrapure water, dropwise adding 4 mu L of Escherichia coli aptamer with one end modified by C-DNA to the surface of the glassy carbon electrode, and incubating for 2-4h at 4 ℃ to obtain the apt/L-DNA/pro/Au/GCE modified reproducible sensor, as shown in FIG. 3 (E). Wherein, the nucleic acid sequence of the aptamer modified by the C-DNA is as follows: 5'-CCGGACGCTTATGCCTTGCCATCTACAGAGCAGGTGTGACGGGCCTGGAG-3' is added.
In order to verify the preparation and detection conditions of the sensor, a 5mM potassium ferricyanide solution was prepared, and the resistance value of the glassy carbon electrode during the preparation process was detected in the solution, and the electrochemical impedance spectrum thereof is shown in FIG. 3. The resistance value of the bare glassy carbon electrode (A) is very small, and after the nano-porous gold is modified, the resistance (B) presents a straight line, because electrons on the glassy carbon electrode modified by the nano-porous gold can be freely transferred, and the resistance value is low. After the adhesion protein is connected to the nano-porous gold modified glassy carbon electrode, electrons are blocked at the electrode and Fe (CN)6 3-/4-The transfer in solution results in an increase in the resistance of the electrode (C). When L-DNA is bound to the adhesion protein, the resistance increases due to the increase of negative charges on the modified electrode, which hinders the transfer of electrons (D). Hybridization of E.coli aptamers modified at one end with C-DNA with L-DNAAfter the double-stranded DNA structure is formed, negative charges on the surface of the modified electrode are increased again, and the resistance of the electrode is further increased (E). When the regenerable electrochemical sensor is contacted with E.coli, the aptamer binds to a specific site on the E.coli cell membrane, causing the electrode resistance to increase again (F). Thus, the method can prepare the aptamer for detecting the Escherichia coli.
The Raman spectrum characterization chart of the protein modification process is shown in FIG. 4. The Raman spectrum curve of the gold electrode (A) modified by the nano-porous gold presents a straight line without a Raman peak. When the adhesion protein is combined with the nano-porous gold, the Raman spectra are respectively 814cm-1、888cm-1And 1188cm-1Has a Raman peak (B). This demonstrates that adhesion proteins are successfully modified on the electrode.
Example 2: detection of renewable electrochemical sensors in E.coli
(1) Drawing a standard curve:
respectively incubating the sensors prepared by the method of example 1 with PBS solutions containing escherichia coli with different concentrations, and incubating for 30-60min at 37 ℃; wherein the PBS solution has a concentration of 0.01M and a pH of 7.0; the concentration of E.coli in the PBS solution was 1.5 x 10, respectively1CFU/mL、7.5*101CFU/mL、1.5*102CFU/mL、7.5*102CFU/mL、1.5*103CFU/mL、1.5*104CFU/mL and 1.5 x 105CFU/mL. FIG. 5 is a graph of electrochemical response signal versus E.coli concentration, where A is the electrochemical response of the electrochemical biosensor to different concentrations of E.coli (curves a-g, E.coli concentration 1.5 x 10)1，7.5*101，1.5*102，7.5*102，1.5*103，1.5*104，1.5*105) And B is a calibration curve of the relation between the electrochemical signal and the concentration of the escherichia coli.
The incubated sensor was washed with PBS solution and measured by ac impedance using an electrochemical workstation and the resistance was recorded as shown in figure 6. As can be observed from FIG. 6(A), the electron transfer resistance value increases with the increase in the concentration of Escherichia coli. With reference to FIG. 6As can be seen from FIG. 6B, the impedance value (Rct) corresponding to each concentration of Escherichia coli is the ordinate, the logarithm value of the concentration of Escherichia coli is the abscissa, and a standard curve is established, and a good linear relationship (R) exists between the electron transfer impedance value of EIS response and the concentration of Escherichia coli20.9954). The regression equation is that y is 310.4231x +287.7254, and the detection limit is 3.719 CFU/mL. (y represents the detected EIS signal and x represents the log of the bacterial concentration) this demonstrates that the present invention can be used to detect the concentration of E.coli in unknown samples to be tested.
(2) Detection of renewable electrochemical sensors in e.coli:
the concentration of 10. mu.L was adjusted to 104And (3) dripping cfu/mL of escherichia coli solution on the surface of the renewable electrochemical sensor obtained in the step (7), and incubating for 30-60min at 37 ℃. Measuring with electrochemical workstation by AC impedance method to obtain concentration of 9.6 x 103cfu/mL. Thus, the renewable electrochemical sensor has higher accuracy.
Example 3: regeneration of renewable electrochemical sensors
And placing the renewable electrochemical sensor after detection in a water bath kettle, and controlling the water bath temperature to be 58 ℃ to release hydrogen bonds between the L-DNA and the C-DNA, so that the conjugate of the aptamer and the bacteria falls off from the electrode, and the L-DNA/pro/Au/GCE modified electrode can be obtained. Dripping 4 μ L of 10 μ M of Escherichia coli aptamer on the surface of the electrode, incubating in a refrigerator at 4 deg.C to obtain a new electrochemical sensor for detecting specific bacteria, and washing the electrode surface with tris-HCL buffer solution for 3-5 times to remove unbound aptamer. The impedance values of apt/L-DNA/pro/Au/GCE modified glassy carbon electrodes before and after the melting of the reproducible sensor are measured by using an alternating-current impedance method through an electrochemical workstation, and the result is shown in FIG. 6.
FIG. 6 shows electron transfer impedances of apt/L-DNA/pro/Au/GCE modified glassy carbon electrodes before (A) and after (B) melting of a regenerable sensor. As can be seen from the figure, the electron transfer impedance values of the apt/L-DNA/pro/Au/GCE modified glassy carbon electrodes obtained by the same modification of the six electrodes before melting are basically the same, and the relative standard deviation value is 2.6%, and when the aptamer on the electrodes is melted with the L-DNA and then the aptamer is modified again, the electron transfer impedance values of the six apt/L-DNA/pro/Au/GCE modified glassy carbon electrodes are slightly changed, but the relative standard deviation value is 3.4%. It can thus be shown that the reproducibility of the electrochemical sensor is possible.
Example 4: renewable electrochemical sensor specificity
The sensor prepared by the method of example 1 was incubated with staphylococcus aureus and salmonella of the same concentration for 30-60min in a 0.01M PBS solution, the electrode was washed with the 0.01M PBS solution after incubation, and the ac impedance measurement was performed by using an electrochemical workstation, and the impedance value was recorded, with the results shown in fig. 7.
FIG. 7 is a specificity diagram of the electrochemical biosensor, wherein A is Escherichia coli, B is Staphylococcus aureus, C is Salmonella, and D is blank PBS solution. As can be seen from the figure, the difference of the electron transfer impedance of the staphylococcus aureus and the salmonella with the PBS solution is far smaller than that of the escherichia coli, which indicates that the electrochemical biosensor has good specificity.
The examples are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any obvious modifications, substitutions or variations can be made by those skilled in the art without departing from the spirit of the present invention.
Claims (10)
1. A renewable electrochemical sensor comprising three parts, a biological recognition element, an intermediate linking element, and a signal transduction element; the biological recognition element is a renewable complementary segment nucleic acid short-chain C-DNA modified escherichia coli aptamer, and the intermediate connecting element is a renewable segment nucleic acid short-chain L-DNA modified adhesion protein.
2. The renewable electrochemical sensor of claim 1 wherein the signal-converting element is nanoporous gold, the sequence of the C-DNA is 5'-CCGGACGCTTATGCCTTGCCATCTACAGAGCAGGTGTGACGGGCCTGGAG-3'; the sequence of the L-DNA is 5' - (SH-C)8)-TCCAGGC-3'。
3. The method of constructing a renewable electrochemical sensor of claim 1, comprising:
(1) preparation of free thiolated L-DNA:
thiolated short-chain L-DNA, tris (2-carboxyethyl) phosphine TCEP and acetic acid were incubated in ultrapure water, and the mixture was then passed through MicroSpinTMG-25Columns to obtain free thiolated L-DNA, and placing the free thiolated L-DNA in PBSE buffer solution for later use;
(2) electrode surface modification:
fixing the pretreated glassy carbon electrode, the cleaned platinum wire electrode and the reference electrode, then soaking the glassy carbon electrode, the cleaned platinum wire electrode and the reference electrode in a chloroauric acid solution, plating gold by adopting a linear scanning voltammetry method, and then putting the glassy carbon electrode, the cleaned platinum wire electrode and the reference electrode into a sulfuric acid solution for activation to obtain a nano porous gold modified glassy carbon electrode;
dropping the adhesive protein solution on the surface of the nanoporous gold modified glassy carbon electrode, naturally airing, then dropping a PBS solution containing 4- (N-maleimide methyl) cyclohexane-1-carboxylic acid sulfonic group succinimide ester sodium salt sSMCC, then performing first incubation at room temperature in a dark place, dropping a PBSE buffer solution containing free thiolated L-DNA after incubation is finished, performing second incubation at room temperature in a dark place, finally dropping an escherichia coli aptamer with end modified C-DNA, performing third incubation, and washing after incubation is finished to obtain the renewable electrochemical sensor.
4. The method for constructing a renewable electrochemical sensor as claimed in claim 3, wherein in the step (1), the mass ratio of thiolated short chain L-DNA, TCEP and acetic acid is 1:1: 5; the initial concentration of TCEP was 10mM and the initial concentration of acetic acid was 500 mM.
5. The method for constructing a renewable electrochemical sensor as claimed in claim 3, wherein in the step (1), the nucleic acid sequence of the thiolated short-chain L-DNA is: 5' - (SH-C)8) -TCCAGGC-3'; the incubation time is 60-90 min.
6. The method for constructing a renewable electrochemical sensor as claimed in claim 3, wherein in the step (2), the volume ratio of the adhesion protein solution, the PBS solution containing sSMCC, the PBSE buffer solution containing free thiolated L-DNA and the aptamer of the end-modified C-DNA is 5:10:10: 4;
the concentration of the adhesion protein solution is 1mg/mL, the concentration of PBS solution containing sSMCC is 0.05mol/L, the concentration of PBSE buffer solution containing free thiolated L-DNA is 10 muM, and the aptamer concentration of the end-modified C-DNA is 10 muM.
7. The method for constructing a renewable electrochemical sensor as defined in claim 3, wherein in the step (2), the nucleic acid sequence of the C-DNA is: 5'-CCGGACGCTTATGCCTTGCCATCTACAGAGCAGGTGTGACGGGCCTGGAG-3' are provided.
8. The method for constructing a renewable electrochemical sensor as claimed in claim 3, wherein in the step (2), the time of the first incubation is 1-1.5 h; the time of the second incubation is 1.5-2 h; the third incubation condition is incubation for 2-4h at 4 ℃.
9. Use of the renewable electrochemical sensor of claim 1 for the detection of a concentration of food-borne pathogenic bacteria.
10. The use according to claim 9, wherein the food-borne pathogenic bacterium is escherichia coli.
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