CN111521664B - Listeria monocytogenes imprinted electrochemical sensor and preparation method thereof - Google Patents
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Abstract
The invention discloses a listeria monocytogenes imprinted electrochemical sensor and a preparation method thereof, and belongs to the technical field of analysis and detection. According to the invention, an ITO electrode is used as a substrate, gold nanoparticles are electro-deposited and modified, Listeria monocytogenes is used as a template, pyrrole is used as a functional monomer, a polypyrrole bacterial membrane is electropolymerized, the template is eluted to obtain an imprinting membrane, and the imprinting membrane is connected with an electrochemical workstation to construct the Listeria monocytogenes imprinting electrochemical sensor. The listeria monocytogenes imprinted electrochemical sensor constructed by the invention can realize the high sensitivity, high selectivity, low cost, simple and quick detection of listeria monocytogenes, is not only suitable for food safety detection, but also can be applied in the fields of environmental monitoring, biological sample analysis and the like.
Description
Technical Field
The invention relates to a Listeria monocytogenes imprinted electrochemical sensor and a preparation method thereof, belonging to the technical field of analysis and detection.
Background
Listeria monocytogenes (abbreviated as Listeria monocytogenes) is a pathogenic bacterium which is possibly infected by both human and animals, and the disease caused by the Listeria monocytogenes is called Listeria disease and is one of the main food-borne diseases. The bacterium causes food poisoning of human by polluting animal food, pregnant women, newborns and adults with low immunity are easy to infect the listeria monocytogenes, and the fatality rate of infecting the bacterium is as high as 30%. In addition, the feed additive has strong reproductive capacity, needs less nutrition and has strong adaptability to the environment, can survive in a refrigerator at 4 ℃, greatly threatens the health of human beings and is listed as one of pathogenic bacteria of four food-borne diseases. Therefore, in order to prevent food contamination and outbreaks of food-borne diseases, it is necessary to detect whether listeria monocytogenes is present in food.
At present, the methods for detecting listeria monocytogenes include traditional detection methods, immunological detection methods, molecular biological detection methods, and the like. However, each of the three methods has advantages and disadvantages: the traditional detection method is a gold standard, but the detection time is long; the immunological detection specificity is good, but the sensitivity is poor, and the antibody preparation cost is high; the molecular biological detection technology has high sensitivity, but the pretreatment is more complex, the technology required in the operation process is higher, and when the detection of a large amount of food samples is met, the batch detection cannot be carried out in a short time. Therefore, a detection method with rapider, more convenient, more accurate, safer and lower cost needs to be explored to ensure that the edible products of people do not contain listeria monocytogenes or to effectively control the spread of pathogenic bacteria in time.
In recent years, various microbial detection technologies are changing day by day, and molecular imprinting technology is emerging from a plurality of emerging technologies and becomes one of the key research objects in the food detection field. Molecular imprinting, also known as template imprinting, refers to the experimental preparation of a molecule (template molecule) that is perfectly matched to form a polymer in its spatial structure and binding site. The technology is a technology simulating a natural antibody and antigen recognition mechanism, and utilizes the characteristic that a polymer can be selectively and specifically recognized, namely, a molecularly imprinted polymer has similar affinity and selectivity with a monoclonal antibody or an enzyme, so that various target molecules can be recognized. The development of molecular imprinting technology is rapid, the application range is wider and wider, the molecular imprinting technology is not limited to the detection of small molecules, and the molecular imprinting technology is oriented to more macromolecular substances such as proteins, bacteria, cells and the like. The bacterial imprinting technology is one type of molecular imprinting technology, and holes in bacterial imprinting polymers can realize specific binding with target bacteria through shape, size matching and chemical bond action.
Disclosure of Invention
In order to solve at least one of the above problems, the present invention provides an electrochemical sensor for listeria monocytogenes blotting, which can be used for detecting listeria monocytogenes, and has the advantages of high sensitivity, strong specificity, low detection cost, and simple operation.
The first purpose of the invention is to provide a method for preparing a listeria monocytogenes imprinted working electrode, which is based on a surface molecular imprinting technology, and the method comprises the steps of taking listeria monocytogenes as a template and pyrrole as a functional monomer, placing an ITO electrode modified with gold nanoparticles in an electropolymerization liquid containing the template, the functional monomer and an electrolyte solution for electrochemical polymerization; and eluting the template to obtain the listeria monocytogenes imprinted working electrode.
In one embodiment of the present invention, the Listeria monocytogenes in the electropolymerization fluid is Listeria monocytogenes ATCC19115, and the concentration is 1.0 × 105~1.0×109CFU/mL。
In one embodiment of the present invention, the concentration of the functional monomer pyrrole in the electropolymerization liquid is 0.05 to 0.4M.
In one embodiment of the present invention, the electrolyte solution in the electropolymerization solution is an aqueous solution of potassium chloride, and the concentration of potassium chloride in the electropolymerization solution is 0.1 to 0.4M.
In one embodiment of the invention, the electrochemical polymerization is cyclic voltammetry, the initial potential is 0V, the final potential is 0.7V, the scanning speed is 100mV/s, and the polymerization cycle number is 1-10 cycles.
In one embodiment of the present invention, the Listeria monocytogenes in the electropolymerization fluid is Listeria monocytogenes ATCC19115, and the concentration is 1.0 × 108CFU/mL。
In one embodiment of the present invention, the concentration of the functional monomer pyrrole in the electropolymerization liquid is 0.2M.
In one embodiment of the present invention, the electrolyte solution in the electropolymerization solution is an aqueous solution of potassium chloride, and the concentration of potassium chloride in the electropolymerization solution is 0.2M.
In one embodiment of the invention, the electrochemical polymerization is cyclic voltammetry, the initial potential is 0V, the final potential is 0.7V, the scanning speed is 100mV/s, and the polymerization cycle number is 8 cycles.
In one embodiment of the present invention, the method comprises the steps of:
(1) placing an ITO electrode in a chloroauric acid solution, and electrodepositing modified gold nanoparticles on the surface of the ITO electrode;
(2) after the electrodeposition is finished, washing the electrode, drying the electrode at room temperature, and placing the ITO electrode modified with the nanogold on an electropolymerization liquid of template Listeria monocytogenes, functional monomer pyrrole and electrolyte potassium chloride solution for electrochemical polymerization;
(3) and after the electrochemical polymerization is finished, washing the electrode, drying the electrode at room temperature, and sequentially immersing the electrode in lysozyme and sodium dodecyl sulfate solution to elute the template to obtain the listeria monocytogenes imprinted working electrode.
In one embodiment of the present invention, the chloroauric acid solution in step (1) is a 0.01M sulfuric acid solution containing 5mM chloroauric acid, and is stored away from light.
In one embodiment of the invention, the electrodeposition in the step (1) is performed by cyclic voltammetry, wherein the initial potential is-1V, the final potential is 0.5V, the scanning rate is 100mV/s, and the number of scanning cycles is 5-15.
In one embodiment of the present invention, the electrodeposition in step (1) is performed by cyclic voltammetry, wherein the initial potential is-1V, the final potential is 0.5V, the scanning rate is 100mV/s, and the number of scanning cycles is 7.
In one embodiment of the present invention, the washing electrode in step (2) is washed with deionized water.
In one embodiment of the present invention, the listeria monocytogenes in the electropolymerization solution in step (2) is listeria monocytogenes ATCC19115, and the concentration is 1.0 × 108CFU/mL。
In one embodiment of the present invention, the concentration of the pyrrole monomer in the electropolymerization liquid in the step (2) is 0.2M.
In one embodiment of the present invention, the concentration of the electrolyte potassium chloride in the electropolymerization liquid in the step (2) is 0.2M.
In an embodiment of the present invention, in the electropolymerization liquid in step (2), listeria monocytogenes (template) and pyrrole (functional monomer) are added into a potassium chloride aqueous solution, and after being uniformly mixed, the mixture is subjected to ultrasonic treatment for 5 s, nitrogen gas is filled for 10min, and the mixture is sealed and kept stand for 10min, such that an electropolymerization liquid is obtained.
In one embodiment of the present invention, the electrochemical polymerization in step (2) is cyclic voltammetry, the initial potential is 0V, the final potential is 0.7V, the scanning speed is 100mV/s, and the number of polymerization cycles is 8.
In one embodiment of the invention, the lysozyme activity in the step (3) is 4000-20000U, and the enzymolysis condition is 25 ℃ for 2 h.
In one embodiment of the present invention, the sodium dodecyl sulfate solution in step (3) is a sodium dodecyl sulfate acetic acid solution with a mass fraction of 5%, and the reaction time is 4 h.
In one embodiment of the present invention, the listeria monocytogenes is cultured by:
culturing the identified Listeria monocytogenes overnight, centrifuging at 6000rpm for 10min, repeatedly blowing and beating the centrifuged precipitate with a proper amount of sterile water to resuspend the precipitate, and repeating for three times to obtain a pure target template Listeria monocytogenes for later use.
In an embodiment of the present invention, the method for preparing a listeria monocytogenes imprinted working electrode specifically comprises: placing an ITO electrode in a 5mM chloroauric acid solution, and completing electrodeposition of gold nanoparticles by adopting cyclic voltammetry, wherein the initial potential is-1V, the final potential is 0.5V, the scanning rate is 100mV/s, and the number of scanning cycles is 5-15 cycles; washing the electrode with deionized water and then drying at room temperature; preparing a template listeria monocytogenes and a functional monomer pyrrole, dissolving the template listeria monocytogenes and the functional monomer pyrrole in an electrolyte potassium chloride aqueous solution to obtain an electropolymerization solution, placing an ITO electrode modified by gold nanoparticles in the electropolymerization solution, and finishing electropolymerization by adopting a cyclic voltammetry method, wherein the initial potential is 0V, the final potential is 0.7V, the scanning speed is 100mV/s, and the number of polymerization turns is 8; washing the electrode with deionized water and then drying at room temperature; immersing the polymerized electrode in 4000-20000U lysozyme, reacting for 2 h at 25 ℃, and dissolving the cell wall of the template listeria monocytogenes; then immersing the sample in a sodium dodecyl sulfate acetic acid solution with the mass fraction of 5%, reacting for 4 hours, and dissolving the cell membrane of the template Listeria monocytogenes; and washing the electrode with deionized water to remove free bacteria, forming imprinted holes, and airing at room temperature to obtain the listeria monocytogenes imprinted working electrode.
The second purpose of the invention is to obtain the listeria monocytogenes imprinted working electrode by the method for preparing the listeria monocytogenes imprinted working electrode.
The third purpose of the invention is to provide a method for preparing a listeria monocytogenes imprinted electrochemical sensor, wherein the method is to connect the listeria monocytogenes imprinted working electrode obtained by the invention with an electrochemical workstation to obtain the listeria monocytogenes imprinted electrochemical sensor.
In one embodiment of the present invention, the method comprises the steps of:
the listeria monocytogenes imprinted working electrode, the platinum sheet counter electrode and the Ag/AgCl (saturated potassium chloride) reference electrode obtained by the method are placed in potassium ferricyanide electrolyte and connected with an electrochemical workstation to obtain the listeria monocytogenes imprinted electrochemical sensor.
The fourth purpose of the invention is to obtain the listeria monocytogenes imprinted electrochemical sensor by the method for preparing the listeria monocytogenes imprinted electrochemical sensor.
The fifth purpose of the invention is to provide the application of the listeria monocytogenes imprinted electrochemical sensor in the detection of listeria monocytogenes.
In one embodiment of the present invention, the application comprises the following steps:
(1) standard curve: immersing the Listeria monocytogenes imprinted membrane/gold-modified ITO electrode into Listeria monocytogenes bacterial liquid with different concentrations, adsorbing for 30min, taking out the electrode, gently washing the surface of the electrode by deionized water, placing the electrode in 2.5mM potassium ferricyanide electrolyte, and carrying out DPV detection at room temperature; drawing a standard curve by taking the logarithm of the concentration of the listeria monocytogenes as an abscissa and taking a peak current value as an ordinate;
(2) testing a sample to be tested: immersing the Listeria monocytogenes imprinted membrane/gold-modified ITO electrode into a sample to be detected, adsorbing for 30min, taking out the electrode, gently washing the surface of the electrode by deionized water, placing the electrode in 2.5mM potassium ferricyanide electrolyte, and carrying out DPV detection at room temperature. And (2) quantifying the concentration of the Listeria monocytogenes according to the standard curve obtained in the step (1).
In one embodiment of the present invention, the peak current value is obtained by setting an optimal electrochemical detection parameter using a prepared listeria monocytogenes imprinted electrochemical sensor and performing DPV measurement.
In one embodiment of the invention, the potassium ferricyanide solution is 100 mL containing 0.0823 g K3[Fe(CN)6]、0.1056 g K4[Fe(CN)6]·3H2O, 0.7455g KCl in 0.1M PB buffer (pH 7.4).
In one embodiment of the present invention, the PB buffer is 100 mL containing 2.1961 g Na2HPO4·12H2O、0.6035 g NaH2PO4·2H2An aqueous solution of O.
In one embodiment of the present invention, the test conditions of the DPV are: the initial potential was-0.2V, the final potential was +0.6V, the potential increment was 4 mV, the pulse width was 50ms, and the pulse period was 500 ms.
In one embodiment of the invention, the listeria monocytogenes is listeria monocytogenes ATCC 19115.
In one embodiment of the present invention, the relationship between the peak current value and the log concentration value of listeria monocytogenes ATCC19115 is plotted with the log concentration value of listeria monocytogenes ATCC19115 as abscissa and the peak current value as ordinate, and the experimental result shows that the peak current value decreases as the concentration of listeria monocytogenes ATCC19115 increases.
In one embodiment of the present invention, the standard curve is at 1 × 102~1×107In the concentration range of CFU/mL, the log value of the concentration of the Listeria monocytogenes is in a linear relation with the peak current value, y = -2.1086x + 156.32 (R)2= 0.9595). The detection limit of Listeria monocytogenes ATCC19115 in the sample to be tested is 50CFU/mL (g).
The invention has the beneficial effects that:
(1) the listeria monocytogenes imprinted electrochemical sensor prepared by the invention is applied to detecting listeria monocytogenes, does not need bacteria increasing, has high detection speed and low equipment requirement, and is expected to be used for on-site instant detection.
(2) The Listeria monocytogenes imprinted electrochemical sensing detection method provided by the invention combines the ITO electrode modified by the gold nanoparticles, has the advantages of high sensitivity, strong specificity, low detection cost and simplicity in operation, and is suitable for the fields of food safety, environmental monitoring, biological sample analysis and the like.
(3) The gold nanoparticles have large active specific surface area, high electrocatalytic activity, good conductivity and biocompatibility, the gold nanoparticles can be used as an electrode modification material to obviously improve the detection sensitivity of the electrochemical sensor, the electrode based on imprinting identification can specifically identify target bacteria and improve the selectivity and the detection efficiency of the electrochemical sensor, and the constructed electrochemical sensor is disclosed as 1 × 102~1×107In the concentration range of CFU/mL, the log value of the concentration of the Listeria monocytogenes is in a linear relation with the peak current value, y = -2.1086x + 156.32 (R)2= 0.9595). The detection limit of Listeria monocytogenes ATCC19115 in the sample to be tested is 50CFU/mL (g).
Drawings
FIG. 1 is a flow chart of the construction of an electrochemical sensor for Listeria monocytogenes.
FIG. 2 is the optimization of electro-deposition gold scanning cycle number; (A) a cyclic voltammogram of gold electrodeposited on the surface of the ITO electrode; (B) scanning differential pulse voltammograms of gold modified electrodes with different circles in 2.5mmol/L potassium ferricyanide electrolyte.
FIG. 3 is a scanning electron microscope characterization of gold nanoparticles.
Figure 4 is the template listeria monocytogenes concentration optimization.
FIG. 5 shows the optimization of the concentration of functional monomeric pyrrole.
Fig. 6 is an optimization of electrolyte potassium chloride concentration.
FIG. 7 shows electrochemical characterization of electrode modification, (A) cyclic voltammetry, (B) differential pulse voltammetry, and (C) AC impedance curve, wherein (a) ITO bare electrode, (B) Au/ITO electrode, (C) PPy + bacteria/Au/ITO electrode (bacteria concentration in electropolymerization solution is 1 × 10)8CFU/mL), (d) BIP/Au/ITO electrode, (e) bacteria/BIP/Au/ITO electrode (adsorbed bacteria concentration is 1 × 10)7CFU/mL), (D) bare electrode, NIP electrode and BIP electrode respectively adsorbing 1 × 107The change of the current value before and after CFU/mL Listeria monocytogenes.
FIG. 8 is a fluorescence characterization diagram of adsorption performance of a Listeria monocytogenes imprinted working electrode; (A) a NIP electrode surface; (B) BIP electrode surface.
FIG. 9 shows the detection of Listeria monocytogenes, (A) the DPV curves of Listeria monocytogenes with different concentrations, the concentration of the bacteria is 1 × 10 from top to bottom1、1×102、1×103、1×104、1×105、1×106、1×107、1×108CFU/mL; (B) a standard curve.
FIG. 10 is a stability characterization of a Listeria monocytogenes imprinted electrochemical sensor.
FIG. 11 is a specific characterization of Listeria monocytogenes imprinted electrochemical sensors.
Detailed Description
In order to better understand the invention, the following embodiments further illustrate the content of the invention, but the content of the invention is not limited to the following implementation.
EXAMPLE 1 preparation of the sensor
A method for preparing a Listeria monocytogenes imprinted electrochemical sensor is disclosed, the preparation process is shown in figure 1, and the method comprises the following steps:
(1) placing an ITO electrode in a 5mM chloroauric acid solution, and completing electrodeposition of gold nanoparticles by adopting cyclic voltammetry, wherein the initial potential is-1V, the final potential is 0.5V, the scanning rate is 100mV/s, and the number of scanning turns is 15 turns;
(2) washing electrode with deionized water after gold electrodeposition, air drying at room temperature, preparing electropolymerization solution with template listeria monocytogenes and functional monomer pyrrole dissolved in electrolyte potassium chloride water solution (the concentration of listeria monocytogenes ATCC19115 in the electropolymerization solution is 1.0 × 108CFU/mL; the concentration of pyrrole monomer was 0.2M; the concentration of potassium chloride is 0.2M), placing the ITO electrode modified by the gold nanoparticles into an electropolymerization liquid, and finishing electropolymerization by adopting a cyclic voltammetry method, wherein the initial potential is 0V, the final potential is 0.7V, the scanning speed is 100mV/s, and the polymerization turns are 8 turns;
(3) washing the electrode after electrochemical polymerization with deionized water, and airing at room temperature; immersing the electrode subjected to electrochemical polymerization in 4000-20000U lysozyme, reacting for 2 h at 25 ℃, and dissolving the cell wall of the template listeria monocytogenes; then immersing the sample in a lauryl sodium sulfate acetic acid solution with the mass fraction of 5% for reaction for 4 hours to dissolve the cell membrane of the template Listeria monocytogenes; washing the electrode with deionized water to remove free bacteria, forming imprinted pores, and drying at room temperature to obtain a Listeria monocytogenes imprinted working electrode (BIP electrode);
(4) and placing the prepared Listeria monocytogenes imprinted working electrode, the platinum sheet counter electrode and the Ag/AgCl (saturated potassium chloride) reference electrode in potassium ferricyanide electrolyte, and connecting an electrochemical workstation to obtain the Listeria monocytogenes imprinted electrochemical sensor.
Comparative example 1
A non-imprinted electrode (NIP electrode) was obtained by omitting the addition of template listeria monocytogenes on the basis of example 1.
Example 2 ITO electrode surface modification
1. Optimization of the number of scanning turns in step (1)
And (3) adjusting the number of scanning turns in the step (1) to be 5-15 turns, and keeping other parameters consistent with those in the embodiment 1 to obtain the ITO electrode for modifying the gold nanoparticles.
Fig. 2A is a graph of cyclic voltammetry scans for 15 cycles. As can be seen from the figure, one reduction peak was observed at E = 0.19V when scanning round 1; when scanning the 2 nd and 3 rd circles, the reduction peak moves to a negative potential; during the 4 th and subsequent rounds of scanning, the reduction peak continuously moves to the positive potential, because the gold nanoparticles deposited on the surface of the ITO electrode promote easier reduction of the chloroauric acid, so that the gold nanoparticles obtained in the last round of scanning are easier than those obtained in the previous round of scanning, and the deposited potential is more and more positive. Fig. 2B shows the DPV signals of the rear electrode after 5, 7, 10, 12 and 15 scans respectively, and it can be seen that the electrode current signal is maximum when the number of scans is 7, so that the number of scans is selected to be 7.
2. Scanning electron microscope characterization of gold nanoparticles in step (1)
The characterization result of the scanning electron microscope is shown in fig. 3, and the surface of the ITO electrode is covered with gold nanoparticles with the particle size of 200-400 nm and uniform distribution.
3. And (3) optimizing the concentration of the template listeria monocytogenes in the step (2).
Adjusting the concentration of the template Listeria monocytogenes in the step (2) to be 1.0 × 105~1.0×109CFU/mL, other parameters were consistent with example 1.
Optimization of template Listeria monocytogenes concentration As shown in FIG. 4, the electrode current decreased with the increase of template Listeria monocytogenes concentration when the concentration of template Listeria monocytogenes was 1.0 × 109The concentration of the listeria monocytogenes and the template is 1.0 × 10 at CFU/mL8Compared with CFU/mL, the electrode current is not changed any more, which indicates that the template Listeria monocytogenes on the surface of the electrode is saturated, therefore, the optimal concentration of the template Listeria monocytogenes is 1.0 × 108CFU/mL。
4. Optimization of pyrrole concentration in step (2)
And (3) adjusting the concentration of the pyrrole in the step (2) to be 0.05-0.4M, and keeping other parameters consistent with those of the embodiment 1.
As shown in FIG. 5, the concentration of pyrrole is optimized, and the electrode current increases with the increase of the concentration of pyrrole in the range of 0.05-0.2M, and decreases with the increase of the concentration of pyrrole in the range of 0.2-0.4M. This is because polypyrrole is rapidly formed due to the high concentration of pyrrole, but the uniformity of the polypyrrole film is poor at this time, which affects the conductivity of the polypyrrole film. The optimum concentration of pyrrole is therefore 0.2M.
5. And (3) optimizing the concentration of the electrolyte potassium chloride in the step (2).
And (3) adjusting the concentration of the electrolyte potassium chloride in the step (2) to be 0.1-0.4M, and keeping other parameters consistent with those of the embodiment 1.
As shown in FIG. 6, the concentration of potassium chloride is optimized, and the electrode current increases with the increase of the concentration of potassium chloride within the range of 0.1-0.2M, and decreases with the increase of the concentration of potassium chloride within the range of 0.2-0.4M. This is because when the potassium chloride concentration reaches a certain level, the electropolymerization rate becomes fast, and the polypyrrole film is formed unevenly, which affects the conductivity. The optimum concentration of potassium chloride is therefore 0.2M.
6. And (3) optimizing the polymerization cycle number in the step (2).
And (3) adjusting the polymerization cycle number of the step (2) to be 1-10 cycles, wherein other parameters are consistent with those of the embodiment 1.
The current decreased with the increase of polymerization cycle number, and when the polymerization was completed to 9 th cycle, the current was not changed any more than that after the polymerization was completed to 8 th cycle. When the number of polymerization turns is less than 8, the polypyrrole film is too thin, and the formed cavity is unstable. When the polymerization cycle number is more than 8, the polypyrrole film is too thick, the mass transfer in the film is slowed, and the electrode conductivity is reduced. Thus, polymerization for 8 cycles is most suitable.
Example 3: characterization of sensors
(1) Electrochemical characterization
The cyclic voltammetry parameter conditions were as follows: the initial potential is-0.2V, the final potential is +0.6V, the scanning speed is 100mV/s, and the sampling interval is 1 mV; the differential pulse voltammetry parameter conditions were as follows: the initial potential is-0.2V, the final potential is +0.6V, the incremental potential is 5mV, the pulse width is 50ms, and the pulse period is 500 ms; the parameter conditions of the alternating-current impedance method are as follows: the initial potential is 0.2V, the minimum frequency is 0.1 Hz, and the maximum frequency is 104Hz, amplitude of 5 mV. The concentration of potassium ferricyanide electrolyte was 2.5 mM.
In fig. 7A and 7B, a curve a is an oxidation reduction peak obtained by scanning the ITO bare electrode in the potassium ferricyanide solution, and the peak current at this time is 42.15 μ a (calculated from a curve a in the DPV diagram of fig. 7B); the curve B is the ITO electrode modified by the gold nanoparticles, and the visible current is obviously increased to 326.02 muA (calculated by the curve B in the DPV diagram of FIG. 7B), which is because the conductivity of the electrode is enhanced after the surface of the electrode is modified by gold. After polymerization of the polypyrrole listeria monocytogenes membranes, the current dropped rapidly to 134.21 μ a (calculated from the c-curve in the DPV plot of fig. 7B). This is because although polypyrrole enhances the conductivity of the electrode, bacteria are non-conductive macromolecules that impair the enhanced conductivity of polypyrrole. After elution of template listeria monocytogenes, the current rose to 188.08 μ a (calculated from the d-curve in the DPV plot of fig. 7B). This is because the BIP electrode has a hole for imprinting, and the potassium ferricyanide probe can freely enter and exit, thereby increasing the current. After adsorption of the target bacteria using the prepared BIP electrode, the current was decreased to 142.16. mu.A (calculated from the e-curve in the DPV graph of FIG. 7B). This is because, after the listeria monocytogenes is adsorbed, the pores are blocked by the bacteria, thereby preventing the potassium ferricyanide probe from entering the imprinted pores. The impedance characterization results (fig. 7C) also correspond to the current characterization results.
(2) Characterization of adsorption Properties
In FIG. 7D, the BIP electrode current change of the adsorbed Listeria monocytogenes was significant, ΔIp =45.92 μ a, while NIP and bare electrode sensors were unchanged. The reason is that the NIP electrode and the surface of the bare electrode are not provided with imprinted holes and cannot adsorb target bacteria, which shows that BIP has a specific adsorption function.
In FIG. 8, no Listeria monocytogenes (SYTO 9 nucleic acid dye labeled) was adsorbed on the NIP electrode, indicating that the NIP could not adsorb Listeria monocytogenes. However, a large number of listeria monocytogenes (SYTO 9 nucleic acid dye labeled) are adsorbed on the BIP electrode, and the result proves that the imprinting holes on the BIP electrode can specifically identify the listeria monocytogenes and have the function of adsorbing the listeria monocytogenes.
Example 4: application of sensor
1. Detection of Listeria monocytogenes
Respectively immersing the listeria monocytogenes imprinted membrane/gold-modified ITO electrode into 1 × 101、1×102、1×103、1×104、1×105、1×106、1×107、1×108Adsorbing in CFU/mL Listeria monocytogenes liquid for 30min, taking out the electrode, gently washing the surface of the electrode with deionized water, placing in 2.5mM potassium ferricyanide electrolyte, and carrying out DPV detection at room temperature.
2. Analysis conditions and methods
Detecting parameters by a DPV method: the initial potential is-0.2V, the final potential is +0.6V, the increment potential is 4 mV, the pulse width is 50ms, and the pulse period is 500 ms.
And (4) plotting the concentration logarithm value and the peak current value of the listeria monocytogenes to obtain a standard curve for detecting the listeria monocytogenes by the listeria monocytogenes imprinted electrochemical sensor.
3. The result of the detection
The experimental results showed that the peak current value decreased with the increase of the concentration of Listeria monocytogenes ATCC19115 (FIG. 9A). As can be seen from FIG. 9B, the peak current value decreased at 1 ×102~1×107In the concentration range of CFU/mL, the log value of the concentration of the Listeria monocytogenes is in a linear relation with the peak current value, y = -2.1086x + 156.32 (R)2= 0.9595). The detection limit of Listeria monocytogenes ATCC19115 in the sample to be tested is 50CFU/mL (g).
Example 5 reproducibility, stability, specificity of the sensor
1. Reproducibility of
The concentration tested was 1 × 10 using the sensor of example 17Peak current values generated by CFU/mL listeria monocytogenes. As can be seen from table 1, the Relative Standard Deviation (RSD) of the peak current values measured using the BIP electrodes prepared three times was 0.39%, and the Relative Standard Deviation (RSD) of the peak current values measured 10 times after repeatedly eluting the target bacteria using the BIP electrodes prepared in the same time was 0.32%, demonstrating that the constructed sensors had good reproducibility.
Table 1 reproducibility testing of electrochemical blot sensors
2. Stability of
The sensor of example 1 was stored at room temperature for 3 months as shown in FIG. 10 at test 1 × 107When CFU/mL Listeria monocytogenes is used, the DPV signals generated by the sensor stored for 3 months and the newly prepared sensor have no significant difference (p> 0.05, n = 3), indicating good stability of the sensor.
3. Specificity of
Respective tests 1 × 10 using the sensor of example 17CFU/mL Listeria monocytogenes, Staphylococcus aureus, Salmonella, and Escherichia coli. As shown in FIG. 11, the peak current values detected in Listeria monocytogenes were very significantly different from those of the blank control group (. lambda.)p< 0.01, n = 3), and the peak current values of other bacteria detected were not different from those of the blank control group (n = 3) (p> 0.05, and n = 3), and the constructed electrochemical sensor is proved to have higher specificity to the listeria monocytogenes.
Example 6 method accuracy verification
Aseptically adding 25 g cooked beef (sterilized) into a homogenizing bag containing 225 mL normal saline, continuously homogenizing for 1 min-2 min on a slapping homogenizer, collecting supernatant, adding 1 mL 5 × 10 into 9mL supernatant2CFU/mL、5×103CFU/mL、5×106CFU/mL Listeria monocytogenes ATCC19115, to a final concentration of 50CFU/mL, 5 × 102CFU/mL、5×105CFU/mL of the solution to be tested. The artificially contaminated sample was tested as described in example 4. Table 2 recovery and relative standard deviation (n = 3) of listeria monocytogenes in artificially contaminated samples. As can be seen from table 2: in the detection of the cooked beef sample of artificially polluted Listeria monocytogenes, the recovery rate of the constructed sensor detection method is within the range of 96-102%, and the RSD value<2.03%, the method is proved to have higher accuracy.
TABLE 2 method accuracy verification results
EXAMPLE 7 actual sample testing
When 150 batches of cooked meat products purchased from the market were tested by the method in example 4, the positive rate of listeria monocytogenes was 4%, and the quantitative test results are shown in table 3.
TABLE 3 actual sample testing
Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined by the appended claims.
Claims (5)
1. A method for preparing a listeria monocytogenes imprinted working electrode is characterized in that the method is based on a surface molecular imprinting technology, and is characterized in that an Indium Tin Oxide (ITO) electrode modified with gold nanoparticles is placed in an electropolymerization liquid containing a template, a functional monomer and an electrolyte solution for electrochemical polymerization by taking listeria monocytogenes as the template and pyrrole as the functional monomer; eluting the template to obtain a listeria monocytogenes imprinted working electrode;
the method comprises the following steps:
(1) placing an ITO electrode in a chloroauric acid solution, and electrodepositing modified gold nanoparticles on the surface of the ITO electrode;
(2) after the electrodeposition is finished, washing the electrode, drying the electrode at room temperature, and placing the ITO electrode modified with the nanogold on an electropolymerization liquid of template Listeria monocytogenes, functional monomer pyrrole and electrolyte potassium chloride solution for electrochemical polymerization;
(3) after the electrochemical polymerization is finished, washing the electrode, drying the electrode at room temperature, and sequentially immersing the electrode in lysozyme and sodium dodecyl sulfate solution to elute the template to obtain a listeria monocytogenes imprinted working electrode;
wherein the Listeria monocytogenes in the electropolymerization solution is Listeria monocytogenes ATCC19115, and the concentration is 1.0 × 108CFU/mL；
Performing the electrodeposition in the step (1) by adopting a cyclic voltammetry, wherein the initial potential is-1V, the final potential is 0.5V, the scanning rate is 100mV/s, and the number of scanning cycles is 7;
the concentration of the functional monomer pyrrole in the electropolymerization liquid in the step (2) is 0.2M;
the concentration of electrolyte potassium chloride in the electropolymerization liquid in the step (2) is 0.2M;
and (3) adopting cyclic voltammetry for electrochemical polymerization in the step (2), wherein the initial potential is 0V, the final potential is 0.7V, the scanning speed is 100mV/s, and the polymerization cycle number is 8.
2. The listeria monocytogenes imprinted working electrode prepared by the method of claim 1.
3. A method for preparing a Listeria monocytogenes imprinted electrochemical sensor, which is characterized in that the method comprises the step of connecting the Listeria monocytogenes imprinted working electrode of claim 2 with an electrochemical workstation to obtain the Listeria monocytogenes imprinted electrochemical sensor.
4. The listeria monocytogenes imprinted electrochemical sensor prepared by the method of claim 3.
5. The use of the listeria monocytogenes imprinted electrochemical sensor of claim 4 for detecting listeria monocytogenes.
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