CN110006984B - Portable AHL molecular imprinting screen printing electrochemical detector - Google Patents
Portable AHL molecular imprinting screen printing electrochemical detector Download PDFInfo
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Abstract
The invention discloses a portable AHL molecular imprinting screen printing electrochemical detector, and belongs to the technical field of rapid detection. The method comprises the steps of dripping gold nano cross on the surface of a working electrode, then taking an AHL molecular analogue as a template, placing a gold-containing electrode in electropolymerization liquid containing a monomer, a cross-linking agent, the template and an initiator by adopting a surface imprinting technology to perform electropolymerization, eluting template molecules to obtain an AHL molecular imprinting screen printing electrode, obtaining a peak current difference value before and after adsorption by matching with a specific electrochemical detection condition based on the electrode, obtaining a standard curve by utilizing the concentration of a substance to be detected and the peak current difference value, and calculating the AHL content. The detector provided by the invention can be used for directly detecting AHL molecules, has the advantages of sensitivity, rapidness, recycling and repeated use, high specificity and the like, is low in price, and is suitable for detecting AHL molecules at the basic level or on site.
Description
Technical Field
The invention relates to a portable AHL molecular imprinting screen printing electrochemical detector, belonging to the technical field of rapid detection.
Background
Quorum sensing is a unique and diverse quorum behavior phenomenon generated by mutual sensing when bacteria grow to a certain density and carrying out gene expression and regulation. N-Acyl Homoserine Lactone (AHL) compounds are the most important signal molecules in gram-negative bacteria quorum sensing and regulate the expression of a plurality of physiological characteristic genes. The method is an important means for deeply researching and knowing bacterial quorum sensing by quickly, simply and effectively detecting whether the bacteria can generate AHL.
The principles of various detection methods of AHL molecules are different, each method has own advantages and disadvantages, but due to the improvement of the sensitivity and accuracy requirements of the detection method in the modern food industry, a plurality of methods are not suitable for the detection. Therefore, the establishment of a sensitive, rapid, simple, convenient, high-specificity and economical detection method is an urgent need of production and operation enterprises, quality control personnel, import and export inspection traders and government management departments and a powerful guarantee of food and environmental safety.
At present, methods for detecting signal molecules AHL generally comprise 3 physical and chemical means of ①, a sample from a liquid culture medium is detected and purified mainly by a High Performance Liquid Chromatography (HPLC) gas chromatography-mass spectrometry (GC-MS) technology, the method can also identify the property of the AHL besides quantification, ② a biological method of sensing bacteria is utilized, namely, a pigment or fluorescence controlled by a quorum sensing system, such as the generation of green fluorescent protein 1 is utilized to detect the existence of the AHL, and ③ a thin layer chromatography combining physical and chemical means and bioluminescence is utilized.
The molecular imprinting is also called as an artificial antibody, has low requirement on the environment, does not need a special preservation mode, and is a convenient and sensitive detection way. However, the molecular imprinting detection methods reported in the existing reports are all based on disposable molecular imprinting membranes for detection, which is portable but has serious resource waste, and the number of the consumed molecular imprinting membranes is large in detection; meanwhile, different detection objects are subjected to electrochemical detection under the molecular imprinting films prepared by different methods, different data prediction rules are expressed, and the constructed data models are different. Therefore, it is of great market significance to develop a non-disposable portable AHL molecularly imprinted screen printed electrochemical detector capable of sensitively detecting AHL.
Disclosure of Invention
Aiming at the defects in the prior art, the invention provides a molecular imprinting screen printing electrochemical detector for quickly detecting AHL. The method comprises the steps of dripping gold nano cross on the surface of a working electrode, then taking an AHL molecular analogue as a template, placing a gold-containing electrode in electropolymerization liquid containing a monomer, a cross-linking agent, the template and an initiator by adopting a surface imprinting technology to perform electropolymerization, eluting template molecules to obtain an AHL molecular imprinting screen printing electrode, obtaining a peak current difference value before and after adsorption by matching with a specific electrochemical detection condition based on the electrode, obtaining a standard curve by utilizing the concentration of a substance to be detected and the peak current difference value, and calculating the AHL content. The detector provided by the invention can be used for directly detecting AHL molecules, has the advantages of sensitivity, rapidness, recycling and repeated use, high specificity and the like, is low in price, and is suitable for detecting AHL molecules at the basic level or on site.
It is a first object of the present invention to provide a method for detecting N-acyl homoserine lactone, which comprises the steps of:
(1) preparing an N-acyl homoserine lactone molecularly imprinted screen printing electrode: dripping gold nano cross on the surface of a working electrode of the screen printing electrode, modifying p-mercaptoaniline through gold mercapto bond, then soaking in template molecular solution, placing in electropolymerization liquid for electroplating polymerization, eluting template molecules, and electroplating a molecular imprinting polymer film on the surface of the working electrode to obtain a molecular imprinting screen printing electrode;
(2) dripping 25-50 mu L of electroconducting liquid on the surface of the N-acyl homoserine lactone molecularly imprinted screen printing electrode, horizontally inserting the N-acyl homoserine lactone molecularly imprinted screen printing electrode into an electrochemical detector for detection, and measuring the current value before adsorption;
(3) adding 25-50 muL of electroconduction liquid containing a sample to be detected to the surface of the N-acyl homoserine lactone molecularly imprinted screen printing electrode for standing adsorption, horizontally inserting the N-acyl homoserine lactone molecularly imprinted screen printing electrode into an electrochemical detector for detection, and measuring the current value after adsorption;
(4) and (4) calculating to obtain a peak current difference value according to the steps (2) and (3), and calculating to obtain the concentration of the N-acyl homoserine lactone in the sample to be detected according to a standard curve of the concentration of the substance to be detected in the sample to be detected and the peak current difference value.
In one embodiment of the present invention, the peak current difference is a post-adsorption current value-a pre-adsorption current value.
In one embodiment of the present invention, the adsorption time in the step (3) is 180 s.
In one embodiment of the invention, the template molecule is furanone.
In one embodiment of the present invention, the standard curve is obtained by: preparing N-acyl homoserine lactone electroconducting liquid and pure electroconducting liquid with different known concentrations, dropwise adding the electroconducting liquid and the pure electroconducting liquid to the surface of an N-acyl homoserine lactone molecularly imprinted screen printing electrode, inserting the N-acyl homoserine lactone molecularly imprinted screen printing electrode into an electrochemical detector, detecting a current peak value formed when electrons flow from a counter electrode, pass through the electroconducting liquid and flow back to a workstation from a working electrode, obtaining peak current difference values under different N-acyl homoserine lactone concentrations, and obtaining a standard curve by taking the N-acyl homoserine internal concentration as a horizontal coordinate and the peak current difference value as a vertical coordinate.
In one embodiment of the present invention, the concentration of N-acylhomoserine in the electric conduction liquid is in the range of 1X 10-13～1×10-9mol·L-1。
In one embodiment of the present invention, the voltage setting range of the electrochemical detector is-0.2V to 0.4V.
In one embodiment of the present invention, the amount of the electric conduction liquid used in the step (2) or the step (3) is preferably 40. mu.L.
The invention provides a portable N-acyl homoserine lactone molecular imprinting screen printing electrochemical detector applicable to the detection method, which comprises an externally-inserted N-acyl homoserine lactone molecular imprinting screen printing electrode and an electrochemical detector, wherein the operation mode is that 25 mu L-50 mu L of electroconducting liquid containing a sample to be detected is dripped on the surface of the N-acyl homoserine lactone molecular imprinting screen printing electrode, the electrode is kept still for adsorption for 180s, then the electrode is inserted into a clamping groove of the electrochemical detector, and the peak current value is detected and read to obtain a peak current difference value; the preparation method of the N-acyl homoserine lactone molecularly imprinted screen printing electrode comprises the following steps: and (2) dripping gold nano crosses on the surface of a working electrode of the screen printing electrode, modifying p-mercaptoaniline through gold mercapto bonds, then soaking the working electrode into a template molecule solution, placing the working electrode into an electropolymerization solution for electroplating polymerization, and eluting template molecules to ensure that the surface of the working electrode is electroplated with a molecularly imprinted polymer membrane, thus obtaining the N-acyl homoserine lactone molecularly imprinted screen printing electrode.
In one embodiment of the invention, the voltage setting range of the electrochemical detector is: -0.2V to 0.4V.
In one embodiment of the invention, the molecularly imprinted screen printing electrode comprises an electrode substrate, a wiring terminal, an electrode connecting wire, a working electrode, a counter electrode and an insulating layer; the surface of the working electrode is electroplated with a molecularly imprinted polymer film.
In an embodiment of the invention, the method for preparing the molecularly imprinted screen printing electrode specifically comprises the following steps: dripping gold nano cross solution (the gold nano layer can enhance the contact specific surface area of the molecular imprinting polymer film which is then electropolymerized, increase the electron transfer rate, thereby increasing the sensitivity) on the surface of a working electrode of a common screen printing electrode, and drying; and modifying p-mercaptoaniline through gold mercapto bonds, immersing the p-mercaptoaniline in a 1 mmol/L-1 furanone solution, incubating for 4 hours at room temperature, then placing the p-mercaptoaniline in an electropolymerization solution, carrying out electroplating polymerization, eluting template molecules, and electroplating a molecular imprinting polymer film on the surface of the working electrode to obtain the molecular imprinting screen printing electrode.
In one embodiment of the invention, the gold nanoparticle cross solution is prepared by using 5ml of 0.5 mmol.L-1 HAuCl4Mixing with 5ml of 0.2 mol.L-1 CTAB, adding 400. mu.l of ultrapure water, and adding 0.6ml of 0.01 mol.L-1 NaBH4Stirring for 2min, and standing for 30min to obtain gold seeds; dissolving 0.9g CTAB and 0.08g sodium salicylate with 25ml ultrapure water at 50 deg.C, cooling to 30 deg.C, adding 0.6ml 4mmol L-1AgNO31028.8. mu.l of 24.28 mmol. multidot.L-1 and 23.9712ml of ultrapure water were stirred for 15min, 700. mu.l of 0.1 mol. multidot.L-1 VC was added, and the mixture was vigorously stirred for 30s until the solution was colorless. Finally, adding gold seeds into the solution, stirring for 30s, and standing and growing for 12h at 30 ℃.
In one embodiment of the invention, the electropolymerization liquid comprises 5-15mmol/L of p-mercaptoaniline, 30-80mol/L of tetrabutylammonium perchlorate, 5-15mol/L of 4, 4' -bipyridine and 0.2-0.6g/L of perchloric acid.
In an optimized embodiment of the invention, the electropolymerization liquid contains 10 mmol.L-1P-mercaptoaniline, 50 mmol.L-1 tetrabutylammonium perchlorate, 1 mmol.L-1 furanone and 0.4g/L perchloric acid.
In an optimized embodiment of the invention, the specific conditions of the electroplating polymerization are as follows: the electropolymerization voltage is-0.3V-1.2V, the scanning speed is 50mv/s, and the optimal number of scanning circles is 10 circles.
In one embodiment of the invention, the template molecule is eluted by immersing the electrode in an aqueous solution (4: 1, v/v) containing ethanol for 10min, and then rinsing the working electrode with ultrapure water.
In one embodiment of the invention, the molecular imprinting screen printing electrode, the electrode substrate can be used for multiple times, and the working electrode, the counter electrode and the reference electrode are led out by the electrode connecting wire to form a convex three-wire socket which can be inserted into a portable electrochemical detector for detecting and reading.
In one embodiment of the invention, the molecularly imprinted screen printed electrode has an electrode substrate printed with a connecting terminal, one electrode substrate with an electrode connecting line integrally connected with a working electrode and a connecting terminal, and the other electrode substrate with an electrode connecting line integrally connected with a counter electrode, wherein the two electrode connecting lines are parallel to each other, the electrode connecting line is arranged in the middle of the electrode substrate, and the surface of the electrode connecting line is coated with a layer of PVC insulator.
In one embodiment of the invention, the molecularly imprinted screen printing electrode has a circular block-shaped working electrode, a semicircular ring-shaped block-shaped counter electrode concentric with and separated from the working electrode, and a polymeric AHL molecularly imprinted membrane is electroplated on the surface of the working electrode.
In one embodiment of the present invention, the electrochemical detector comprises a simplified electrochemical workstation equipped with an LED display screen, and the standard curve of the patulin concentration and the current peak value difference is built in the portable electrochemical detector program, so that the concentration of the target substance in the sample to be detected can be displayed on the LED display screen.
A third object of the present invention is to provide an application of the detector, wherein the application is as follows: firstly, inserting standard substance molecularly imprinted screen-printed electrodes (a standard substance before adsorption and a standard substance after adsorption), correcting a detection result of the portable detector according to a standard curve of a peak current difference value and AHL sample concentration, and then inserting the electrodes to complete the molecularly imprinted screen-printed electrode test of the adsorption of a sample to be tested.
In one embodiment of the invention, the detector is calibrated by inserting more than two concentrations of standard molecularly imprinted screen-printed electrodes (the electrode corresponding to each concentration comprises a standard before adsorption and a standard after adsorption).
The preparation method of the standard curve is, in one embodiment of the invention, that: and (3) dropwise adding an electric conduction liquid on the eluted molecular imprinting screen printing electrode, measuring the peak current value, preparing aqueous solution (ultrapure water) containing the detected substance with different known concentrations, dropwise adding the aqueous solution into the molecular imprinting screen printing electrode of the detected substance (the detected substance with the standard concentration is used for setting a standard curve), adsorbing for a certain time, washing, dropwise adding the electric conduction liquid, detecting by a portable detector, recording data, and calculating the standard curve between the substance concentration in the solution of the detected substance and the peak current difference (the current value before adsorption minus the current value after adsorption). And (4) carrying out calibration according to the standard curve, and carrying out technical reference inside the system when the calibration is used for detecting and analyzing the actual sample.
The application, in one embodiment of the present invention, is for detecting AHL. The molecular imprinting screen printing electrode is an AHL molecular imprinting screen printing electrode, and the standard curve is that y is 0.56439lgx +8.57752 (R)20.94637) detection limit of 10-11mol·L-1。
The fourth purpose of the invention is to apply the detection method or the detection instrument to the bacterial quorum sensing aspect.
The invention has the following beneficial effects:
(1) the portable detector is small and exquisite and rapid, and accurate data are detected after 180s of adsorption; meanwhile, the detector of the invention has high sensitivity and detection limit of 10-11mol·L-1And the method is recycled for 5 times, the data accuracy is still high, and the difference between the real value and the measured value is not more than 8%. The detector has the advantages of sensitivity, rapidness, repeated recycling, high specificity and the like, is low in price, and is suitable for detecting AHL molecules at the basic level or on site.
(2) The surface of the screen printing electrode is coated with gold nano-crosses in a dropping mode, so that the contact specific surface area of a molecularly imprinted polymeric membrane subjected to electroplating polymerization is enhanced, the electron transfer rate is increased, the sensitivity is increased, the response signal of the electrode is enhanced, the difference between the measured value and the true value is not more than 10%, and the data accuracy is high.
(4) The detection method of the invention adopts the molecularly imprinted polymeric membrane as the recognition target spot, has strong detection specificity, and simultaneously, the molecularly imprinted electrochemical sensor has the advantages of realizing on-site detection, being not influenced by the color and the turbidity of the sample, not needing complex treatment and separating the sample. By combining an electrochemical analysis method capable of quickly and sensitively detecting and the low cost and high performance of the screen printing electrode, the AHL molecules in the sample can be quickly and conveniently detected (after the extracting solution of the sample to be detected is obtained, the detection can be completed within a few minutes, the peak current value before adsorption can be detected and recorded in advance, and thus, only the peak current value after adsorption needs to be obtained again).
(5) The detector of the invention is a rapid detection product which can really solve the market demand and meet the detection demands of the departments of industry and commerce, quality inspection, research and colleges and the like, can be used for production and operation enterprises, quality control personnel, import and export inspection traders, government management departments, hospitals and even individual families, and is suitable for the fields of food industry, feed industry, environmental protection, medical drug screening, biochemistry and the like.
Drawings
FIG. 1: a schematic diagram of preparing an AHL molecularly imprinted screen-printed electrode plate;
FIG. 2: a portable AHL molecular imprinting electrochemical detector structure diagram;
FIG. 3: detecting a standard curve of the portable AHL molecularly imprinted electrochemical detector;
FIG. 4: and (3) dropwise adding a cyclic voltammetry curve chart of front and rear electrodes of the gold nano cross.
Detailed Description
In order to clearly understand the technical contents of the present invention, the following examples are given in detail for the purpose of better understanding the contents of the present invention and are not intended to limit the scope of the present invention.
Example 1 preparation of AHL molecularly imprinted screen printed electrode
The preparation schematic diagram of the AHL molecular imprinting screen printing electrode plate is shown in figure 1. The preparation method comprises the following steps:
(1) dripping gold nano cross on the surface of a working screen printing electrode, wherein the dripping method comprises the following steps: and (3) dripping the prepared gold nano cross solution on the surface of the working electrode, and drying under an infrared lamp to finish the dripping of the gold nano cross. The peak current value of the electrode on the CV diagram can be increased by dropping the gold nano-cross, namely, the sensitivity of the electrode is increased, as shown in FIG. 3.
(2) P-mercaptoaniline is modified through gold mercapto bond, and the modification method comprises the following steps: dipping the screen printing electrode coated with gold nano particles into 50 mmol.L < -1 > p-mercaptoaniline ethanol solution, soaking for 24h at room temperature, then thoroughly cleaning with ethanol and water to remove unadsorbed p-mercaptoaniline, and drying with nitrogen for later use;
(4) soaking the screen-printed electrode modified with p-mercaptoaniline in 1 mmol.L-1 furanone solution, incubating at room temperature for 4h, washing with ethanol and ultrapure water, and drying with nitrogen for later use; then immersing the screen printing electrode after electrostatic adsorption into 10mL of the screen printing electrode containing 10 mmol.L-1 p-mercaptoaniline, 50 mmol.L-1 tetrabutylammonium perchlorate, 1 mmol.L-1 furanone and 0.4 g.L-1 perchloric acid, scanning by adopting a cyclic voltammetry method, and electropolymerization voltage is as follows: -0.3V-1.2V, scan rate: 50mv/s, number of scan cycles: 10;
(5) and finally, eluting template molecules, soaking the screen printing electrode of the electro-polymerized AHL molecularly imprinted membrane in an ethanol aqueous solution (4: 1, v/v) for 10min, and then leaching the screen printing electrode with ultrapure water to finish the preparation of the screen printing electrode of the AHL molecularly imprinted membrane.
Embodiment 2 Portable AHL molecular imprinting screen printing electrochemical detector
The structure diagram of the portable AHL molecular imprinting electrochemical detector is shown in figure 2, and the structure is as follows:
the molecularly imprinted screen-printed electrode obtained in example 1 includes an electrode substrate, a connection terminal, an electrode connection wire, a working electrode, a counter electrode, and an insulating layer; the surface of the working electrode is electroplated with a molecularly imprinted polymer film; the electrode substrate is a disposable consumption substrate, the working electrode, the counter electrode and the reference electrode are led out by an electrode connecting line to form a convex three-wire socket which can be inserted into a portable electrochemical detector for detecting and reading. The electrode substrate is printed with a connecting terminal, an electrode matrix and a connecting terminal, wherein the electrode matrix and the connecting terminal are connected into a whole, the electrode matrix and the counter electrode are connected into a whole, the two electrode connecting lines are parallel to each other, the electrode connecting line is arranged in the middle of the electrode matrix, and the surface of the electrode matrix is coated with a layer of PVC insulator. The working electrode is in a round block shape, the counter electrode is in a semicircular annular block shape concentric with and separated from the working electrode, and the surface of the working electrode is electroplated with a polymeric AHL molecularly imprinted membrane.
The portable electrochemical detector is a simple electrochemical workstation with set voltage, and is provided with an LED display screen for recording the current value formed when electrons flowing out of the counter electrode flow through a sample or an electric conduction liquid and then flow back to the workstation from the working electrode. The current is recorded by an electrochemical detector, calculated by a standard curve prepared by an AHL molecular imprinting screen printing electrode with standard concentration, and displayed on an LED display screen in a concentration mode.
The sample testing procedure was as follows:
the detection results of the portable detector were first corrected by inserting the screen-printed electrodes into the portable detector using standard (AHL of known concentration) molecular imprinting. Then preparing the purified AHL sample into aqueous solution (ultrapure water), dripping 40 mu L of aqueous solution of the AHL sample on the surface of a screen-printed electrode of the prepared AHL molecularly imprinted membrane, and standing for adsorption time: 180s, washing after the adsorption is finished, then dropwise adding the electric conduction liquid, and configuring the electric conduction liquid: 2.5 mmol. L-1[Fe(CN)6]3/4And 0.1 mol. L-1KCl solution, voltage setting range: -0.2V-0.4V, recording the current peak value in scanning, calculating according to the standard curve between the AHL concentration and the current peak difference value built in the AHL special portable detector, and finally displaying the measured AHL concentration in the sample on an LED screen.
Example 3 Standard Curve
In one portable detector of the present invention, which is an AHL-specific portable detector, the standard curve between AHL concentration and peak current difference has been built into the detector program.
The standard curve drawing method comprises the following steps: preparation of different known concentrations (concentration range: 1X 10)-13～1×10-9mol·L-1) AHL molecular imprinting screen printing electrode plugAnd (3) detecting a current peak value formed when electrons flow from the counter electrode, pass through the conductive liquid and then flow back to the workstation from the working electrode in a detector, and obtaining a standard curve by taking the AHL concentration as a horizontal coordinate and taking a peak current value as a vertical coordinate, wherein the standard curve is shown in figure 3. The standard curve equation is obtained as y-0.56439 lgx +8.57752 (R)20.94799) detection limit of 10-11mol·L-1。
And (4) embedding the obtained standard curve in a detector through a computer program to obtain the portable detector special for the AHL.
When a sample to be detected is detected, more than 2 AHL standard substance molecular imprinting screen printing electrodes are used for correction, and then a sample solution to be detected is detected. The portable detector can finally and directly display the concentration of the AHL in the sample to be detected on an LED display screen of the detector according to an internally set standard curve.
Example 4 Standard Curve
In a portable detector of the present invention, the calibration curve is not built into the detector program.
For different measured substances, preparing molecular imprinting screen printing electrodes (including electrodes before adsorption and electrodes after adsorption) of the measured substances with different known concentrations, inserting the electrodes into a detector, inputting the concentrations into the detector on an operation interface of the detector respectively, calculating by a detector system according to the measured current value difference and received concentration information to obtain a standard curve of the current value difference and the substance concentration, detecting a sample to be detected by an operator, and finally directly displaying the concentration of the target substance in the sample to be detected according to the measured current value difference and the calculated standard curve in the system.
The standard curve equation is obtained as y-0.56439 lgx +8.57752 (R)20.94799) detection limit of 10- 11mol·L-1。
Example 5 optimization of assay conditions
1) Optimizing standing adsorption time:
referring to example 2, the standing adsorption time was changed to 80s and 280s, and the other conditions were not changed to obtain the detection results, thereby preparing a calibration curve. As a result, it was found that: when the adsorption time is 80s, the peak current value corresponding to each concentration standard sample is generally low, which indicates that the adsorption is insufficient and is not beneficial to final reading; when the adsorption time is 280s, the peak current value corresponding to each concentration standard sample is basically the same as that when the adsorption time is 180s, but the dropwise added standard sample solution is observed to be obviously reduced, which indicates that the solution is volatilized, so that the properly selected standing adsorption time is 180 s.
2) Optimization of the amount of AHL sample solution:
referring to the test method of example 2, the amounts of the AHL sample solutions were changed to 20 μ L, 25 μ L, 50 μ L, and 60 μ L, and the other conditions were not changed to obtain the test results, thereby preparing a standard curve. As a result, it was found that: when the using amount is 20 muL, the AHL sample solution is obviously reduced due to volatilization, and AHL molecules have certain volatility, so that a part of AHL molecules can not be adsorbed, the peak current detection value is lower than 10% of the true value, and the data accuracy is poor; gradually increasing to 25 and 40, improving the accuracy, wherein the difference between the actual value and the measured value is not more than 5% of the actual value, especially the difference is less than 1% when the volume is 40 muL; the difference value at 50. mu.L was increased to about 3.5% by further increasing the dose. When the detection dosage is increased to 60 muL, the AHL sample solution is easy to flow away from the surface of the working electrode during dripping, and the adsorption is insufficient, so that the peak current detection value is lower than the true value, and the difference is about 15%.
3) Optimizing the detection voltage:
referring to the test method of example 2, the voltage range was expanded to-0.6V-0.6V, and the other conditions were not changed to obtain the test results, and a standard curve was prepared. As a result, it was found that: within the voltage range of-0.6V to-0.2V and 0.4V to 0.6V, the DPV curve of the electrode has more mixed peaks and small data significance, within the range of-0.2V to 0.4V, the DPV curve of the electrode is smooth, the reading of the peak current value is accurate, and the detection time is saved. Therefore, we select the detection voltage range to be-0.2V-0.4V.
(4) The number of recycling times:
referring to the test method in example 2, the electrode used in the test after adsorption is eluted, and then the electrode is continuously used as an electrode to test the object to be tested, and the result shows that the electrode of the present invention has good stability and can be recycled for 5 times, wherein the data difference of the first 4 times does not exceed 5%, the accuracy of the detection data is high, and the data difference of the 5 th time is 10%, and the decrease starts to occur. Therefore, the electrode overcomes the defect of one-time use, can be recycled for multiple times and has better data accuracy.
Comparative example 1:
referring to example 1, an AHL molecularly imprinted screen-printed electrode was prepared by replacing the dispensing method in step (1) with the following plating method without changing other conditions.
The electroplating method comprises the following steps: gold nanoparticles are electroplated and deposited on the surface of the working screen printing electrode, and the electroplating method comprises the following steps: the working electrode is placed in the gold nanometer solution, and the electroplating time is 120s under the potential of-0.2V voltage, so as to complete the electroplating;
when a sample test was performed using the AHL molecularly imprinted screen-printed electrode obtained by the above method with reference to example 2, it was found that: the difference of the obtained electrode data is 5%, but when the electrode data is recycled for the 2 nd time, the data difference is increased to 12%, the accuracy is poor, and the electrode data cannot be used for accurately detecting the AHL. Thus, the electrode is a single use electrode.
Comparative example 2:
referring to example 2, electrochemical measurements were carried out using the electrode of example 1, and only the ordinate used for the standard curve construction was replaced by the peak current difference value after adsorption, and the measurement data was read, and as a result, it was found that: the peak current value after adsorption is taken as a curve of a vertical coordinate, and the peak current value after adsorption and the concentration of the measured sample have no good linear relation, so that the peak current value after adsorption is difficult to be used for detection. And the relation between the concentration of the measured sample and the electric signal can be better reflected by a curve taking the peak current difference value as a vertical coordinate, and the correlation coefficient can reach 0.94799 after fitting. Therefore, different molecularly imprinted electrodes are matched with the respective suitable data models, different molecularly imprinted electrodes cannot directly share the same prediction model, and specific research and analysis are required for the imprinted electrode with specific molecular characteristics.
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 in the appended claims.
Claims (7)
1. A method for detecting N-acyl homoserine lactone, comprising the steps of:
(1) preparing an N-acyl homoserine lactone molecularly imprinted screen printing electrode: dripping gold nano cross on the surface of a working electrode of the screen printing electrode, modifying p-mercaptoaniline through gold mercapto bond, then soaking in template molecular solution, placing in electropolymerization liquid for electroplating polymerization, eluting template molecules, and electroplating a molecular imprinting polymer film on the surface of the working electrode to obtain a molecular imprinting screen printing electrode;
(2) dripping 25-50 mu L of electroconducting liquid on the surface of the N-acyl homoserine lactone molecularly imprinted screen printing electrode, horizontally inserting the N-acyl homoserine lactone molecularly imprinted screen printing electrode into an electrochemical detector for detection, and measuring the current value before adsorption;
(3) dropwise adding 25-50 mu L of an electric conduction liquid containing a sample to be detected to the surface of the N-acyl homoserine lactone molecularly imprinted screen printing electrode for standing adsorption, horizontally inserting the N-acyl homoserine lactone molecularly imprinted screen printing electrode into an electrochemical detector for detection, and measuring the current value after adsorption;
(4) calculating to obtain a peak current difference value according to the steps (2) and (3), and calculating to obtain the concentration of the N-acyl homoserine lactone in the sample to be detected according to a standard curve of the concentration of the substance to be detected in the sample to be detected and the peak current difference value;
the adsorption time in the step (3) is 180 s;
the voltage setting range of the electrochemical detector is-0.2V-0.4V;
the template molecule is furanone.
2. The method of claim 1, wherein the standard curve is obtained by: preparing N-acyl homoserine lactone electroconducting liquid and pure electroconducting liquid with different known concentrations, dropwise adding the electroconducting liquid and the pure electroconducting liquid to the surface of an N-acyl homoserine lactone molecularly imprinted screen printing electrode, inserting the N-acyl homoserine lactone molecularly imprinted screen printing electrode into an electrochemical detector, detecting a current peak value formed when electrons flow from a counter electrode, pass through the electroconducting liquid and flow back to a workstation from a working electrode, obtaining peak current difference values under different N-acyl homoserine lactone concentrations, and obtaining a standard curve by taking the N-acyl homoserine internal concentration as a horizontal coordinate and the peak current difference value as a vertical coordinate.
3. The method as claimed in claim 2, wherein the concentration of N-acylhomoserine in the electroconducting fluid is in the range of 1X 10-13～1×10-9mol•L-1。
4. A portable N-acyl homoserine lactone molecularly imprinted screen printing electrochemical detector is characterized in that the detector is applied to the detection method according to any one of claims 1 to 3, and comprises an externally-inserted N-acyl homoserine lactone molecularly imprinted screen printing electrode and an electrochemical detector, wherein the operation mode of the detector is that 25-50 muL of electroconducting liquid containing a sample to be detected is dripped on the surface of the N-acyl homoserine lactone molecularly imprinted screen printing electrode, standing and adsorption is carried out for 180s, then the electrode is inserted into a clamping groove of the electrochemical detector, and a peak current value is detected and read to obtain a peak current difference value; the preparation method of the N-acyl homoserine lactone molecularly imprinted screen printing electrode comprises the following steps: and (2) dripping gold nano crosses on the surface of a working electrode of the screen printing electrode, modifying p-mercaptoaniline through gold mercapto bonds, then soaking the working electrode into a template molecule solution, placing the working electrode into an electropolymerization solution for electroplating polymerization, and eluting template molecules to ensure that the surface of the working electrode is electroplated with a molecularly imprinted polymer membrane, thus obtaining the N-acyl homoserine lactone molecularly imprinted screen printing electrode.
5. The meter of claim 4, wherein the electrochemical detector has a voltage setting range of: -0.2V to 0.4V.
6. The detector of claim 4, wherein the electrochemical detector comprises a simplified electrochemical workstation equipped with an LED display screen, and the standard curve of the concentration of N-acyl homoserine lactone and the current peak value difference is built in the portable electrochemical detector program, and the concentration of the target substance in the sample to be detected can be displayed on the LED display screen.
7. Use of the assay according to any one of claims 1 to 3 or the detector according to any one of claims 4 to 6 for bacterial quorum sensing.
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