CN112345607A - Triazophos biosensor based on two-dimensional nano material - Google Patents
Triazophos biosensor based on two-dimensional nano material Download PDFInfo
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Abstract
Triazophos, a substitute for methamidophos, is a broad-spectrum organic pesticide widely used for controlling pests. However, triazophos has strong stomach toxicity and contact toxicity, and its residue has important influence on environment and human health. According to the invention, the acetylcholinesterase is fixed on the electrode by adopting a two-dimensional nano material co-modification method, so that the detection sensitivity is improved, and the quantitative detection of the acetylcholinesterase inhibitor triazophos is realized.
Description
Technical Field
The invention belongs to the field of organophosphorus detection, and particularly relates to a triazophos biosensor based on a two-dimensional nano material.
Background
Triazophos, a substitute for methamidophos, is a broad-spectrum organic pesticide widely used for controlling pests. However, triazophos has strong stomach toxicity and contact toxicity, and its residue has important influence on environment and human health. The traditional method comprises the steps of analyzing and testing the organophosphorus pesticide triazophos by gas chromatography and high performance liquid chromatography, has higher sensitivity and accuracy, but takes longer time and needs large-scale equipment. To improve detection efficiency, various methods have been developed including chemiluminescence, fluorescence, and electrochemistry.
Triazophos has an inhibitory effect on acetylcholinesterase, and the biosensor based on acetylcholinesterase has the advantages of quick response, high sensitivity and simple instrument. By immobilizing acetylcholinesterase on the electrode surface, the acetylcholinesterase catalyzes the substrate and generates an electrochemical signal. However, at present, reports on how to firmly modify acetylcholinesterase on electrodes are few, and reports on how to modify acetylcholinesterase by two-dimensional nano materials such as graphite-phase carbon nitride and the like are not yet found.
Disclosure of Invention
Aiming at the defects, the acetylcholinesterase is fixed on the electrode in a mode of co-modifying the graphite-phase carbon nitride two-dimensional nano material, so that quantitative detection of the acetylcholinesterase inhibitor triazophos is realized, and the method is simple, convenient and sensitive.
The technical scheme of the invention is as follows:
a triazophos biosensor based on a two-dimensional nanomaterial is prepared by the following steps:
1) preparing a graphite phase carbon nitride solution: mixing graphite-phase carbon nitride with water to prepare a suspension, centrifuging to remove large particles, and taking a supernatant;
2) preparing Tris-HCl buffer solution: preparing a Tris-HCl buffer solution containing bovine serum albumin;
3) preparation of acetylcholinesterase solution: dissolving acetylcholinesterase in the Tris-HCl buffer solution obtained in the step (2) to obtain an acetylcholinesterase solution;
4) preparing a sensor: and (3) dropwise adding the graphite-phase carbon nitride solution obtained in the step (1) onto the surface of the polished glassy carbon electrode, naturally airing, immersing into a cysteine solution, then washing the electrode with double distilled water, dropwise adding the acetylcholine solution obtained in the step (3), and washing the electrode with a phosphoric acid buffer solution after airing to obtain the triazophos biosensor.
Further, in the triazophos biosensor based on the two-dimensional nanomaterial, the concentration of the suspension prepared by mixing the graphite-phase carbon nitride and water in the step 1 is 50-150 mg/ml.
Further, in the triazophos biosensor based on the two-dimensional nanomaterial, the concentration of the Tris-HCl buffer solution containing bovine serum albumin in the step 2 is that each L of the buffer solution contains 0.1-0.3g of bovine serum albumin.
Further, in the triazophos biosensor based on the two-dimensional nanomaterial described above, the concentration of acetylcholinesterase in the acetylcholinesterase solution in step 3 is in the range of 1-6U/ml.
Further, in the triazophos biosensor based on the two-dimensional nanomaterial, the volume of the graphite-phase carbon nitride solution is equal to that of the acetylcholine solution in step 4, and the volume of the graphite-phase carbon nitride solution is 5-15 ul.
Further, the triazophos biosensor based on the two-dimensional nanomaterial is prepared by the following specific steps:
1) preparing a graphite phase carbon nitride solution: mixing graphite-phase carbon nitride with water to prepare 50-150mg/L suspension, centrifuging to remove large particles, and taking supernatant;
2) preparing Tris-HCl buffer solution: preparing a Tris-HCl buffer solution containing bovine serum albumin; each L of buffer solution contains 0.1-0.3g of bovine serum albumin;
3) preparation of acetylcholinesterase solution: dissolving acetylcholinesterase in the Tris-HCl buffer solution obtained in the step (2) to obtain an acetylcholinesterase solution with the concentration range of 2-6U/ml;
4) preparing a sensor: dropwise adding the graphite-phase carbon nitride solution obtained in the step 1 to the surface of the polished glassy carbon electrode, naturally airing, immersing in a cysteine solution, then washing the electrode with double distilled water, dropwise adding the acetylcholine solution obtained in the step 3, and washing the electrode with a phosphoric acid buffer solution after airing to obtain the triazophos biosensor; the volume of the graphite phase carbon nitride solution is equal to that of the acetylcholine solution, and the volume of the graphite phase carbon nitride solution is 5-15 ul.
Further, the triazophos biosensor based on the two-dimensional nanomaterial is prepared by the following preferred steps:
1) preparing a graphite phase carbon nitride solution: mixing graphite-phase carbon nitride with water to prepare 100mg/ml of floating liquid, centrifuging to remove large particles, and taking supernatant;
2) preparing Tris-HCl buffer solution: preparing a Tris-HCl buffer solution containing bovine serum albumin; each L of the buffer solution contains 0.2g of bovine serum albumin and 50mM of Tris-HCl;
3) preparation of acetylcholinesterase solution: dissolving acetylcholinesterase in the Tris-HCl buffer solution obtained in the step (2) to obtain an acetylcholinesterase solution with the concentration of 4U/ml;
4) preparing a sensor: dropwise adding the graphite-phase carbon nitride solution obtained in the step 1 to the surface of the polished glassy carbon electrode, naturally airing, immersing in a cysteine solution, then washing the electrode with double distilled water, dropwise adding the acetylcholine solution obtained in the step 3, and washing the electrode with a phosphoric acid buffer solution after airing to obtain the triazophos biosensor; the volume of the graphite-phase carbon nitride solution is equal to that of the acetylcholine solution, and the volume of the graphite-phase carbon nitride solution is 10 ul.
Further, the measuring method of the triazophos biosensor based on the two-dimensional nanomaterial comprises the following steps:
5) preparing 0.01-0.03M phosphate buffer solution with pH 7-8, and dissolving acetylthiocholine chloride with the solution to make its final concentration be 0.1-0.4 mM;
6) and (5) preparing the solution in the step (5) to prepare phosphate standard solutions of the triazophos with different concentrations for detecting electrochemical signals, drawing a standard curve through the change of peak current between 0.6 and 0.7V, and contrasting the standard curve according to corresponding electric signals of the sample to be detected to obtain the concentration of the triazophos in the sample to be detected.
Further, the measurement method of the triazophos biosensor based on the two-dimensional nanomaterial comprises the following preferred steps:
5) preparing 0.02M phosphate buffer solution with pH of 7.4, and dissolving acetylthiocholine chloride with the solution to make the final concentration of the acetylthiocholine chloride be 0.2 mM;
6) and (5) preparing the solution in the step (5) to prepare phosphate standard solutions of the triazophos with different concentrations for detecting electrochemical signals, drawing a standard curve through the change of about 0.65V peak current, and contrasting the standard curve according to corresponding electric signals of the sample to be detected to obtain the concentration of the triazophos in the sample to be detected.
Further, in the measuring method of the triazophos biosensor based on the two-dimensional nanomaterial, the detection limit of the triazophos is 0.07 uM.
Further, in the measuring method of the triazophos biosensor based on the two-dimensional nanomaterial, in the step 6, the concentration of the triazophos in the phosphate standard solutions with different concentrations of the triazophos is 0.05, 0.1, 0.25, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, and 4.0 uM, respectively.
Furthermore, the triazophos biosensor based on the two-dimensional nanomaterial and the measurement method thereof are suitable for being widely applied to the fields of environmental protection, agriculture, biology and the like.
The invention has the beneficial effects that:
(1) the triazophos biosensor based on the two-dimensional nano material utilizes the two-dimensional nano material graphite phase carbon nitride to firmly modify acetylcholinesterase on the glassy carbon electrode, and has the advantages of simple preparation method, mild preparation conditions and low cost.
(2) The detection method of the triazophos biosensor based on the two-dimensional nanomaterial is simple, has high corresponding speed, is convenient and quick to process data, does not need large-scale equipment, and has high sensitivity.
(3) The triazophos biosensor based on the two-dimensional nanomaterial can be used for quantitatively detecting triazophos, has the detection limit as low as 0.07uM, has good application and popularization values, and can be widely used for rapidly detecting the residues of the triazophos in the fields of environmental protection, agriculture, biology and the like.
Drawings
FIG. 1 is a Fourier infrared spectrum of graphite-phase carbon nitride obtained by preparation;
FIG. 2 is a graph of the electrochemical response of a sensor according to the present invention;
FIG. 3 is a graph showing the response of the sensor of the present invention to different concentrations of triazophos.
Detailed Description
The technical solution of the present invention is further explained by the following embodiments. It should be understood by those skilled in the art that the examples are only for the understanding of the present invention and should not be construed as the specific limitations of the present invention.
The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.
Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
Example 1
(1): according to the reference literature (catalytic science, 2015, 36(12): 2089-2094), graphite phase carbon nitride is obtained by a high-temperature nitriding thermal polymerization method, and a Fourier infrared spectrum of the prepared graphite phase carbon nitride is shown in figure 1; weighing the graphite phase carbon nitride, adding a certain amount of water, performing ultrasonic treatment to obtain a suspension with the final concentration of the graphite phase carbon nitride of 100mg/mL, and then performing centrifugal treatment to remove large granular substances.
(2): weighing 121.1g of Tris (hydroxymethyl) aminomethane (Tris), and carrying out constant volume to 1L to obtain 1M Tris-HCl stock solution. 50mL of 1M Tris-HCl solution was taken, 0.2g of Bovine Serum Albumin (BSA) was added, 800mL of double distilled water was added, pH was adjusted to 8.0, and a volume of 1L was determined to obtain a 50mM Tris-HCl buffer solution (pH 8.0) containing 0.2g of (BSA).
(3): 2 mg of acetylcholinesterase (500U/mg) was weighed, dissolved in the above 50mM Tris-HCl buffer solution and made up to 250mL to give a 4U/mL acetylcholine solution.
(4): and (3) dropwise adding 10 mu L of the graphite-phase carbon nitride solution on the surface of the polished glassy carbon electrode, naturally airing, immersing in a cysteine solution, washing the electrode with double distilled water, dropwise adding 10 mu L of 4U/mL acetylcholine solution, washing the electrode with a phosphoric acid buffer solution with the pH value of 7.0 after airing, and then performing an electrochemical response test of the electrode, wherein the response is linear as shown in FIG. 2.
(5): A0.02M phosphate buffer solution having a pH of 7.4 was prepared, and acetylthiocholine chloride was dissolved in this solution to give a final concentration of 0.2 mM.
(6): taking 10mL of the solution, preparing the solution with different concentrationsThe phosphate standard solution of triazophos (the concentration of triazophos in the phosphate standard solutions of triazophos with different concentrations is 0.05, 0.1, 0.25, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0 uM respectively), 10ml is taken for detecting electrochemical signals, a standard curve is drawn by the change of peak current about 0.65V, as shown in FIG. 3, the response of acetylcholinesterase to triazophos with different concentrations is linearly related in the concentration range of 0.2-3 μ M, and R is2=0.998, good linearity, detection limit of 0.07 μ M; and detecting the sample to be detected, and obtaining the concentration of the triazophos in the sample according to the corresponding electric signal.
Example 2
(1): according to the literature (catalytic science 2015, 36(12): 2089-2094), graphite-phase carbon nitride is obtained by a high-temperature nitridation thermal polymerization method; weighing the graphite phase carbon nitride, adding a certain amount of water, performing ultrasonic treatment to obtain a suspension with the final concentration of the graphite phase carbon nitride of 50mg/mL, and then performing centrifugal treatment to remove large granular substances.
(2): weighing 121.1g of Tris (hydroxymethyl) aminomethane (Tris), and carrying out constant volume to 1L to obtain 1M Tris-HCl stock solution. 50mL of 1M Tris-HCl solution was taken, 0.1g of Bovine Serum Albumin (BSA) was added, 800mL of double distilled water was added, pH was adjusted to 8.0, and a volume of 1L was determined to obtain a 50mM Tris-HCl buffer solution (pH 8.0) containing 0.1g of (BSA).
(3): 0.5mg of acetylcholinesterase (500U/mg) was weighed, dissolved in the above 50mM Tris-HCl buffer solution and made up to 250mL to give a 1U/mL acetylcholine solution.
(4): and (3) dropwise adding 5 mu L of the graphite-phase carbon nitride solution on the surface of the polished glassy carbon electrode, naturally airing, immersing in a cysteine solution, washing the electrode with double distilled water, dropwise adding 5 mu L of 1U/mL acetylcholine solution, washing the electrode with a phosphoric acid buffer solution with the pH value of 7.0 after airing, and then carrying out an electrochemical response test on the electrode, wherein the response is linear.
(5): A0.01M phosphate buffer solution having a pH of 7 was prepared, and acetylthiocholine chloride was dissolved in this solution to give a final concentration of 0.1 mM.
(6): taking 10mL of the solution, preparing phosphate standard solutions of the triazophos with different concentrations, taking 10mL of the solution for detecting electrochemical signals, drawing a standard curve through the change of 0.6V peak current, then detecting a sample to be detected, and obtaining the concentration of the triazophos in the sample according to corresponding electric signals.
Example 3
(1): the graphite phase carbon nitride is obtained by a high-temperature nitriding thermal polymerization method in reference documents (catalytic science 2015, 36(12): 2089-2094); weighing the graphite phase carbon nitride, adding a certain amount of water, performing ultrasonic treatment to obtain a suspension with the final concentration of the graphite phase carbon nitride of 150mg/mL, and then performing centrifugal treatment to remove large granular substances.
(2): weighing 121.1g of Tris (hydroxymethyl) aminomethane (Tris), and carrying out constant volume to 1L to obtain 1M Tris-HCl stock solution. 50mL of 1M Tris-HCl solution was taken, 0.3g of Bovine Serum Albumin (BSA) was added, 800mL of double distilled water was added, pH was adjusted to 8.0, and a volume of 1L was determined to obtain a 50mM Tris-HCl buffer solution (pH 8.0) containing 0.3g of (BSA).
(3): 3 mg of acetylcholinesterase (500U/mg) was weighed, dissolved in the above 50mM Tris-HCl buffer solution and made up to 250mL to give a 6U/mL acetylcholine solution.
(4): and (3) dropwise adding 15 mu L of the graphite-phase carbon nitride solution on the surface of the polished glassy carbon electrode, naturally airing, immersing in a cysteine solution, washing the electrode with double distilled water, dropwise adding 15 mu L of 6U/mL acetylcholine solution, washing the electrode with a phosphoric acid buffer solution with the pH value of 7.0 after airing, and then carrying out an electrochemical response test on the electrode.
(5): A0.03M phosphate buffer solution having a pH of 8 was prepared, and acetylthiocholine chloride was dissolved in this solution to give a final concentration of 0.4 mM.
(6): taking 10mL of the solution, preparing phosphate standard solutions of the triazophos with different concentrations (the concentration of the triazophos in the phosphate standard solutions of the triazophos with different concentrations is respectively 0.05, 0.1, 0.25, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5 and 4.0 uM), taking 10mL of the solution for detecting electrochemical signals, drawing a standard curve through the change of 0.7V peak current, detecting a sample to be detected, and obtaining the concentration of the triazophos in the sample according to corresponding electric signals.
According to the experimental data, the acetylcholinesterase is fixed on the electrode by adopting a two-dimensional nano material co-modification method, so that the detection sensitivity is improved, and the quantitative detection of the acetylcholinesterase inhibitor triazophos is realized.
Claims (10)
1. A triazophos biosensor based on a two-dimensional nanomaterial is characterized by being prepared by the following steps:
1) preparing a graphite phase carbon nitride solution: mixing graphite-phase carbon nitride with water to prepare a suspension, centrifuging to remove large particles, and taking a supernatant;
2) preparing Tris-HCl buffer solution: preparing a Tris-HCl buffer solution containing bovine serum albumin;
3) preparation of acetylcholinesterase solution: dissolving acetylcholinesterase in the Tris-HCl buffer solution obtained in the step (2) to obtain an acetylcholinesterase solution;
4) preparing a sensor: and (3) dropwise adding the graphite-phase carbon nitride solution obtained in the step (1) onto the surface of the polished glassy carbon electrode, naturally airing, immersing into a cysteine solution, then washing the electrode with double distilled water, dropwise adding the acetylcholine solution obtained in the step (3), and washing the electrode with a phosphoric acid buffer solution after airing to obtain the triazophos biosensor.
2. The triazophos biosensor based on two-dimensional nanomaterial of claim 1, wherein; in the step 1, the concentration of the suspension prepared by mixing the graphite-phase carbon nitride with water is 50-150 mg/ml.
3. The biosensor of claim 1, wherein the concentration of the Tris-HCl buffer solution containing bovine serum albumin in step 2 is 0.1-0.3g of bovine serum albumin per L of buffer solution.
4. The two-dimensional nanomaterial-based triazophos biosensor according to claim 1, wherein the concentration of acetylcholinesterase in the acetylcholinesterase solution in step 3 is in the range of 1-6U/ml.
5. The two-dimensional nanomaterial-based triazophos biosensor in accordance with claim 1, wherein the volume of the graphite phase carbon nitride solution and the volume of the acetylcholine solution in step 4 are equal, each being 5-15 ul.
6. The triazophos biosensor based on two-dimensional nanomaterial of claim 1, prepared by the following steps:
1) preparing a graphite phase carbon nitride solution: mixing graphite-phase carbon nitride with water to prepare 50-150mg/L suspension, centrifuging to remove large particles, and taking supernatant;
2) preparing Tris-HCl buffer solution: preparing a Tris-HCl buffer solution containing bovine serum albumin; each L of buffer solution contains 0.1-0.3g of bovine serum albumin;
3) preparation of acetylcholinesterase solution: dissolving acetylcholinesterase in the Tris-HCl buffer solution obtained in the step (2) to obtain an acetylcholinesterase solution with the concentration range of 2-6U/ml;
4) preparing a sensor: dropwise adding the graphite-phase carbon nitride solution obtained in the step 1 to the surface of the polished glassy carbon electrode, naturally airing, immersing in a cysteine solution, then washing the electrode with double distilled water, dropwise adding the acetylcholine solution obtained in the step 3, and washing the electrode with a phosphoric acid buffer solution after airing to obtain the triazophos biosensor; the volume of the graphite phase carbon nitride solution is equal to that of the acetylcholine solution, and the volume of the graphite phase carbon nitride solution is 5-15 ul.
7. The measuring method of the triazophos biosensor based on two-dimensional nanometer material as claimed in claim 6, comprising the following steps:
5) preparing 0.01-0.03M phosphate buffer solution with pH 7-8, and dissolving acetylthiocholine chloride with the solution to make its final concentration be 0.1-0.4 mM;
6) and (5) preparing the solution in the step (5) to prepare phosphate standard solutions of the triazophos with different concentrations for detecting electrochemical signals, drawing a standard curve through the change of peak current between 0.6 and 0.7V, and contrasting the standard curve according to corresponding electric signals of the sample to be detected to obtain the concentration of the triazophos in the sample to be detected.
8. The measurement method of the triazophos biosensor based on two-dimensional nanometer material as claimed in claim 7, wherein the limit of detection of triazophos is 0.07 uM.
9. The method for measuring the triazophos biosensor based on two-dimensional nanomaterial according to claim 7, wherein in step 6, the concentration of triazophos in the standard solution of phosphate with different concentration of triazophos is 0.05, 0.1, 0.25, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0 uM.
10. Use of the two-dimensional nanomaterial-based triazophos biosensor of any one of claims 1-6 in pesticide residue detection.
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