CN110794016B - Preparation method and application of immunosensor based on nickel-cobalt MOFs loaded molybdenum disulfide doped palladium nanoparticles - Google Patents
Preparation method and application of immunosensor based on nickel-cobalt MOFs loaded molybdenum disulfide doped palladium nanoparticles Download PDFInfo
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Abstract
The invention relates to preparation and application of an electrochemical immunosensor based on nickel-cobalt MOFs loaded molybdenum disulfide doped palladium nanoparticles, and belongs to the technical field of nano materials and electrochemical analysis. The electrochemical immunosensor prepared by adopting the modified nickel-cobalt MOFs doped palladium nanoparticles is used for sensitive detection of procalcitonin.
Description
Technical Field
The invention relates to preparation and application of an electrochemical immunosensor based on nickel-cobalt MOFs loaded molybdenum disulfide doped palladium nanoparticles, and belongs to the field of nano materials and electrochemical analysis.
Background
Procalcitonin (PCT) is a protein secreted by thyroid C cells, and the concentration of PCT in humans reflects the activity of the systemic inflammatory response, a sensitive biomarker of inflammation. PCT levels are elevated when there is severe infection, fungal, parasitic infection, as well as sepsis and multi-organ failure in the organism. The PCT content in the serum also has a guiding effect on the diagnosis of the sepsis, and the PCT higher than 2 ng/mL indicates that the patient is infected with the sepsis. Therefore, accurate detection of the content of PCT in serum is important for early diagnosis of sepsis, and has important significance for further guidance of therapy, and realization of efficient and accurate detection of procalcitonin is a problem to be solved at present.
The electrochemical immunosensor has received wide attention due to the advantages of simple operation, high sensitivity, strong specificity and the like, can directly monitor the combination process of antigen-antibody reaction, avoids the interference caused by the contact of a coupling marker and harmful substances, and is widely applied to the tumor marker detection in clinical examination at present.
The invention prepares a functionalized nickel-cobalt MOFs electrochemical immunosensor, and particularly adopts nickel-cobalt MOFs loaded molybdenum disulfide doped palladium nanoparticles as a sensor platform to construct a label-free electrochemical immunosensor which is used for detecting carcinoembryonic antigens. The nickel-cobalt MOFs have a uniform cubic shape and provide a carrier for the attachment of molybdenum disulfide. The molybdenum disulfide is of a layered structure and presents a triangular prism shape, and the edge surface of the molybdenum disulfide can also be used as a catalytic active center. Studies have shown that molybdenum disulfide has catalytically active sites with high surface energy on the edge planes. Molybdenum disulfide is considered a potentially desirable alternative to platinum group catalysts due to its high degree of electrocatalytic activity and binding energy with atomic hydrogen. The molybdenum disulfide related derivative with a large specific surface area can load a more active probe and an active domain to combine with biomolecules, and has a remarkable effect on amplifying electrochemical signals. In addition, the molybdenum disulfide can be effectively combined with other nano materials, and shows excellent compatibility. In the experiment, the nickel-cobalt MOFs-loaded molybdenum disulfide is synthesized by a hydrothermal synthesis method, so that the contact specific surface area is increased, more active sites can be provided, and the catalytic activity is increased. Secondly, palladium nanoparticles are doped on the surface of the nickel-cobalt MOFs loaded molybdenum disulfide, the palladium nanoparticles have strong adsorption capacity, particularly, the palladium nanoparticles are in closer contact with the surface of an electrode than other nanoparticles in shapes, the electron transfer capacity between an electrolyte and the surface of the electrode is greatly improved, and meanwhile, the loading capacity of an antibody can be greatly improved by a large number of palladium nanoparticles. The nickel-cobalt MOFs loaded molybdenum disulfide doped palladium nanoparticles prepared by the invention have the following advantages: the cationic surfactant cetyl trimethyl ammonium bromide is introduced to ensure that the molybdenum disulfide nanospheres have good dispersibility and anion adsorption capacity, more chloropalladate can be adsorbed on the surfaces of the molybdenum disulfide nanospheres, and the palladium nanoparticles can be uniformly distributed on the surfaces of the molybdenum disulfide nanospheres under the reduction action of ascorbic acid. The nickel-cobalt MOF loads the synergistic effect between the molybdenum disulfide and the palladium nanoparticles, so that the synthesized material has more excellent catalytic performance.
Disclosure of Invention
One purpose of the invention is to provide a method for preparing nickel-cobalt MOFs loaded molybdenum disulfide based on a hydrothermal method;
the second purpose of the invention is to prepare the nickel-cobalt MOFs loaded molybdenum disulfide doped palladium nanoparticles, wherein the nickel-cobalt MOFs loaded molybdenum disulfide is modified by utilizing hexadecyl trimethyl ammonium bromide, and then the added palladium chloride acid is reduced into the palladium nanoparticles under the action of ascorbic acid. According to the method, palladium nanoparticles are uniformly loaded on the surface of the molybdenum disulfide loaded with the nickel-cobalt MOF, so that the catalytic capability of the molybdenum disulfide on the hydrogen peroxide solution is improved;
the invention also aims to use the immunosensor prepared based on nickel-cobalt MOFs loaded molybdenum disulfide doped palladium nanoparticles for high-sensitivity and specificity detection of carcinoembryonic antigen. The palladium nano particles can be directly connected with the procalcitonin antibody, so that the experimental steps are simplified, and the detection of procalcitonin is widened.
The technical scheme of the invention is as follows:
1. preparation method of electrochemical immunosensor constructed based on nickel-cobalt MOFs loaded molybdenum disulfide doped palladium nanoparticles
(1) Preparation of nickel-cobalt MOFs
1) Dissolving 150-240 mg of nickel nitrate and 260-340 mg of sodium citrate in 20-80 mL of water, and recording the solution as solution A;
2) dissolving 120-200 mg of potassium cobalt cyanide in 40-100 mL of water, and recording the solution as a solution B;
3) mixing and stirring the solution A and the solution B for 1 min, then placing the mixture in the shade for aging for 18 h, finally washing the solution with distilled water and absolute ethyl alcohol for multiple times, and drying the product at 70 ℃ for 12 h;
(2) preparation of nickel-cobalt MOFs loaded molybdenum disulfide
1) Dissolving 30-90 mg of nickel-cobalt MOFs and 10-60 mg of ammonium tetrathiomolybdate in 30 mL of N, N-dimethylformamide solution, and carrying out ultrasonic treatment for 15 min;
2) continuously transferring the solution into a 50 mL high-pressure reaction kettle, and reacting for 20 h at 210 ℃;
3) after the reaction is finished, cooling the solution to room temperature, washing the solution with ultrapure water and absolute ethyl alcohol for three times respectively, and drying the collected product at the temperature of 70 ℃ for 12 hours;
(3) preparation of nickel-cobalt MOFs loaded molybdenum disulfide doped palladium nanoparticles
1) 2-12 mg of the synthesized nickel-cobalt MOFs loaded molybdenum disulfide is dissolved in an aqueous solution containing 0.1-0.8 g of hexadecyl trimethyl ammonium bromide, and ultrasonic treatment is carried out for 20 min;
2) centrifuging the solution, dispersing the solution in 10 mL of distilled water again, adding 10-60 mL of chloropalladate solution, and carrying out ultrasonic treatment for 10 min;
3) continuously and rapidly adding 1-8 mL of ascorbic acid solution into the solution, oscillating for 30 min, transferring the solution to a dark place, standing and aging for 6 h;
4) after the reaction is finished, centrifuging the solution, washing the solution with ultrapure water and absolute ethyl alcohol for three times respectively, and drying the collected product at room temperature for 12 hours;
(4) preparation of electrochemical immunosensor
1) Polishing the glassy carbon electrode to a mirror surface by using 0.05 mm, 0.3 mm and 1.0 mm of aluminum oxide powder in sequence, and cleaning by using ultrapure water;
2) dripping 6.0 mu L of 0.5-2.5 mg/mL of nickel-cobalt MOFs loaded molybdenum disulfide doped palladium nanoparticle composite solution on the surface of an electrode, and storing in a refrigerator at 4 ℃ until the solution is dried;
3) continuously dropwise adding 6 mu L of procalcitonin antibody of 6-12 mu g/mL to modify the surface of the electrode, and storing in a refrigerator of 4 ℃ until the mixture is dried;
4) continuously dropwise adding 3 mu L of bovine serum albumin solution with the mass fraction of 0.5% -2%, sealing nonspecific active sites on the surface of the electrode, washing the surface of the electrode with phosphate buffer solution with the pH =7.38, and storing in a refrigerator at 4 ℃ until the electrode is dried;
5) respectively dropwise adding 6 mu L of procalcitonin with different concentrations to modify the surfaces of the electrodes, incubating for 1 h at the temperature of 4 ℃, washing the surfaces of the electrodes by using a phosphate buffer solution with the pH =7.38, and storing in a refrigerator at the temperature of 4 ℃ until the surfaces are dried.
2. The method comprises the steps of synthesizing nickel-cobalt MOFs with uniform appearance through simple reaction at room temperature, then loading molybdenum disulfide on the surface of the nickel-cobalt MOFs through hydrothermal reaction, finally modifying the surface of the nickel-cobalt MOFs through a cationic surfactant, and enabling palladium nanoparticles to be uniformly distributed on the molybdenum disulfide loaded on the nickel-cobalt MOFs through in-situ reduction at room temperature.
3. The electrochemical immunosensor based on nickel-cobalt MOFs loaded molybdenum disulfide doped palladium nanoparticles is applied to detection of procalcitonin.
4. The electrochemical immunosensor is applied to detecting procalcitonin, and the detection steps are as follows:
(1) the test in the experimental process adopts a three-electrode system, wherein a saturated calomel electrode is used as a reference electrode, a platinum wire electrode is used as an auxiliary electrode, the constructed sensor is a working electrode, and the whole experiment is carried out in 10 mL of phosphate buffer solution with the pH = 6.24-8.04 of 50 mmol/L;
(2) detecting the analyte by a time-lapse current method, wherein the input voltage is-0.4V, and the running time is 400 s;
(3) when the background current tends to be stable, injecting 10 mu L of hydrogen peroxide solution with the concentration of 5 mol/L into 10 mL of phosphate buffer solution with the pH = 6.24-8.04 of 50 mmol/L every 90 s, and recording the current change;
(4) the method comprises the steps of replacing a procalcitonin standard solution with a procalcitonin sample solution to be detected for detection, and using the electrochemical immunosensor based on nickel-cobalt MOFs loaded molybdenum disulfide doped palladium nanoparticles for procalcitonin detection, wherein the detection range is 0.01-50.0 ng/mL, and the detection limit is 2.13 pg/mL.
Advantageous results of the invention
(1) According to the method, nickel-cobalt MOFs is synthesized at room temperature, molybdenum disulfide is loaded on the surface of the nickel-cobalt MOFs under a hydrothermal condition, and palladium nanoparticles are fixed through the modification effect of cetyl trimethyl ammonium bromide and the reduction effect of ascorbic acid;
(2) according to the invention, nickel-cobalt MOFs loaded molybdenum disulfide doped palladium nanoparticles are used as a substrate, and the nickel-cobalt MOFs has a cubic shape and provides a supporting template for loading molybdenum disulfide. Molybdenum disulfide has a large specific surface area and has a certain catalytic activity on hydrogen peroxide. The palladium nano particles have excellent conductivity and good biocompatibility, the loading capacity of the antibody is greatly improved, and the sensitivity of the immunosensor is enhanced by the synergistic effect of the materials;
(3) the invention discloses an electrochemical immunosensor of nickel-cobalt MOFs loaded molybdenum disulfide doped palladium nanoparticles, which is used for detecting procalcitonin. The whole experimental process has simple operation steps, high current response speed and wide signal response range, is linear in the range of 0.01-50.0 ng/mL, has the detection limit of 2.13 pg/mL, and realizes simple, quick, high-sensitivity and specific detection of procalcitonin.
Detailed description of the preferred embodiments
Example 1 preparation method of nickel-cobalt MOFs loaded molybdenum disulfide doped palladium nanoparticles
(1) Preparation of nickel-cobalt MOFs
1) 150 mg of nickel nitrate and 260 mg of sodium citrate were dissolved in 20 mL of water and recorded as solution A;
2) 120 mg of potassium cobalt cyanide was dissolved in 50 mL of water and designated as solution B;
3) mixing and stirring the solution A and the solution B for 1 min, then placing the mixture in the shade for aging for 18 h, finally washing the solution with distilled water and absolute ethyl alcohol for multiple times, and drying the product at 70 ℃ for 12 h;
(2) preparation of nickel-cobalt MOFs loaded molybdenum disulfide
1) Dissolving 40 mg of nickel-cobalt MOFs and 20 mg of ammonium tetrathiomolybdate in 30 mL of N, N-dimethylformamide solution, and carrying out ultrasonic treatment for 15 min;
2) continuously transferring the solution into a 50 mL high-pressure reaction kettle, and reacting for 20 h at 210 ℃;
3) after the reaction is finished, cooling the solution to room temperature, washing the solution with ultrapure water and absolute ethyl alcohol for three times respectively, and drying the collected product at the temperature of 70 ℃ for 12 hours;
(3) preparation of nickel-cobalt MOFs loaded molybdenum disulfide doped palladium nanoparticles
1) 2 mg of the synthesized nickel-cobalt MOFs loaded molybdenum disulfide is dissolved in an aqueous solution containing 0.2 g of hexadecyl trimethyl ammonium bromide, and ultrasonic treatment is carried out for 20 min;
2) centrifuging the solution, dispersing the solution in 10 mL of distilled water again, adding 20 mL of chloropalladate solution, and carrying out ultrasonic treatment for 10 min;
3) continuously and rapidly adding 2 mL of ascorbic acid solution into the solution, oscillating for 30 min, transferring the solution to a dark place, standing and aging for 6 h;
4) after the reaction was completed, the solution was centrifuged and washed three times with ultrapure water and anhydrous ethanol, respectively, and the collected product was dried at room temperature for 12 hours.
Embodiment 2 preparation method of nickel-cobalt MOFs loaded molybdenum disulfide doped palladium nanoparticles
(1) Preparation of nickel-cobalt MOFs
1) 170 mg of nickel nitrate and 280 mg of sodium citrate were dissolved in 40 mL of water and recorded as solution A;
2) 150 mg of potassium cobalt cyanide was dissolved in 60 mL of water and designated as solution B;
3) mixing and stirring the solution A and the solution B for 1 min, then placing the mixture in the shade for aging for 18 h, finally washing the solution with distilled water and absolute ethyl alcohol for multiple times, and drying the product at 70 ℃ for 12 h;
(2) preparation of nickel-cobalt MOFs loaded molybdenum disulfide
1) Dissolving 50 mg of nickel-cobalt MOFs and 30 mg of ammonium tetrathiomolybdate in 30 mL of N, N-dimethylformamide solution, and carrying out ultrasonic treatment for 15 min;
2) continuously transferring the solution into a 50 mL high-pressure reaction kettle, and reacting for 20 h at 210 ℃;
3) after the reaction is finished, cooling the solution to room temperature, washing the solution with ultrapure water and absolute ethyl alcohol for three times respectively, and drying the collected product at the temperature of 70 ℃ for 12 hours;
(3) preparation of nickel-cobalt MOFs loaded molybdenum disulfide doped palladium nanoparticles
1) 4 mg of the synthesized nickel-cobalt MOFs loaded molybdenum disulfide is dissolved in an aqueous solution containing 0.4 g of hexadecyl trimethyl ammonium bromide, and ultrasonic treatment is carried out for 20 min;
2) centrifuging the solution, dispersing the solution in 10 mL of distilled water again, adding 30 mL of chloropalladate solution, and carrying out ultrasonic treatment for 10 min;
3) continuously and rapidly adding 2 mL of ascorbic acid solution into the solution, oscillating for 30 min, transferring the solution to a dark place, standing and aging for 6 h;
4) after the reaction was completed, the solution was centrifuged and washed three times with ultrapure water and anhydrous ethanol, respectively, and the collected product was dried at room temperature for 12 hours.
Embodiment 3 preparation method of nickel-cobalt MOFs loaded molybdenum disulfide doped palladium nanoparticles
(1) Preparation of nickel-cobalt MOFs
1) Dissolving 220 mg of nickel nitrate and 340 mg of sodium citrate in 80 mL of water, and recording the solution as solution A;
2) dissolve 200 mg potassium cobalt cyanide in 90 mL water, record as solution B;
3) mixing and stirring the solution A and the solution B for 1 min, then placing the mixture in the shade for aging for 18 h, finally washing the solution with distilled water and absolute ethyl alcohol for multiple times, and drying the product at 70 ℃ for 12 h;
(2) preparation of nickel-cobalt MOFs loaded molybdenum disulfide
1) Dissolving 80 mg of nickel-cobalt MOFs and 50 mg of ammonium tetrathiomolybdate in 30 mL of N, N-dimethylformamide solution, and carrying out ultrasonic treatment for 15 min;
2) continuously transferring the solution into a 50 mL high-pressure reaction kettle, and reacting for 20 h at 210 ℃;
3) after the reaction is finished, cooling the solution to room temperature, washing the solution with ultrapure water and absolute ethyl alcohol for three times respectively, and drying the collected product at the temperature of 70 ℃ for 12 hours;
(3) preparation of nickel-cobalt MOFs loaded molybdenum disulfide doped palladium nanoparticles
1) Dissolving 10 mg of the synthesized nickel-cobalt MOFs loaded molybdenum disulfide in an aqueous solution containing 0.7 g of hexadecyl trimethyl ammonium bromide, and carrying out ultrasonic treatment for 20 min;
2) centrifuging the solution, dispersing the solution in 10 mL of distilled water again, adding 60 mL of chloropalladate solution, and carrying out ultrasonic treatment for 10 min;
3) continuously and rapidly adding 8 mL of ascorbic acid solution into the solution, oscillating for 30 min, transferring the solution to a dark place, standing and aging for 6 h;
4) after the reaction was completed, the solution was centrifuged and washed three times with ultrapure water and anhydrous ethanol, respectively, and the collected product was dried at room temperature for 12 hours.
Example 4
1. Preparation of immunosensor based on functionalized nickel-cobalt MOFs
(1) Polishing the glassy carbon electrode to a mirror surface by using 0.05 mm, 0.3 mm and 1.0 mm of aluminum oxide powder in sequence, and cleaning by using ultrapure water;
(2) dripping 6.0 mu L of 0.5 mg/mL nickel-cobalt MOFs loaded molybdenum disulfide doped palladium nanoparticle composite solution on the surface of an electrode, and storing in a refrigerator at 4 ℃ until the solution is dried;
(3) continuously dropwise adding 6 mu L of procalcitonin antibody of 6 mu g/mL to modify the surface of the electrode, and storing in a refrigerator of 4 ℃ until the mixture is dried;
(4) continuously dropwise adding 3 mu L of bovine serum albumin solution with the mass fraction of 0.5%, sealing non-specific active sites on the surface of the electrode, washing the surface of the electrode by phosphate buffer solution with the pH =7.38, and storing in a refrigerator of 4 ℃ until the electrode is dried;
(5) respectively dropwise adding 6 mu L of procalcitonin with different concentrations to modify the surfaces of the electrodes, incubating for 1 h at the temperature of 4 ℃, washing the surfaces of the electrodes by using a phosphate buffer solution with the pH =7.38, and storing in a refrigerator at the temperature of 4 ℃ until the electrodes are dried;
2. the application of the electrochemical immunosensor for detecting carcinoembryonic antigen comprises the following detection steps:
(1) the test in the experimental process adopts a three-electrode system, wherein a saturated calomel electrode is used as a reference electrode, a platinum wire electrode is used as an auxiliary electrode, the constructed sensor is a working electrode, and the whole experiment is carried out in 10 mL of 50 mmol/L phosphate buffer solution with pH = 6.24;
(2) detecting the analyte by a time-lapse current method, wherein the input voltage is-0.4V, and the running time is 400 s;
(3) when the background current tended to stabilize, 10 μ L of 5 mol/L hydrogen peroxide solution was injected into 10 mL of 50 mmol/L phosphate buffer solution at intervals of 90 s, pH =6.24, and the change in current was recorded.
Example 5
1. Preparation of immunosensor based on functionalized nickel-cobalt MOFs
(1) Polishing the glassy carbon electrode to a mirror surface by using 0.05 mm, 0.3 mm and 1.0 mm of aluminum oxide powder in sequence, and cleaning by using ultrapure water;
(2) dripping 6.0 mu L of 1.5 mg/mL nickel-cobalt MOFs loaded molybdenum disulfide doped palladium nanoparticle composite solution on the surface of an electrode, and storing in a refrigerator at 4 ℃ until the solution is dried;
(3) continuously dropwise adding 6 mu L of procalcitonin antibody of 10 mu g/mL to modify the surface of the electrode, and storing in a refrigerator of 4 ℃ until the mixture is dried;
(4) continuously dropwise adding 3 mu L of bovine serum albumin solution with the mass fraction of 1.5%, sealing non-specific active sites on the surface of the electrode, washing the surface of the electrode by phosphate buffer solution with the pH =7.38, and storing in a refrigerator of 4 ℃ until the electrode is dried;
(5) respectively dropwise adding 6 mu L of procalcitonin with different concentrations to modify the surfaces of the electrodes, incubating for 1 h at the temperature of 4 ℃, washing the surfaces of the electrodes by using a phosphate buffer solution with the pH =7.38, and storing in a refrigerator at the temperature of 4 ℃ until the electrodes are dried;
2. the electrochemical immunosensor is applied to detecting procalcitonin, and the detection steps are as follows:
(1) the test in the experimental process adopts a three-electrode system, wherein a saturated calomel electrode is used as a reference electrode, a platinum wire electrode is used as an auxiliary electrode, the constructed sensor is a working electrode, and the whole experiment is carried out in 10 mL of phosphate buffer solution with pH =7.38 of 50 mmol/L;
(2) detecting the analyte by a time-lapse current method, wherein the input voltage is-0.4V, and the running time is 400 s;
(3) when the background current tended to stabilize, 10 μ L of 5 mol/L hydrogen peroxide solution was injected into 10 mL of 50 mmol/L phosphate buffer solution at 90 s intervals, and the change in current was recorded.
Example 6
1. Preparation of immunosensor based on functionalized nickel-cobalt MOFs
(1) Polishing the glassy carbon electrode to a mirror surface by using 0.05 mm, 0.3 mm and 1.0 mm of aluminum oxide powder in sequence, and cleaning by using ultrapure water;
(2) dripping 6.0 mu L of 2.5 mg/mL nickel-cobalt MOFs loaded molybdenum disulfide doped palladium nanoparticle composite solution on the surface of an electrode, and storing in a refrigerator at 4 ℃ until the solution is dried;
(3) continuously dropwise adding 6 mu L of procalcitonin antibody of 12 mu g/mL to modify the surface of the electrode, and storing in a refrigerator of 4 ℃ until the mixture is dried;
(4) continuously dropwise adding 3 mu L of bovine serum albumin solution with the mass fraction of 2%, sealing non-specific active sites on the surface of the electrode, washing the surface of the electrode by using phosphate buffer solution with the pH =7.38, and storing in a refrigerator at 4 ℃ until the surface is dried;
(5) respectively dropwise adding 6 mu L of procalcitonin with different concentrations to modify the surfaces of the electrodes, incubating for 1 h at the temperature of 4 ℃, washing the surfaces of the electrodes by using a phosphate buffer solution with the pH =7.38, and storing in a refrigerator at the temperature of 4 ℃ until the electrodes are dried;
2. the electrochemical immunosensor is applied to detecting procalcitonin, and the detection steps are as follows:
(1) the test in the experimental process adopts a three-electrode system, wherein a saturated calomel electrode is used as a reference electrode, a platinum wire electrode is used as an auxiliary electrode, the constructed sensor is a working electrode, and the whole experiment is carried out in 10 mL of phosphate buffer solution with the pH =8.04 of 50 mmol/L;
(2) detecting the analyte by a time-lapse current method, wherein the input voltage is-0.4V, and the running time is 400 s;
(3) when the background current tended to stabilize, 10 μ L of 5 mol/L hydrogen peroxide solution was injected into 10 mL of 50 mmol/L phosphate buffer solution at 90 s intervals, and the change in current was recorded.
Example 7
The methods described in examples 3 and 6 detect procalcitonin, exhibit linearity in the range of 0.01-50.0 ng/mL, and have a detection limit of 2.13 pg/mL, thereby realizing simple, rapid, highly sensitive and specific detection of procalcitonin.
Claims (3)
1. A preparation method of an electrochemical immunosensor based on nickel-cobalt MOFs loaded molybdenum disulfide doped palladium nanoparticles is characterized by comprising the following preparation steps:
preparation of nickel cobalt MOF
1) Dissolving 150-240 mg of nickel nitrate and 260-340 mg of sodium citrate in 20-80 mL of water, and recording the solution as solution A;
2) dissolving 120-200 mg of potassium cobalt cyanide in 40-100 mL of water, and recording the solution as a solution B;
3) mixing and stirring the solution A and the solution B for 1 min, then placing the solution in the shade for aging for 18 h, finally washing the solution with distilled water and absolute ethyl alcohol for multiple times, and drying the product for 12 h at 70 ℃;
preparation of nickel-cobalt MOFs loaded molybdenum disulfide
1) Dissolving 30-90 mg of nickel-cobalt MOFs and 10-60 mg of ammonium tetrathiomolybdate in 30 mL of N, N-dimethylformamide solution, and carrying out ultrasonic treatment for 15 min;
2) continuously transferring the solution into a 50 mL high-pressure reaction kettle, and reacting for 20 h at 210 ℃;
3) after the reaction is finished, cooling the solution to room temperature, washing the solution with ultrapure water and absolute ethyl alcohol for three times respectively, and drying the collected product for 12 hours at the temperature of 70 ℃;
preparation of nickel-cobalt MOFs loaded molybdenum disulfide doped palladium nanoparticles
1) Dissolving 2-12 mg of synthesized nickel-cobalt MOFs loaded molybdenum disulfide in an aqueous solution containing 0.1-0.8 g of hexadecyl trimethyl ammonium bromide, and carrying out ultrasonic treatment for 20 min;
2) centrifuging the solution, dispersing the solution in 10 mL of distilled water again, adding 10-60 mL of chloropalladate solution, and carrying out ultrasonic treatment for 10 min;
3) continuously and rapidly adding 1-8 mL of ascorbic acid solution into the solution, oscillating for 30 min, transferring the solution to a dark place, standing and aging for 6 h;
4) after the reaction is finished, centrifuging the solution, washing the solution with ultrapure water and absolute ethyl alcohol for three times respectively, and drying the collected product at room temperature for 12 hours;
preparation of electrochemical immunosensor
1) Polishing the glassy carbon electrode to a mirror surface by using 0.05 mm, 0.3 mm and 1.0 mm of aluminum oxide powder in sequence, and cleaning by using ultrapure water;
2) dripping 6.0 mu L of 0.5-2.5 mg/mL of nickel-cobalt MOFs loaded molybdenum disulfide doped palladium nanoparticle solution on the surface of an electrode, and storing in a refrigerator at 4 ℃ until the solution is dried;
3) continuously dropwise adding 6 mu L of procalcitonin antibody of 6-12 mu g/mL to modify the surface of the electrode, and storing in a refrigerator of 4 ℃ until the mixture is dried;
4) continuously dropwise adding 3 mu L of bovine serum albumin solution with the mass fraction of 0.5% -2%, sealing nonspecific active sites on the surface of the electrode, washing the surface of the electrode with phosphate buffer solution with the pH =7.38, and storing in a refrigerator at 4 ℃ until the electrode is dried;
5) respectively dropwise adding 6 mu L of procalcitonin with different concentrations to modify the surfaces of the electrodes, incubating at the temperature of 4 ℃ for 1 h, washing the surfaces of the electrodes by using a phosphate buffer solution with the pH =7.38, and storing in a refrigerator of 4 ℃ until the surfaces are dried.
2. The application of the electrochemical immunosensor prepared by the preparation method according to claim 1, wherein the electrochemical immunosensor is applied to detection of procalcitonin.
3. Use of an electrochemical immunosensor according to claim 2, wherein the procalcitonin is detected by the following steps:
(1) the test in the experimental process adopts a three-electrode system, wherein a saturated calomel electrode is used as a reference electrode, a platinum wire electrode is used as an auxiliary electrode, the constructed sensor is a working electrode, and the whole experiment is carried out in 10 mL of phosphate buffer solution with the pH = 6.24-8.04 of 50 mmol/L;
(2) detecting the analyte by a time-lapse current method, wherein the input voltage is-0.4V, and the running time is 400 s;
(3) when the background current tends to be stable, 10 mu L of hydrogen peroxide solution with the concentration of 5 mol/L is injected into 10 mL of phosphate buffer solution with the pH = 6.24-8.04 of 50 mmol/L every 90 s, and procalcitonin standard solutions with different concentrations are used as recognition antigens to record current change;
(4) and replacing the procalcitonin sample solution to be detected with the procalcitonin standard solution for detection.
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