Preparation and detection method of a three-dimensional photosensitive electrode for detecting phospholipids in crude oil
By applying a photoelectrochemical biosensor prepared by a three-dimensional photosensitive electrode in crude oil, the problem that existing detection methods are difficult to quickly and accurately monitor the residual phosphorus in crude oil is solved, and a high sensitivity, rapid and accurate detection effect is achieved.
Patent Information
- Application Number
- CN202011494723.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2020-12-17
- Publication Date
- 2025-05-06
- Estimated Expiration
- 2040-12-17
AI Technical Summary
The existing detection methods are difficult to quickly and accurately monitor the residual phosphorus in crude oil, resulting in the oil refining process lag behind the production process.
A photoelectrochemical biosensor prepared using three-dimensional photosensitive electrodes was synthesized by hydrothermal method, and thionithium was deposited on its surface, combining choline oxidase and glutaraldehyde to form a high-sensitivity detection system.
It realizes rapid and accurate detection of phospholipids in crude oil, with high selectivity, stability and repeatability, a wide linear range and ultra-low detection limit, which significantly improves detection efficiency and accuracy.
Smart Images
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Figure HDA0002841794210000012
Abstract
Description
Technical Field
[0001] The invention relates to a preparation method of a three-dimensional photosensitive electrode, in particular to a detection method for phospholipids in crude oil. Background Art
[0002] my country is the world's largest producer and consumer of edible vegetable oils. The quality and safety of oils and fats are related to the health of the people. With the implementation of the "China Good Grain and Oil Action Plan" strategy, people's requirements for the quality of vegetable oils and fats are getting higher and higher.
[0003] In the oil refining and degumming process, not only chemical refining involves the inspection and control of residual phosphorus in crude oil, but also the evaluation of residual phosphorus in crude oil during bioenzymatic refining is the main parameter that determines the progress of bioenzymatic hydrolysis reaction, which can effectively prevent excessive enzymatic hydrolysis of oil. The residual phosphorus in oil affects the quality and processing cost of oil. The existing detection methods generally have the problem of slow analysis speed and lag behind the production process. Therefore, it is necessary to quickly detect and monitor the residual phosphorus in crude oil to achieve accurate and appropriate processing of oil.
[0004] In recent years, electrochemical analysis has gradually attracted attention in the field of vegetable oil quality detection, and has a good development space, but there are still problems such as low sensitivity and poor selectivity that restrict its application in practice. Photoelectrochemical biosensor is a new detection technology developed by combining photoelectrochemical analysis and biosensing. It has lower background noise, higher sensitivity and lower detection limit than traditional electrochemical methods. Enzyme-modified electrodes can further enhance the special biological affinity between reaction molecules and simplify the detection process. The construction of three-dimensional enzyme electrodes can break through the bottleneck problem of low photoelectric conversion efficiency, making practical application possible. Summary of the invention
[0005] The purpose of the present invention is to provide a photoelectrochemical biosensor and a detection method for detecting phospholipids in crude oil in view of the deficiencies of the prior art. The photoelectrochemical biosensor can conveniently and quickly measure phospholipids in crude oil, and the PC content of the photoelectrochemical biosensor is linearly related to the photocurrent in the range of 5 to 25 mg / L. The detection limit of the method is 2 mg / L (S / N=3). Compared with the detection value of the HPLC method, the linear correlation coefficient is high. It has satisfactory selectivity, stability and repeatability, a wide linear range and an ultra-low detection limit; it makes up for the defects of the traditional detection method that is cumbersome to operate and expensive, which shows its great prospects in practical applications. The technical solution adopted by the present invention to solve the above-mentioned technical problems is: a preparation and detection method of a three-dimensional photosensitive electrode for detecting phospholipids in crude oil, comprising the following steps:
[0006] (1) Preparation of three-dimensional SnO2 nanoarrays
[0007] The indium tin oxide (ITO) conductive glass was ultrasonically cleaned and dried. The three-dimensional SnO2 nanoarray was synthesized by hydrothermal method. After the reaction was completed, the autoclave was naturally cooled to room temperature. Then the deposited substrate was repeatedly rinsed with deionized water and then dried in air. Figure 1 This is a characterization diagram of tin dioxide nanomaterials.
[0008] (2) Preparation of polythiophene photosensitive electrode
[0009] The treated ITO electrode was placed in a thionine (Th) electrolytic cell for electrodeposition. After deposition at a potential of 1.2 V for 2400 s, the Th monomer adsorbed on the surface was washed off with water, and then washed with PBS at pH 5.5. The obtained electrode was the prepared polythionine (PTh) photosensitive electrode.
[0010] (3) Preparation of photoelectrochemical biosensors
[0011] Chitosan was dissolved in 1% acetic acid solution and stirred at room temperature for one hour until completely dissolved to obtain a transparent chitosan colloid. Choline oxidase (CHOx) was dissolved in Tris-Hcl at pH 8.0, stirred and dissolved until it became a transparent solution, and stored in a refrigerator at 4°C. 5% glutaraldehyde was diluted to 0.25% and stored for later use. Use a microsyringe to transfer the chitosan solution and apply it dropwise on the surface of the polythiocyanine photosensitive electrode, and dry it naturally at room temperature to form a film, wash it with PBS, and dry it. Take glutaraldehyde and drop it on the electrode surface to react for 30 minutes. After washing with PBS, add 5-10μL CHOx enzyme solution, react at room temperature for 1 hour, and dry it to obtain the prepared three-dimensional photosensitive electrode.
[0012] Figure 2 This is a characterization diagram of choline oxidase;
[0013] DETAILED DESCRIPTION Specific implementation method one:
[0015] The three-dimensional SnO2 nanoarray of the present invention is prepared by the following method: ITO conductive glass is cut into strips of 5.0×1.0 cm, ultrasonically cleaned with ethanol, acetone and water for 5 minutes respectively, and dried. Then, a 1.0 cm section is reserved at one end as an electrode terminal, and the other section is sealed with insulating paint and a blank with a diameter of 6.0 mm is reserved on the surface as an ITO electrode. The three-dimensional SnO2 nanoarray is synthesized by a hydrothermal method. 0.03 mol·L -1 SnCl4·5H2O and 0.3 mol L -1 NaOH is used to make 30-40 ml of precursor solution, and 5-10 ml of NaOH with a concentration of 0.12 mol·L is added. -1NaCl and 0.6-0.8g polyvinyl pyrrolidone (PVP). The precursor solution was stirred for 1h, and then the ITO glass substrate was immersed in the precursor solution in a sealed polytetrafluoroethylene-lined autoclave and hydrothermally grown at 200°C for 12h. After the reaction was completed, the autoclave was naturally cooled to room temperature. The deposited substrate was then repeatedly rinsed with deionized water and then dried in air. Specific implementation method 2:
[0017] Preparation of polythionine photoelectrode: 3.9 mg of polythionine was weighed and dissolved in 3 mL of 4.4 mol·L sodium hydroxide solution with a pH of 1.9. -1 The thionine concentration prepared in acetic acid solution is 4mmol·L -1 The ITO electrode treated in the first embodiment was placed in a solution containing 4 mmol·L -1 After electro-deposition in a thionine electrolytic cell at 1.2 V for 2400 s, the Th monomer adsorbed on the surface was washed off with water, and then washed with PBS at pH 5.5. The obtained electrode is the prepared polythionine photosensitive electrode. Specific implementation method three:
[0019] Preparation of photoelectrochemical biosensor: Accurately weigh 10.0 mg chitosan and dissolve it in 1% acetic acid solution. Stir at room temperature for one hour until it is completely dissolved to obtain a transparent chitosan colloid with a concentration of 10.0 mg mL -1 . Dilute 5% glutaraldehyde to 0.25% and store for later use. Use a microsyringe to transfer 5-10 μL of chitosan solution and apply it on the surface of the polysulfide pine photosensitive electrode, and let it dry naturally at room temperature to form a film, then wash with PBS and dry. Take 5-10 μL of 0.25% glutaraldehyde and drop it on the electrode surface to react for 30 minutes. After washing with PBS, add 5-10 μL of 0-1.7 g·L -1 Choline oxidase was reacted at room temperature for 1 hour and dried to obtain the prepared three-dimensional photosensitive electrode. Specific implementation method four:
[0021] The difference between this embodiment and the third embodiment is that the amount of choline oxidase added is 0 to 1.7 g·L -1 The other steps of preparing the photoelectrochemical biosensor under these conditions are the same as those in the third specific implementation method.
[0022] Specific implementation method five: Photoelectrochemical detection of PC content: Photoelectrochemical detection is performed using a homemade photoelectrochemical system. All photoelectrochemical experiments are performed on the chi660b electrochemical workstation. PEC detection uses a classic three-electrode system. The three-dimensional photosensitive electrode is used as the working electrode, the Ag / AgCl electrode is used as the reference electrode, and the platinum wire electrode is used as the counter electrode. All electrochemical tests are performed in a room temperature photoelectrochemical reaction cell. 30mL of a certain concentration of pH 5.5-7.5 phosphate buffer is used as the electrolyte, a 50W iodine tungsten lamp is used as the irradiation source, and the light intensity is 6mW·cm -2 ~15mW·cm -2 , apply a bias voltage of 0.10V to 0.50V to the photoelectric interface. Inject PC into the electrolytic cell and start current detection at the same time. When the catalytic reaction lasts for 8 minutes, open the light gate, switch once every 20 seconds, form an instantaneous photocurrent-time spectrum, and obtain a linear response in the concentration range of 5 to 25 mg / L.
[0023] Specific implementation example 6: The difference between this implementation example and specific implementation example 5 is that the applied bias voltage range is 0.10V to 0.50V. Under this condition, the other steps of photoelectrochemical detection are the same as those of specific implementation example 5.
[0024] Specific embodiment 7: The difference between this embodiment and specific embodiment 5 is that the light intensity is 6mW·cm -2 ~15mW·cm -2 Within this range, the other steps of photoelectrochemical detection under this condition are the same as those in the fifth specific implementation method.
[0025] Specific embodiment eight: The difference between this embodiment and specific embodiment five is that the pH of the PC buffer solution is in the range of 5.5 to 7.5. The other steps of photoelectrochemical detection under this condition are the same as those of specific embodiment five.
[0026] Specific implementation method 9: Determination of PC content in crude soybean oil: 40-60 mg PC is added to a container containing 90-110 mL of first-grade soybean oil, and stirred thoroughly to prepare crude soybean oil. Then, the sample is diluted into PBS buffer of different concentrations. A crude soybean oil sample with a certain PC content is prepared, and after adding 90-110 μL of 3 mg / mL phospholipase D solution, 1% Triton-X100 is added as an emulsifier, and stirred at 37°C for 30 minutes. Under the optimal conditions, a three-dimensional photosensitive electrode is added to 10-30 mL of crude soybean oil to measure its photocurrent to obtain the PC content of the sample.
Claims
1. A preparation and detection method of a three-dimensional photosensitive electrode for detecting phospholipids in crude oil, characterized in that The preparation of the three-dimensional photosensitive electrode and the detection method of phospholipids in crude oil are achieved by the following steps: Step 1: Cut the ITO conductive glass into 5.0×1.0 cm strips, clean them with ethanol, acetone and water for 5 min, and dry them. Then, leave a 1.0 cm section at one end as the electrode terminal, seal the other end with insulating paint and leave a blank with a diameter of 6.0 mm on the surface as the ITO electrode. Synthesize the three-dimensional SnO2 nanoarray by hydrothermal method. - 1 SnCl4·5H2O and 0.3 mol L -1 NaOH is used to make a 30-40 ml precursor solution, and 5-10 ml of a 0.12 mol·L - 1 NaCl and 0.6-0.8g polyvinyl pyrrolidone (PVP); the precursor solution was stirred for 1h, and then the ITO glass substrate was immersed in the precursor solution in a sealed polytetrafluoroethylene-lined autoclave, and hydrothermal growth was carried out at 200°C for 12h; after the reaction was completed, the autoclave was naturally cooled to room temperature; then the deposited substrate was repeatedly rinsed with deionized water and then dried in air; Step 2: Weigh 3.9 mg of thionine and dissolve it in 3 mL of 4.4 mol·L sodium hydroxide solution with a pH of 1.
9. -1 The thionine concentration prepared in acetic acid solution is 4mmol·L -1 ; Place the treated ITO electrode in a solution containing 4mmol·L -1 Electrodeposition was performed in a thionine electrolytic cell at 1.2 V for 2400 s, and then the Th monomer adsorbed on the surface was washed off with water, and then washed with PBS at pH 5.5; the obtained electrode was the prepared polythionine photosensitive electrode; Step 3: Accurately weigh 10.0 mg chitosan and dissolve it in 1% acetic acid solution. Stir at room temperature for one hour until it is completely dissolved to obtain a transparent chitosan colloid with a concentration of 10.0 mg mL -1 ; 5% glutaraldehyde was diluted to 0.25% and stored for future use; 5-10 μL chitosan solution was transferred with a microsyringe and applied to the surface of the polysulfide pine photosensitive electrode, and dried naturally at room temperature to form a film, washed with PBS, and dried; 5-10 μL 0.25% glutaraldehyde was dropped on the electrode surface for 30 minutes, washed with PBS, and then 5-10 μL of 0-1.7 g·L -1 Choline oxidase was reacted at room temperature for 1 h and dried to obtain a prepared three-dimensional photosensitive electrode; Step 4: Photoelectric detection was performed using a self-made photoelectrochemical system; all photoelectrochemical experiments were performed on a chi660b electrochemical workstation; PEC detection used a classic three-electrode system; a three-dimensional photosensitive electrode was used as the working electrode, an Ag / AgCl electrode was used as the reference electrode, and a platinum wire electrode was used as the counter electrode; all electrochemical tests were performed in a room temperature photoelectrochemical reaction cell; 20-40 mL, 10 mmol / L pH 5.5-7.5 phosphate buffer was used as the electrolyte, a 50 W iodine tungsten lamp was used as the irradiation source, and the light intensity was 6 mW cm -2 ~15mW·cm -2 , apply a bias voltage of 0.10V to 0.50V to the photoelectric interface; inject PC into the electrolytic cell and start current detection at the same time, open the light gate when the catalytic reaction lasts for 8 minutes, switch once every 20 seconds, form an instantaneous photocurrent-time spectrum, and obtain a linear response in the concentration range of 5 to 25 mg / L; Step 5: Add 40-60 mg PC into a container containing 90-110 mL of first-grade soybean oil and stir thoroughly to prepare crude soybean oil; then dilute the sample into PBS buffer of different concentrations; prepare a crude soybean oil sample with a certain PC content, add 90-110 μL of 3 mg / mL phospholipase D solution, add 1% Triton-X100 as an emulsifier, stir at 37°C for 30 minutes, and under optimal conditions, add the three-dimensional photosensitive electrode into 10-30 mL of crude soybean oil to measure its photocurrent to obtain the PC content of the sample.
2. The preparation and detection method of a three-dimensional photosensitive electrode for detecting phospholipids in crude oil according to claim 1, characterized in that: The concentration of choline oxidase in step 3 is 0-1.7 g·L -1 .
3. The preparation and detection method of a three-dimensional photosensitive electrode for detecting phospholipids in crude oil according to claim 1, characterized in that: The bias voltage applied in step 4 is 0.10V to 0.50V.
4. The preparation and detection method of a three-dimensional photosensitive electrode for detecting phospholipids in crude oil according to claim 1, characterized in that: The illumination intensity in step 4 is 6 mW·cm -2 ~15mW·cm -2 .
5. The preparation and detection method of a three-dimensional photosensitive electrode for detecting phospholipids in crude oil according to claim 1, characterized in that: The pH of the PC buffer solution in step 4 is 5.5-7.5.
Citation Information
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