Molecularly imprinted sensing electrode for detecting cortisol and preparation method thereof

Abstract

The invention provides a molecularly imprinted sensing electrode for detecting cortisol and a preparation method of the molecularly imprinted sensing electrode. The molecularly imprinted sensing electrode for detecting cortisol comprises a screen-printed electrode, and MXene / CNF / Ag NWs aerogel, a chitosan protective layer and a molecularly imprinted polymer film which are sequentially loaded on the surface of the screen-printed electrode. The MXene / CNF / Ag NWs aerogel enables the surface of the electrode to obtain a porous structure, a high specific surface area and high conductivity, so that the performance of the sensing electrode is more excellent. The cortisol sensing electrode is prepared by electropolymerization through an electrochemical method, so that sensitive detection of cortisol with different concentrations can be realized, and the application value of the sensing electrode in the field of continuous detection is shown.

Description

Technical Field

[0001] The invention belongs to the field of biosensor detection, and in particular relates to a molecular imprinting sensing electrode for detecting cortisol and a preparation method thereof. Background Art

[0002] Cortisol is the most common glucocorticoid in clinical practice. Due to its anti-inflammatory, antitoxin and immunosuppressive effects, it is widely used to treat severe skin allergies and inflammation in humans and animals. In addition to the external intake mentioned above, cortisol can also be obtained through the body's secretion. Cortisol plays a vital role in emotional responses such as stress: it can keep blood pressure stable and prevent excessive inflammation under stressful conditions, so it is considered a stress biomarker. However, if people are under long-term stress or consume excessive cortisol, their cortisol levels will be high for a long time. The harm of cortisol to the human body will become obvious, manifested as osteoporosis, weight gain, muscle weakness, growth inhibition in children, and aggravation of diabetes. Therefore, regular monitoring of cortisol levels in the human body can effectively prevent the occurrence of diseases.

[0003] At present, the analysis of cortisol mainly relies on high performance liquid chromatography, liquid chromatography-tandem mass spectrometry and enzyme-linked immunosorbent assay. Although these traditional technologies have achieved good sensitivity and accuracy, they require expensive equipment, a large amount of organic solvents, a long experimental time and professional technicians, which limits their application in actual field tests. Electrochemical sensors have the advantages of short response time, high cost-effectiveness, easy operation, miniaturization and excellent sensitivity. Some functional materials are usually modified on the functional interface of the sensing electrode as molecular recognition elements to achieve high selectivity. Compared with other molecular recognition elements, MIP is widely used in many fields due to its low price and stable chemical and mechanical properties. However, most MIPs are made of non-conductive or poorly conductive materials, which greatly hinders the transfer of electrons and reduces the detection sensitivity. In order to effectively improve the sensitivity of MIP electrochemical sensing electrodes, a variety of new conductive materials such as aerogels have been used to construct composite electrochemical sensing electrodes.

[0004] So far, researchers at home and abroad have explored various MIP sensors for cortisol detection, but the detection of different concentrations of cortisol is still not satisfactory. Therefore, it is necessary to develop sensors with larger specific surface area and better performance to meet practical needs. Summary of the invention

[0005] In view of this, the present invention aims to propose a molecular imprinting sensing electrode for detecting cortisol and a preparation method thereof, so as to solve the problems of poor electrode sensitivity, narrow detection range and low electrical signal response in the prior art.

[0006] To achieve the above object, the technical solution of the present invention is achieved as follows: A first aspect of the present invention provides a molecular imprinting sensing electrode for detecting cortisol, comprising a screen-printed electrode, and a MXene / CNF / Ag NWs aerogel, a chitosan protective layer and a molecular imprinting polymer film sequentially loaded on the surface of the screen-printed electrode.

[0007] The second aspect of the present invention provides a method for preparing a molecular imprinting sensing electrode for detecting cortisol according to the first aspect of the present invention, comprising the following steps: (1) Using MXene / CNF / Ag NWs aerogel to modify the screen-printed electrode to obtain MXene / CNF / Ag NWs aerogel / screen-printed electrode; (2) Modifying the MXene / CNF / Ag NWs aerogel / screen-printed electrode with chitosan solution to obtain a MXene / CNF / Ag NWs aerogel / chitosan / screen-printed electrode; (3) Using a polymerization solution, an electrochemical polymerization reaction is carried out on the surface of the MXene / CNF / Ag NWs aerogel / chitosan / screen-printed electrode by cyclic voltammetry, and the cortisol molecules are eluted in an elution solution to leave a cortisol molecular cavity, and finally a cortisol molecular imprinting sensing electrode is obtained.

[0008] Furthermore, the specific preparation method of step (1) is: (1) Add MXene / CNF / Ag NWs aerogel into ethanol and stir to obtain a dispersion; (2) Take 8-12 μL of the dispersion obtained in step (1), drop it onto the screen-printed electrode, and dry it in an oven at 40-50 °C to obtain MXene / CNF / Ag NWs aerogel / screen-printed electrode.

[0009] Preferably, the amount of the dispersion is 10 μL.

[0010] Furthermore, the preparation method of the MXene / CNF / Ag NWs aerogel comprises the following steps: MXene and CNF are added to Ag NWs to obtain a MXene / CNF / Ag NWs dispersion, the MXene / CNF / AgNWs dispersion is filled into a vial, the vial is fixed on a copper plate, the bottom of the copper plate is immersed in liquid nitrogen and frozen, and then placed in a vacuum freeze dryer to obtain a MXene / CNF / Ag NWs aerogel.

[0011] Preferably, the mass ratio of the Mxene, CNF and Ag NWs is 2:1:1.

[0012] Furthermore, the specific preparation method of step (2) is: drop-coating the chitosan solution onto the MXene / CNF / Ag NWs aerogel / screen-printed electrode obtained in step (1), and drying in an oven at 45° C. to obtain a MXene / CNF / AgNWs aerogel / chitosan / screen-printed electrode.

[0013] Furthermore, the preparation method of the chitosan solution is: prepare a 0.05M hydrochloric acid solution, place chitosan powder in the hydrochloric acid solution, and prepare a 4 mg / mL chitosan solution.

[0014] Furthermore, the polymerization solution in step (3) includes 1-2 mM o-phenylenediamine, 0.1-0.2 mM cortisol, and 0.1 M (PH=5) acetate buffer solution.

[0015] Furthermore, the specific preparation method of step (3) is: fixing the electrode after the electrochemical polymerization reaction in the eluent, placing the eluent in a beaker, pouring 40-70 ml of anhydrous ethanol into the beaker for magnetic stirring, and removing the cortisol molecules in the polymer on the electrode surface.

[0016] Furthermore, the parameters of the electrochemical polymerization reaction are: scanning speed 25 to 100 mV / s, potential range -1 to 1 V, and cycle number 20 to 40 times.

[0017] Furthermore, the eluent is anhydrous ethanol.

[0018] Compared with the prior art, the molecular imprinting sensing electrode for detecting cortisol and the preparation method thereof described in the present invention have the following advantages: (1) The present invention uses MXene / CNF / Ag NWs aerogel to modify the screen-printed electrode, so that the modified electrode not only has a large specific surface area, high conductivity, and abundant element groups, but also the porous structure of the aerogel is conducive to the attachment of imprinted molecules during the electropolymerization process.

[0019] (2) Through comprehensive comparison of test data, the present invention shows that when the MXene / CNF / Ag NWs aerogel is selected as 2:1:1, the electrical signal response is the largest and the sensing performance is the best. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] The accompanying drawings constituting a part of the present invention are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention. In the accompanying drawings: Figure 1 This is a SEM image of the aerogel prepared by MXene / CNF / Ag NWs at a ratio of 4:2:1 in Example 1 of the present invention; Figure 2 This is a SEM image of the aerogel prepared by MXene / CNF / Ag NWs at a ratio of 2:1:1 in Example 1 of the present invention; Figure 3 This is a SEM image of the aerogel prepared by MXene / CNF / Ag NWs at a ratio of 2:1:2 in Example 1 of the present invention; Figure 4 CV test curves of MXene / CNF / Ag NWs aerogel modified electrodes prepared from materials of different mass ratios in Example 2 of the present invention; Figure 5 The DPV test curve of the aerogel modified electrode prepared by MXene / CNF / Ag NWs at a ratio of 4:2:1 in Example 3 of the present invention to detect different cortisol concentrations (PBS solution); Figure 6 The DPV test curve of the aerogel modified electrode prepared by MXene / CNF / Ag NWs at a ratio of 2:1:1 in Example 3 of the present invention to detect different cortisol concentrations (PBS solution); Figure 7 This is the DPV test curve of the aerogel modified electrode prepared with MXene / CNF / Ag NWs at a ratio of 2:1:2 in Example 3 of the present invention for detecting different cortisol concentrations (PBS solution). DETAILED DESCRIPTION

[0021] It should be noted that, in the absence of conflict, the embodiments of the present invention and the features in the embodiments may be combined with each other.

[0022] The present invention will be described in detail below with reference to the accompanying drawings and in conjunction with embodiments.

[0023] Example 1 1. Preparation of MXene / CNF / Ag NWs aerogel: 10 mg of MXene and 5 mg of CNF were added to 1 mL of Ag NWs (2.5 mg / mL), Ag NWs (5 mg / mL), and Ag NWs (10 mg / mL), respectively, to obtain MXene / CNF / AgNWs dispersions with different proportions. The MXene / CNF / Ag NWs dispersions with different proportions were placed in vials, and the vials were fixed on a copper plate, and the bottom of the copper plate was immersed in liquid nitrogen and frozen. Finally, the MXene / CNF / Ag NWs dispersions with different proportions were placed in a vacuum freeze dryer and dried for 48 hours to obtain MXene / CNF / Ag NWs aerogels.

[0024] 2. Place the MXene / CNF / Ag NWs aerogel obtained in step 1 into ethanol and stir to disperse it to obtain a MXene / CNF / Ag NWs aerogel dispersion.

[0025] 3. Rinse the screen-printed electrode with deionized water and dry it in an oven at 45°C for later use. Take 10 μL of the dispersion obtained in step 2 and drop it on the screen-printed electrode. Dry the electrode in an oven at 45°C for later use.

[0026] 4. Prepare 0.05M hydrochloric acid solution, place chitosan powder in the hydrochloric acid solution to prepare a 4 mg / mL chitosan solution.

[0027] 5. Take 10 μL of the chitosan solution obtained in step 4 and drop it onto the screen-printed electrode obtained in step 3. Dry the electrode in an oven at 45° C. for later use.

[0028] 6. Prepare 0.016g o-phenylenediamine, 0.005g cortisol, and 100ml of 0.1M (PH=5) acetate buffer solution (sodium acetate + acetic acid) as the polymerization solution. Prepare the molecular imprinting membrane using the electropolymerization method. Electropolymerization conditions: scanning speed 25-100mV / s, potential range 0-1V, cycle number 10-30 times, and perform CV polymerization.

[0029] 7. Fix the electrode obtained after electropolymerization in the eluent, put the eluent into a beaker, pour 50 ml of anhydrous ethanol into the beaker for magnetic stirring to remove the cortisol molecules in the polymer on the electrode surface.

[0030] 8. Dry the electrode in an oven at 45°C and observe its morphology using SEM.

[0031] like Figure 1-Figure 3 As shown, MXene / CNF / Ag NWs aerogel makes the electrode surface rough, and the aerogel prepared at a ratio of MXene / CNF / AgNWs of 2:1:1 has the most porous structure and can provide more cortisol binding sites.

[0032] Example 2 1. Preparation of MXene / CNF / Ag NWs aerogel: 10 mg of MXene and 5 mg of CNF were added to 1 mL of Ag NWs (2.5 mg / mL), Ag NWs (5 mg / mL), and Ag NWs (10 mg / mL), respectively, to obtain MXene / CNF / AgNWs dispersions with different proportions. The MXene / CNF / Ag NWs dispersions with different proportions were placed in vials, and the vials were fixed on a copper plate, and the bottom of the copper plate was immersed in liquid nitrogen and frozen. Finally, the MXene / CNF / Ag NWs dispersions with different proportions were placed in a vacuum freeze dryer and dried for 48 hours to obtain MXene / CNF / Ag NWs aerogels.

[0033] 2. Place the MXene / CNF / Ag NWs aerogel obtained in step 1 into ethanol and stir to disperse it to obtain a MXene / CNF / Ag NWs aerogel dispersion.

[0034] 3. Rinse the screen-printed electrode with deionized water and dry it in an oven at 45°C for later use. Take 10 μL of the dispersion obtained in step 2 and drop it on the screen-printed electrode. Dry the electrode in an oven at 45°C for later use.

[0035] 4. Prepare 0.05M hydrochloric acid solution, place chitosan powder in the hydrochloric acid solution to prepare a 4 mg / mL chitosan solution.

[0036] 5. Take 10 μL of the chitosan solution obtained in step 4 and drop it onto the screen-printed electrode obtained in step 3. Dry the electrode in an oven at 45° C. for later use.

[0037] 6. Prepare 0.016g o-phenylenediamine, 0.005g cortisol, and 100ml of 0.1M (PH=5) acetate buffer solution (sodium acetate + acetic acid) as the polymerization solution. Prepare the molecular imprinting membrane using the electropolymerization method. Electropolymerization conditions: scanning speed 25-100mV / s, potential range 0-1V, cycle number 10-30 times, and perform CV polymerization.

[0038] 7. Fix the electrode obtained after electropolymerization in the eluent, put the eluent into a beaker, pour 50 ml of anhydrous ethanol into the beaker for magnetic stirring to remove the cortisol molecules in the polymer on the electrode surface.

[0039] 8. Prepare 0.041 g potassium ferrocyanide, 0.046 g potassium ferrocyanide, 0.373 g potassium chloride, and 50 ml deionized water as the redox probe solution.

[0040] 9. Take 50 μL of the redox probe solution obtained in step 8 and drop it onto the screen-printed electrode to test CV and its sensing ability.

[0041] like Figure 4 As shown, the electrical signal response is maximum when the MXene / CNF / Ag NWs weight ratio is 2:1:1.

[0042] Example 3 1. Preparation of MXene / CNF / Ag NWs aerogel: 10 mg of MXene and 5 mg of CNF were added to 1 mL of Ag NWs (2.5 mg / mL), Ag NWs (5 mg / mL), and Ag NWs (10 mg / mL), respectively, to obtain MXene / CNF / AgNWs dispersions with different proportions. The MXene / CNF / Ag NWs dispersions with different proportions were placed in vials, and the vials were fixed on a copper plate, and the bottom of the copper plate was immersed in liquid nitrogen and frozen. Finally, the MXene / CNF / Ag NWs dispersions with different proportions were placed in a vacuum freeze dryer and dried for 48 hours to obtain MXene / CNF / Ag NWs aerogels.

[0043] 2. Place the MXene / CNF / Ag NWs aerogel obtained in step 1 into ethanol and stir to disperse it to obtain a MXene / CNF / Ag NWs aerogel dispersion.

[0044] 3. Rinse the screen-printed electrode with deionized water and dry it in an oven at 45°C for later use. Take 10 μL of the dispersion obtained in step 2 and drop it on the screen-printed electrode. Dry the electrode in an oven at 45°C for later use.

[0045] 4. Prepare 0.05M hydrochloric acid solution, place chitosan powder in the hydrochloric acid solution to prepare a 4 mg / mL chitosan solution.

[0046] 5. Take 10 μL of the chitosan solution obtained in step 4 and drop it onto the screen-printed electrode obtained in step 3. Dry the electrode in an oven at 45° C. for later use.

[0047] 6. Prepare 0.016g o-phenylenediamine, 0.005g cortisol, and 100ml of 0.1M (PH=5) acetate buffer solution (sodium acetate + acetic acid) as the polymerization solution. Prepare the molecular imprinting membrane using the electropolymerization method. Electropolymerization conditions: scanning speed 25-100mV / s, potential range 0-1V, cycle number 10-30 times, and perform CV polymerization.

[0048] 7. Fix the electrode obtained after electropolymerization in the eluent, put the eluent into a beaker, pour 50 ml of anhydrous ethanol into the beaker for magnetic stirring to remove the cortisol molecules in the polymer on the electrode surface.

[0049] 8. Take 50 μL of cortisol solution of different concentrations and drop it on the screen-printed electrode, incubate for 10 minutes, then rinse the electrode with deionized water and dry the electrode in an oven at 45°C for later use.

[0050] 9. Prepare 0.041 g potassium ferrocyanide, 0.046 g potassium ferrocyanide, 0.373 g potassium chloride, and 50 ml deionized water as the redox probe solution.

[0051] 10. Take 10 μL of the redox probe solution obtained in step 9 and drop it onto the screen-printed electrode to test DPV and its sensing ability.

[0052] like Figure 5-Figure 7 As shown, MXene / CNF / Ag NWs aerogel increases the specific surface area of ​​the electrode and improves the electrical properties of the electrode, enabling the molecular imprinting sensing electrode to detect cortisol solutions of different concentrations.

[0053] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A molecular imprinting sensing electrode for detecting cortisol, characterized in that: The invention comprises a screen-printed electrode, and a MXene / CNF / Ag NWs aerogel, a chitosan protective layer and a molecular imprinting polymer film which are sequentially loaded on the surface of the screen-printed electrode.

2. A method for preparing a molecular imprinting sensing electrode for detecting cortisol according to claim 1, characterized in that: The following steps are involved: (1) Using MXene / CNF / Ag NWs aerogel to modify the screen-printed electrode to obtain MXene / CNF / Ag NWs aerogel / screen-printed electrode; (2) Modifying the MXene / CNF / Ag NWs aerogel / screen-printed electrode with chitosan solution to obtain a MXene / CNF / AgNWs aerogel / chitosan / screen-printed electrode; (3) Using a polymerization solution, an electrochemical polymerization reaction is carried out on the surface of the MXene / CNF / Ag NWs aerogel / chitosan / screen-printed electrode by cyclic voltammetry, and the cortisol molecules are eluted in an elution solution to leave a cortisol molecular cavity, and finally a cortisol molecular imprinting sensing electrode is obtained.

3. The preparation method according to claim 2, characterized in that: The specific preparation method of step (1) is: (1) Add MXene / CNF / Ag NWs aerogel into ethanol and stir to obtain a dispersion; (2) Take 8-12 μL of the dispersion obtained in step (1), drop it onto the screen-printed electrode, and dry it in an oven at 40-50 °C to obtain MXene / CNF / Ag NWs aerogel / screen-printed electrode.

4. The preparation method according to claim 2, characterized in that: The preparation method of the MXene / CNF / Ag NWs aerogel comprises the following steps: MXene and CNF are added to Ag NWs to obtain a MXene / CNF / Ag NWs dispersion, the MXene / CNF / Ag NWs dispersion is filled into a vial, the vial is fixed on a copper plate, the bottom of the copper plate is immersed in liquid nitrogen and frozen, and then placed in a vacuum freeze dryer to obtain a MXene / CNF / Ag NWs aerogel.

5. The preparation method according to claim 2, characterized in that: The specific preparation method of step (2) is: drop-coating the chitosan solution onto the MXene / CNF / Ag NWs aerogel / screen-printed electrode obtained in step (1), and drying in an oven at 45° C. to obtain a MXene / CNF / Ag NWs aerogel / chitosan / screen-printed electrode.

6. The preparation method according to claim 2, characterized in that: The preparation method of the chitosan solution is as follows: prepare a 0.05M hydrochloric acid solution, place chitosan powder in the hydrochloric acid solution, and prepare a 4 mg / mL chitosan solution.

7. The preparation method according to claim 2, characterized in that: The polymerization solution in step (3) includes 1-2 mM o-phenylenediamine, 0.1-0.2 mM cortisol, and 0.1 M acetate buffer solution.

8. The preparation method according to claim 2, characterized in that: The specific preparation method of step (3) is: fix the electrode after the electrochemical polymerization reaction in the eluent, put the eluent into a beaker, pour 40-70 ml of anhydrous ethanol into the beaker for magnetic stirring, and remove the cortisol molecules in the polymer on the electrode surface.

9. The preparation method according to claim 2, characterized in that: The parameters of the electrochemical polymerization reaction are: scanning speed 25-100 mV / s, potential range -1-1 V, and cycle number 20-40 times.

10. The preparation method according to claim 2, characterized in that: The eluent is anhydrous ethanol.

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