A molecularly imprinted sensing electrode for detecting cortisol and a preparation method thereof

Abstract

The application provides a molecular imprinting sensing electrode for detecting cortisol and a preparation method thereof, wherein the molecular imprinting sensing electrode for detecting cortisol comprises a screen-printed electrode, and an MXene / CNF / Ag NWs aerogel, a chitosan protective layer and a molecular imprinting polymer film loaded on the surface of the screen-printed electrode in sequence. The MXene / CNF / Ag NWs aerogel of the application makes the electrode surface obtain a porous structure, a high specific surface area and high conductivity, and makes the sensing electrode performance more excellent. The cortisol sensing electrode is prepared by electrochemical method, and the sensing electrode can realize sensitive detection on different concentrations of cortisol, and shows application value of the sensing electrode in the field of continuous detection.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biosensing detection, and particularly relates to a molecular imprinting sensing electrode for detecting cortisol and a preparation method thereof. BACKGROUND

[0002] Cortisol is the most common glucocorticoid in clinical practice. Due to its anti-inflammatory, anti-toxin and immunosuppressive effects, it is widely used to treat severe skin allergies and inflammation in humans and animals. In addition to the above-mentioned external intake, cortisol can also be obtained through the secretion of the human body. Cortisol plays a crucial role in emotional responses such as stress: it can maintain blood pressure stable and prevent excessive inflammation in stress situations, and is therefore considered a stress biomarker. However, if people are under stress for a long time or take too much cortisol, the cortisol level in their body will be high for a long time. The harm of cortisol to the human body will become apparent, manifested as osteoporosis, weight gain, muscle weakness, inhibition of child growth and aggravation of diabetes, etc. 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 immunoassay. Although these traditional techniques have achieved good sensitivity and accuracy, they require expensive equipment, large amounts of organic solvents, long experimental time and professional technicians, which limits their application in actual field testing. 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, MIPs are widely used in many fields due to their 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, various new conductive materials such as aerogels are used to construct composite electrochemical sensing electrodes.

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

[0005] Therefore, the present application aims to provide 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] In order to achieve the above object, the technical scheme of the present application is as follows:

[0007] The first aspect of the present application provides a molecular imprinting sensing electrode for detecting cortisol, comprising a screen-printed electrode, and MXene / CNF / Ag NWs aerogel, a chitosan protective layer and a molecular imprinting polymer film loaded on the surface of the screen-printed electrode in sequence.

[0008] The second aspect of the present application provides a preparation method of the molecular imprinting sensing electrode for detecting cortisol according to the first aspect of the present application, comprising the following steps:

[0009] (1) modifying the screen-printed electrode with MXene / CNF / Ag NWs aerogel to obtain MXene / CNF / Ag NWs aerogel / screen-printed electrode;

[0010] (2) modifying the MXene / CNF / Ag NWs aerogel / screen-printed electrode with chitosan solution to obtain MXene / CNF / Ag NWs aerogel / chitosan / screen-printed electrode;

[0011] (3) performing electrochemical polymerization reaction on the surface of the MXene / CNF / Ag NWs aerogel / chitosan / screen-printed electrode by cyclic voltammetry using polymerization solution, and then performing elution in eluent to remove cortisol molecules and leave cortisol molecule cavities, so as to finally obtain a cortisol molecular imprinting sensing electrode.

[0012] Further, the specific preparation method of step (1) is as follows:

[0013] (1) adding MXene / CNF / Ag NWs aerogel into ethanol and stirring to obtain a dispersion;

[0014] (2) taking 8-12 μL of the dispersion obtained in step (1) and dropping it on the screen-printed electrode, and then drying it in an oven at 40-50℃ to obtain MXene / CNF / Ag NWs aerogel / screen-printed electrode.

[0015] Preferably, the amount of the dispersion is 10 μL.

[0016] Further, the preparation method of the MXene / CNF / Ag NWs aerogel comprises the following steps:

[0017] The MXene and CNF are added to the Ag NWs to obtain a MXene / CNF / Ag NWs dispersion liquid, the MXene / CNF / Ag NWs dispersion liquid is loaded into a vial, the vial is fixed on a copper disc, the bottom of the copper disc is immersed in liquid nitrogen for freezing, and the vial is placed in a vacuum freeze dryer for drying to obtain a MXene / CNF / Ag NWs aerogel.

[0018] Preferably, the mass ratio of the MXene, the CNF and the Ag NWs is 2:1:1.

[0019] Further, the specific preparation method of the step (2) is that a chitosan solution is drop-coated on the MXene / CNF / Ag NWs aerogel / silk screen printing electrode obtained in the step (1), and the MXene / CNF / Ag NWs aerogel / silk screen printing electrode is dried in an oven at 45 DEG C to obtain a MXene / CNF / Ag NWs aerogel / chitosan / silk screen printing electrode.

[0020] Further, the preparation method of the chitosan solution is that a 0.05M hydrochloric acid solution is prepared, chitosan powder is placed in the hydrochloric acid solution, and a 4mg / mL chitosan solution is prepared.

[0021] Further, the polymerization liquid in the step (3) comprises 1-2mM o-phenylenediamine, 0.1-0.2mM cortisol, and 0.1M (PH=5) acetate buffer solution.

[0022] Further, the specific preparation method of the step (3) is that the electrode after the electrochemical polymerization reaction is fixed in an eluent, the eluent is loaded into a beaker, 40-70ml anhydrous ethanol is poured into the beaker for magnetic stirring, and the cortisol molecules in the polymer on the surface of the electrode are removed.

[0023] Further, the parameters of the electrochemical polymerization reaction are as follows: a scanning speed is 25-100mV / s, a potential range is -1-1V, and a cycle number is 20-40 times.

[0024] Further, the eluent is anhydrous ethanol.

[0025] Compared with the prior art, the molecularly imprinted sensing electrode for detecting cortisol and the preparation method thereof have the following advantages:

[0026] (1) The MXene / CNF / Ag NWs aerogel is used to modify the silk screen printing electrode, so that the modified electrode has a large specific surface area, high conductivity, rich element groups, and the porous structure of the aerogel is beneficial to the attachment of the imprinted molecules in the electro-polymerization process.

[0027] (2) The comprehensive comparison of the test data shows that the MXene / CNF / Ag NWs aerogel selected at 2:1:1 has the largest electrical signal response and the best sensing performance. BRIEF DESCRIPTION OF DRAWINGS

[0028] The accompanying drawings, which form a part of the present application, are intended to provide further understanding of the present application and are incorporated herein for explanation by way of the non-limiting examples. In the drawings:

[0029] Figure 1 SEM image of the aerogel prepared by MXene / CNF / Ag NWs at a ratio of 4:2:1 in Example 1 of the present application;

[0030] Figure 2 SEM image of the aerogel prepared by MXene / CNF / Ag NWs at a ratio of 2:1:1 in Example 1 of the present application;

[0031] Figure 3 SEM image of the aerogel prepared by MXene / CNF / Ag NWs at a ratio of 2:1:2 in Example 1 of the present application;

[0032] Figure 4 CV test curve of the MXene / CNF / Ag NWs aerogel modified electrode prepared by materials with different mass ratios in Example 2 of the present application;

[0033] Figure 5 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 application for detecting different cortisol concentrations (PBS solution);

[0034] Figure 6 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 application for detecting different cortisol concentrations (PBS solution);

[0035] Figure 7 DPV test curve of the aerogel modified electrode prepared by MXene / CNF / Ag NWs at a ratio of 2:1:2 in Example 3 of the present application for detecting different cortisol concentrations (PBS solution). DETAILED DESCRIPTION

[0036] It should be noted that the examples in the present application and the features in the examples can be combined with each other without conflict.

[0037] The present application will be described in detail below with reference to the accompanying drawings and in combination with the examples.

[0038] Example 1

[0039] 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 / Ag NWs dispersions with different proportions. The MXene / CNF / Ag NWs dispersions with different proportions were loaded into vials, which were fixed on a copper plate immersed in liquid nitrogen for freezing. Finally, the MXene / CNF / Ag NWs dispersions with different proportions were placed in a vacuum freeze dryer for 48 h to obtain MXene / CNF / Ag NWs aerogels.

[0040] 2. The MXene / CNF / Ag NWs aerogel obtained in step 1 was placed in ethanol and stirred to disperse, obtaining a MXene / CNF / Ag NWs aerogel dispersion.

[0041] 3. The screen-printed electrode was rinsed with deionized water and dried in an oven at 45°C for use. 10 μL of the dispersion obtained in step 2 was dropped onto the screen-printed electrode, and the electrode was dried in an oven at 45°C for use.

[0042] 4. A 0.05 M hydrochloric acid solution was prepared, and chitosan powder was placed in the hydrochloric acid solution to prepare a 4 mg / mL chitosan solution.

[0043] 5. 10 μL of the chitosan solution obtained in step 4 was dropped onto the screen-printed electrode obtained in step 3, and the electrode was dried in an oven at 45°C for use.

[0044] 6. A 0.016 g o-phenylenediamine, 0.005 g cortisol, 0.1 M (PH=5) acetate buffer solution (sodium acetate + acetic acid) 100 ml was prepared as a polymerization solution. A molecularly imprinted film was prepared by using an electro-polymerization method. The electro-polymerization conditions were as follows: scan speed 25-100 mV / s, potential range 0-1 V, cycle number 10-30 times, and CV method polymerization.

[0045] 7. The electrode obtained after electro-polymerization was fixed in an eluent, the eluent was loaded into a beaker, 50 ml of anhydrous ethanol was poured into the beaker, and magnetic stirring was performed to remove the cortisol molecules in the polymer on the surface of the electrode.

[0046] 8. The electrode was dried in an oven at 45°C, and SEM was used to observe the morphology.

[0047] As Figures 1-3As shown, the MXene / CNF / Ag NWs aerogel makes the electrode surface rough, and the aerogel prepared at a ratio of 2:1:1 has the most porous structure, which can provide more cortisol binding sites.

[0048] Example 2

[0049] 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 / Ag NWs dispersions with different ratios. The MXene / CNF / Ag NWs dispersions with different ratios were loaded into vials, which were fixed on a copper plate, and the bottom of the copper plate was immersed in liquid nitrogen for freezing. Finally, the MXene / CNF / Ag NWs dispersions with different ratios were placed in a vacuum freeze dryer for drying for 48 h to obtain MXene / CNF / Ag NWs aerogel.

[0050] 2. The MXene / CNF / Ag NWs aerogel obtained in step 1 was placed in ethanol and stirred to disperse, obtaining a MXene / CNF / Ag NWs aerogel dispersion.

[0051] 3. The screen-printed electrode was rinsed with deionized water and dried in an oven at 45°C for use. 10 μL of the dispersion obtained in step 2 was dropped onto the screen-printed electrode, and the electrode was dried in an oven at 45°C for use.

[0052] 4. A 0.05 M hydrochloric acid solution was prepared, and chitosan powder was placed in the hydrochloric acid solution to prepare a 4 mg / mL chitosan solution.

[0053] 5. 10 μL of the chitosan solution obtained in step 4 was dropped onto the screen-printed electrode obtained in step 3, and the electrode was dried in an oven at 45°C for use.

[0054] 6. A 0.016 g o-phenylenediamine, 0.005 g cortisol, and 0.1 M (PH=5) acetate buffer solution (sodium acetate + acetic acid) 100 ml were prepared as a polymerization solution. A molecularly imprinted film was prepared by an electro-polymerization method. The electro-polymerization conditions were: scan speed 25-100 mV / s, potential range 0-1 V, cycle number 10-30 times, and CV method polymerization.

[0055] 7. The electrode obtained after electro-polymerization was fixed in an eluent, the eluent was loaded into a beaker, 50 ml of anhydrous ethanol was poured into the beaker, and magnetic stirring was performed to remove the cortisol molecules in the polymer on the surface of the electrode.

[0056] 8. 0.041 g of potassium ferricyanide, 0.046 g of potassium ferrocyanide, 0.373 g of potassium chloride, 50 ml of deionized water were configured as a redox probe solution.

[0057] 9. Take 50 μL of the redox probe solution obtained in step 8, drop it on the screen-printed electrode to test the CV, and test its transmission ability.

[0058] As shown in Figure 4 The electrical signal response is the largest when the weight ratio of MXene / CNF / Ag NWs is 2:1:1.

[0059] Example 3

[0060] 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 / Ag NWs dispersions with different proportions. The MXene / CNF / Ag NWs dispersions with different proportions were loaded into vials, which were fixed on a copper plate, and the bottom of the copper plate was immersed in liquid nitrogen for freezing. Finally, the MXene / CNF / Ag NWs dispersions with different proportions were placed in a vacuum freeze dryer for 48 h to obtain MXene / CNF / Ag NWs aerogel.

[0061] 2. The MXene / CNF / Ag NWs aerogel obtained in step 1 was placed in ethanol and stirred to obtain a MXene / CNF / Ag NWs aerogel dispersion.

[0062] 3. The screen-printed electrode was rinsed with deionized water and placed in an oven at 45°C for drying, 10 μL of the dispersion obtained in step 2 was dropped onto the screen-printed electrode, and the electrode was dried in an oven at 45°C for use.

[0063] 4. A 0.05 M hydrochloric acid solution was configured, and chitosan powder was placed in the hydrochloric acid solution to configure a 4 mg / mL chitosan solution.

[0064] 5. 10 μL of the chitosan solution obtained in step 4 was dropped onto the screen-printed electrode obtained in step 3, and the electrode was dried in an oven at 45°C for use.

[0065] 6. 0.016 g of o-phenylenediamine, 0.005 g of cortisol, and 0.1 M (pH = 5) acetate buffer solution (sodium acetate + acetic acid) 100 ml were configured as a polymerization solution. A molecularly imprinted film was prepared by using an electro-polymerization method. The electro-polymerization conditions were as follows: scan speed 25-100 mV / s, potential range 0-1 V, cycle number 10-30 times, and CV method polymerization.

[0066] 7. The electrode obtained after electro-polymerization is fixed in the eluent, the eluent is loaded into a beaker, 50 ml of anhydrous ethanol is poured into the beaker for magnetic stirring, and the cortisol molecules in the polymer on the surface of the electrode are removed.

[0067] 8. 50 μL of cortisol solution of different concentrations is dropped on the screen-printed electrode, incubated for 10 minutes, then the electrode is washed with deionized water, and dried in an oven at 45°C for standby.

[0068] 9. 0.041 g of potassium ferricyanide, 0.046 g of potassium ferrocyanide, 0.373 g of potassium chloride, and 50 ml of deionized water are configured as a redox probe solution.

[0069] 10. 10 μL of the redox probe solution obtained in step 9 is dropped on the screen-printed electrode for DPV test to test the sensing ability.

[0070] As shown in Figures 5-7 , the MXene / CNF / Ag NWs aerogel increases the specific surface area of the electrode, improves the electrical performance of the electrode, and enables the molecularly imprinted sensing electrode to detect cortisol solutions of different concentrations.

[0071] The above only describes the preferred embodiments of the present application and is not intended to limit the present application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A method for preparing a molecularly imprinted sensing electrode for detecting cortisol, characterized in that: Includes the following steps: (1) The screen-printed electrode was modified with MXene / CNF / Ag NWs aerogel to obtain the MXene / CNF / Ag NWs aerogel / screen-printed electrode; (2) MXene / CNF / Ag NWs aerogel / screen printed electrode was modified with chitosan solution to obtain MXene / CNF / Ag NWs aerogel / chitosan / screen printed electrode; (3) Electrochemical polymerization reaction was carried out on the surface of the MXene / CNF / Ag NWs aerogel / chitosan / screen printed electrode using cyclic voltammetry with the polymerization liquid. Cortisol molecules were removed by elution in the eluent, leaving cortisol molecule cavities, and finally cortisol molecule imprinted sensing electrode was obtained. The molecularly imprinted sensing electrode for detecting cortisol includes a screen-printed electrode and MXene / CNF / Ag NWs aerogel, a chitosan protective layer, and a molecularly imprinted polymer film sequentially loaded on the surface of the screen-printed electrode. The specific preparation method for step (1) is as follows: a: Add MXene / CNF / Ag NWs aerogel to ethanol and stir to obtain a dispersion; b: Take 8-12 μL of the dispersion obtained in step a, drop it onto the screen-printed electrode, and dry it in an oven at 40-50℃ to obtain the MXene / CNF / Ag NWs aerogel / screen-printed electrode. The preparation method of the MXene / CNF / Ag NWs aerogel includes the following steps: MXene and CNF were added to Ag NWs to obtain an MXene / CNF / Ag NWs dispersion. The MXene / CNF / Ag NWs dispersion was then placed into a vial, which was fixed on a copper disk. The bottom of the copper disk was immersed in liquid nitrogen for freezing. The vial was then placed in a vacuum freeze dryer for drying to obtain an MXene / CNF / Ag NWs aerogel. The specific preparation method of step (2) is as follows: the chitosan solution is drop-coated onto the MXene / CNF / AgNWs aerogel / screen-printed electrode obtained in step (1), and dried in an oven at 45°C to obtain the MXene / CNF / Ag NWs aerogel / chitosan / screen-printed electrode.

2. The preparation method according to claim 1, characterized in that: The chitosan solution is prepared by: preparing a 0.05M hydrochloric acid solution, placing chitosan powder in the hydrochloric acid solution, and preparing a 4 mg / mL chitosan solution.

3. The preparation method according to claim 1, 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.

4. The preparation method according to claim 1, characterized in that: The specific preparation method of step (3) is as follows: the electrode after electrochemical polymerization reaction is fixed in the eluent, the eluent is put into a beaker, 40-70 ml of anhydrous ethanol is poured into the beaker and magnetically stirred to remove cortisol molecules in the polymer on the electrode surface.

5. The preparation method according to claim 1, characterized in that: The parameters for the electrochemical polymerization reaction are: scan rate 25–100 mV / s, potential range -1–1 V, and number of cycles 20–40.

6. The preparation method according to claim 1, characterized in that: The eluent is anhydrous ethanol.

Images