Preparation method of molecularly imprinted sensing electrode for detecting cortisol

Abstract

The invention relates to the technical field of biosensing, in particular to a preparation method of a carboxylated cellulose nanofiber and carboxylated multi-walled carbon nanotube composite material and gold nanoparticle modified cortisol molecularly imprinted (MIP) sensing electrode. The carboxylated cellulose nanofibers and the carboxylated multi-walled carbon nanotubes are combined, and then the gold nanoparticles are electrically deposited, so that the surface of the electrode becomes rough, the performance of the sensing electrode is more excellent due to the high specific surface area and the high conductivity, and the electrode is more stable due to the chitosan protective layer. The cortisol sensing electrode prepared by electropolymerization through an electrochemical method can realize sensitive detection of cortisol with different concentrations, and shows the application value of the sensing electrode in the field of continuous detection.

Description

[Technical field]

[0001] The invention belongs to the field of biosensor detection, and specifically relates to a method for preparing a molecular imprinting (MIP) sensing electrode modified by a composite material of carboxylated cellulose nanofibers, carboxylated multi-walled carbon nanotubes and gold nanoparticles for detecting cortisol of different concentrations.

Technical Background

[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 stress for a long time 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: 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 multi-walled carbon nanotubes have been used to construct composite electrochemical sensing electrodes.

[0004] So far, researchers at home and abroad have explored various MIP-type sensors for cortisol detection. Some researchers, such as Kunling Wei et al. (Kunling Wei, Huimin Tang, Guangfei Qu, et al. Selective detection of hydroxyl radicals by molecularly imprinted electrochemical sensors constructed with MWCNTs-COOH-modified electrodes, Microchemical Journal, Volume 205, 2024, 111194) increased the electrode electrical signal response by modifying multi-walled carbon nanotubes to improve the sensitivity of the electrode, and achieved good results. However, the detection of cortisol at different concentrations is still not satisfactory, and long-term detection will cause the electrode modification material to fall off, affecting the stability of the electrode. [Summary of the invention]

[0005] The invention aims to synthesize a composite material of carboxylated cellulose nanofibers and carboxylated multi-walled carbon nanotubes, and use the composite material to modify a screen-printed electrode, and then electrodeposit gold nanoparticles on the surface of the electrode to increase the electrode sensitivity, detection range and electrical signal response. A chitosan solution is used to provide a protective layer for the modified electrode to prevent the modified material from falling off. At the same time, the modified screen-printed electrode is electropolymerized to form a MIP-type cortisol sensing electrode, which can achieve a wide detection range and high electrical signal response.

[0006] To achieve the above purpose, the specific preparation scheme adopted by the present invention is as follows:

[0007] Carboxylated cellulose nanofiber / carboxylated multi-walled carbon nanotube composites were synthesized and used to modify screen-printed electrodes; gold nanoparticles were electrodeposited on the electrode surface using chloroauric acid; the modified electrode was protected with a chitosan protective layer; molecular imprinting polymers were polymerized on the electrode surface using an electrochemical method; and the target molecule cortisol was eluted with anhydrous ethanol to prepare a sensing electrode.

[0008] Required materials: chloroauric acid, carboxylated cellulose nanofibers, carboxylated multi-walled carbon nanotubes, deionized water, hydrochloric acid, anhydrous ethanol, o-phenylenediamine, cortisol solution, potassium ferrocyanide, potassium ferrocyanide, potassium chloride, sodium acetate, acetic acid, PBS buffer solution, screen-printed electrodes, chitosan.

[0009] The specific steps include:

[0010] The raw materials are calculated by weight:

[0011] Step 1: Preparation of carboxylated cellulose nanofiber / carboxylated multi-walled carbon nanotube composite materials: prepare 0.015g carboxylated cellulose nanofiber, 0.03g carboxylated multi-walled carbon nanotube, 3ml deionized water mixed solution, 0.015g carboxylated cellulose nanofiber, 0.015g carboxylated multi-walled carbon nanotube, 3ml deionized water mixed solution, 0.015g carboxylated cellulose nanofiber, 0.0075g carboxylated multi-walled carbon nanotube, 3ml deionized water mixed solution respectively to obtain carboxylated cellulose nanofiber / carboxylated multi-walled carbon nanotube composite materials with different proportions.

[0012] Step 2: Rinse the screen-printed electrode with deionized water and dry it in an oven at 50°C for later use. Take 15 μL of the composite solution obtained in step 1, drop it on the screen-printed electrode, and dry the electrode in an oven at 50°C for later use.

[0013] Step 3: Prepare 5 mM chloroauric acid solution, take 50 μL of chloroauric acid solution and drop it on the electrode, deposit it at a constant potential of -0.2 V for 120 s, and dry the electrode in an oven at 50°C.

[0014] Step 4: Prepare 0.05M hydrochloric acid solution, place chitosan powder in the hydrochloric acid solution, and prepare a 4 mg / mL chitosan solution.

[0015] Step 5: Take 15 μL of the chitosan solution obtained in step 4 and drop it onto the screen-printed electrode, and dry the electrode in an oven at 50° C. for later use.

[0016] Step 6: Prepare 54 mg of o-phenylenediamine, 18 mg of cortisol, and 100 ml of 0.1 M 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 50 mV / s, potential range 0-1 V, cycle number 25 times, and perform CV polymerization.

[0017] Step 7: Fix the electrode obtained after electropolymerization in the eluent, pour 50 ml of anhydrous ethanol into a beaker for magnetic stirring to remove the cortisol molecules in the polymer on the electrode surface.

[0018] By adopting the above process route, high-performance and durable MIP-type cortisol sensing electrodes can be prepared.

[0019] In summary, this application has the following beneficial effects:

[0020] 1. The present application adopts carboxylated cellulose nanofibers and carboxylated multi-walled carbon nanotubes as composites and uses them to modify screen-printed electrodes, and then electrodeposit gold nanoparticles on the electrode surface, so that the modified electrode not only has a large specific surface area, high conductivity, and abundant element groups, but also the rough electrode surface is conducive to the attachment of imprinted molecular polymers during the electropolymerization process.

[0021] 2. Protecting the modified electrode with a chitosan protective layer can prevent the modified material from falling off during the detection process and improve the stability of the electrode.

[0022] 3. Comprehensive comparison of test data shows that when the ratio of carboxylated cellulose nanofibers to carboxylated multi-walled carbon nanotubes modified materials is 1:2, the electrical signal response is the largest and the sensing performance is the best.

[0023] 4. The preparation method of the present invention is simple, easy to operate, low in cost and environmentally friendly.

Brief Description of the Drawings

[0024] The embodiments of the present invention are described in detail below with reference to the accompanying drawings, wherein:

[0025] Figure 1 SEM images of composite modified electrodes prepared at a ratio of 1:2 between carboxylated cellulose nanofibers and carboxylated multi-walled carbon nanotubes (i.e., Example 1), with chitosan protective layer on the left and without chitosan protective layer on the right

[0026] Figure 2 CV test curves of electrodes modified by carboxylated cellulose nanofibers and carboxylated multi-walled carbon nanotubes in different mass ratios (i.e., Example 2)

[0027] Figure 3 DPV test curve (ie, Example 3) and fitting line of the composite modified electrode prepared at a ratio of 1:2 for detecting different cortisol concentrations (PBS solution)

[0028] Figure 4 DPV test curve (ie, Example 3) and fitting line of the composite modified electrode prepared at a ratio of 1:1 for carboxylated cellulose nanofibers and carboxylated multi-walled carbon nanotubes to detect different cortisol concentrations (PBS solution)

[0029] Figure 5 DPV test curve (ie, Example 3) and fitting line of the composite modified electrode prepared at a ratio of 2:1 for detecting different cortisol concentrations (PBS solution)

[0030] Figure 6DPV test curves of the composite modified electrode prepared at a ratio of 1:2 for detecting different cortisol concentrations (PBS solution) (ie, Example 4), without chitosan protective layer [Specific implementation method]

[0031] The present invention is described in detail below in conjunction with the accompanying drawings and embodiments.

[0032] Embodiment 1:

[0033] (1) A mixed solution of 0.015 g of carboxylated cellulose nanofibers, 0.03 g of carboxylated multi-walled carbon nanotubes, and 3 ml of deionized water was prepared and stirred to obtain a carboxylated cellulose nanofiber / carboxylated multi-walled carbon nanotube composite material.

[0034] (2) Rinse the screen-printed electrode with deionized water and dry it in an oven at 50°C for later use. Take 15 μL of the composite solution obtained in step 1 and drop it onto the screen-printed electrode. Dry the electrode in an oven at 50°C for later use.

[0035] (3) Prepare a 5 mM chloroauric acid solution, take 50 μL of the chloroauric acid solution and drop it onto the electrode, deposit it at a constant potential at a voltage of -0.2 V for 120 s, and dry the electrode in an oven at 50°C.

[0036] (4) Prepare a 0.05 M hydrochloric acid solution, place the chitosan powder in the hydrochloric acid solution, and prepare a 4 mg / mL chitosan solution.

[0037] (5) Take 15 μL of the chitosan solution obtained in step 4 and apply it dropwise onto the screen-printed electrode. Dry the electrode in an oven at 50°C for later use. (The control group does not add chitosan)

[0038] (6) Prepare 54 mg of o-phenylenediamine, 18 mg of cortisol, and 100 ml of 0.1 M pH = 5 acetate buffer solution (sodium acetate + acetic acid) as the polymerization solution. Prepare the molecular imprinting membrane by electropolymerization. Electropolymerization conditions: scanning speed 50 mV / s, potential range 0-1 V, cycle number 25 times, and perform CV polymerization.

[0039] (7) The electrode obtained after electropolymerization is fixed in the eluent, and 50 ml of anhydrous ethanol is poured into a beaker for magnetic stirring to remove the cortisol molecules in the polymer on the electrode surface.

[0040] (8) Dry the electrode in an oven at 50 °C and observe its morphology using SEM.

[0041] Through this example Figure 1It can be seen that carboxylated cellulose nanofibers, carboxylated multi-walled carbon nanotubes and gold nanoparticles make the electrode surface rough, and the electrode modification materials without chitosan protective layer fall off.

[0042] Embodiment 2:

[0043] (1) Prepare a mixed solution of 0.015 g of carboxylated cellulose nanofibers, 0.03 g of carboxylated multi-walled carbon nanotubes, and 3 ml of deionized water, a mixed solution of 0.015 g of carboxylated cellulose nanofibers, 0.015 g of carboxylated multi-walled carbon nanotubes, and 3 ml of deionized water, and a mixed solution of 0.015 g of carboxylated cellulose nanofibers, 0.0075 g of carboxylated multi-walled carbon nanotubes, and 3 ml of deionized water, respectively, to obtain carboxylated cellulose nanofiber / carboxylated multi-walled carbon nanotube composite materials in different proportions.

[0044] (2) Rinse the screen-printed electrode with deionized water and dry it in an oven at 50°C for later use. Take 15 μL of the composite solution obtained in step 1 and drop it onto the screen-printed electrode. Dry the electrode in an oven at 50°C for later use.

[0045] (3) Prepare a 5 mM chloroauric acid solution, take 50 μL of the chloroauric acid solution and drop it onto the electrode, deposit it at a constant potential at a voltage of -0.2 V for 120 s, and dry the electrode in an oven at 50°C.

[0046] (4) Prepare a 0.05 M hydrochloric acid solution, place the chitosan powder in the hydrochloric acid solution, and prepare a 4 mg / mL chitosan solution.

[0047] (5) Take 15 μL of the chitosan solution obtained in step 4 and drop it onto the screen-printed electrode. Dry the electrode in an oven at 50° C. for later use.

[0048] (6) Prepare 54 mg of o-phenylenediamine, 18 mg of cortisol, and 100 ml of 0.1 M pH = 5 acetate buffer solution (sodium acetate + acetic acid) as the polymerization solution. Prepare the molecular imprinting membrane by electropolymerization. Electropolymerization conditions: scanning speed 50 mV / s, potential range 0-1 V, cycle number 25 times, and perform CV polymerization.

[0049] (7) The electrode obtained after electropolymerization is fixed in the eluent, and 50 ml of anhydrous ethanol is poured into a beaker for magnetic stirring to remove the cortisol molecules in the polymer on the electrode surface.

[0050] (8) Prepare 0.041 g of potassium ferrocyanide, 0.046 g of potassium ferrocyanide, 0.373 g of potassium chloride, and 50 ml of deionized water as a redox probe solution.

[0051] (9) Take 50 μL of the redox probe solution obtained in step 2 and drop it onto the screen-printed electrode to test CV and its sensing ability.

[0052] Through this example Figure 4 It can be seen that the electrical signal response is maximum when the weight ratio of carboxylated cellulose nanofibers to carboxylated multi-walled carbon nanotubes is 1:2.

[0053] Embodiment 3:

[0054] (1) The steps are the same as those in Example 2.

[0055] (2) 50 μL of cortisol solution of different concentrations was drop-coated on the screen-printed electrode and incubated for ten minutes. The electrode was then rinsed with deionized water and dried in an oven at 50°C for later use.

[0056] (3) Take 50 μL of the redox probe solution and drop it onto the screen-printed electrode to test the DPV and its sensing ability.

[0057] Through this example Figure 3-5 It can be seen that carboxylated cellulose nanofibers, carboxylated multi-walled carbon nanotubes and gold nanoparticles increase the specific surface area and electrical properties of the electrode, enabling the MIP sensing electrode to detect cortisol solutions of different concentrations.

[0058] Embodiment 4:

[0059] (1) The steps are the same as those in Example 1. (without chitosan protective layer)

[0060] (2) 50 μL of cortisol solution of different concentrations was drop-coated on the screen-printed electrode and incubated for ten minutes. The electrode was then rinsed with deionized water and dried in an oven at 50°C for later use.

[0061] (3) Prepare 0.041 g of potassium ferrocyanide, 0.046 g of potassium ferrocyanide, 0.373 g of potassium chloride, and 50 ml of deionized water as a redox probe solution.

[0062] (4) Take 50 μL of the redox probe solution obtained in step 3 and drop it onto the screen-printed electrode to test DPV and its sensing ability.

[0063] Through this example Figure 6 It can be seen that the electrode modification material without the chitosan protective layer has fallen off, resulting in unstable test current and inability to test different concentrations of cortisol.

[0064] Obviously, the above are only preferred embodiments of the present invention and are not intended to limit the present invention. As long as they are within the principles of the present invention, any modifications, equivalent substitutions, improvements, etc., 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 carboxylated cellulose nanofibers and a carboxylated multi-walled carbon nanotube composite material, gold nanoparticles, 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 the cortisol molecular imprinting sensing electrode according to claim 1, characterized in that: The method comprises the following steps: Step 1: modifying the screen-printed electrode with a composite material of carboxylated cellulose nanofibers and carboxylated multi-walled carbon nanotubes to obtain carboxylated cellulose nanofibers / carboxylated multi-walled carbon nanotubes / screen-printed electrodes; Step 2: electroplating the surface of carboxylated cellulose nanofibers / carboxylated multi-walled carbon nanotubes / screen-printed electrodes with chloroauric acid to obtain carboxylated cellulose nanofibers / carboxylated multi-walled carbon nanotubes / gold nanoparticles / screen-printed electrodes; Step 3: Modify carboxylated cellulose nanofibers / carboxylated multi-walled carbon nanotubes / gold nanoparticles / screen-printed electrodes using chitosan solution to obtain carboxylated cellulose nanofibers / carboxylated multi-walled carbon nanotubes / gold nanoparticles / chitosan / screen-printed electrodes Step 4: providing a mixed solution containing functional monomers and cortisol molecules, performing an electrochemical polymerization reaction on the surface of the carboxylated cellulose nanofibers / carboxylated multi-walled carbon nanotubes / gold nanoparticles / chitosan / screen-printed electrodes by cyclic voltammetry, eluting and removing the cortisol molecules in an eluent, leaving a cortisol molecule cavity, and finally obtaining a cortisol molecular imprinting sensing electrode.

3. The preparation method according to claim 2, characterized in that: The steps of modifying the screen-printed electrode with carboxylated cellulose nanofibers and carboxylated multi-walled carbon nanotubes in step 1 are as follows: Step 1: Place carboxylated multi-walled carbon nanotube powder in deionized water to prepare a 5 mg / mL solution. Step 2: Add carboxylated cellulose nanofiber powder to the solution obtained in step 1, stir magnetically for 2 hours, and control the weight ratio of cellulose nanofiber to multi-walled carbon nanotube to be between 2:1 and 1:

2. Step 3: Take 14-16 μL of the composite solution obtained in step 2, drop it onto the screen-printed electrode, and dry the electrode in an oven at 45-55°C.

4. The preparation method according to claim 2, characterized in that: The steps of carboxylated cellulose nanofibers / carboxylated multi-walled carbon nanotubes / gold nanoparticles / screen-printed electrodes in step 2 are as follows: Step 1: Place chloroauric acid powder in deionized water to prepare a 4-6 mM solution. Step 2: Take 40-60 μL of the solution obtained in step 1, drop it onto the electrode, deposit it at a constant potential of -0.2 V for 110-130 s, and dry the electrode in an oven at 45-55°C.

5. The preparation method according to claim 2, characterized in that: In step 4, 4.5-5.5 mM o-phenylenediamine, 0.45-0.55 mM cortisol, and 0.1 M acetate buffer solution (PH=5) are prepared as the polymerization solution.

6. The preparation method according to claim 2, characterized in that: The parameters of the electrochemical polymerization reaction in step 4 are: scanning speed 30 to 70 mV / s, potential range -1 to 1 V, and cycle number 20 to 30 times.

7. The preparation method according to claim 2, characterized in that: In step 4, anhydrous ethanol is used as an eluent, the electrode obtained in claim 6 is fixed in the eluent, 40 to 60 ml of anhydrous ethanol is poured into a beaker for magnetic stirring to remove cortisol molecules in the polymer on the electrode surface.

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